Posts Tagged ‘protein misfolding’

Is CRISPR a Solution to Familial Amyloid Polyneuropathy?

Author and Curator: Larry H. Bernstein, MD, FCAP



What FAP is

Familial amyloid polyneuropathy (FAP), also called transthyretin-related hereditary amyloidosis, transthyretin amyloidosis abbreviated also as ATTR ( hereditary form), or Corino de Andrade’s disease,[1] is an autosomal dominant[2] neurodegenerative disease. It is a form of amyloidosis, and was first identified and described by Portugueseneurologist Mário Corino da Costa Andrade, in 1952.[3] FAP is distinct from senile systemic amyloidosis (SSA), which is not inherited, and which was determined to be the primary cause of death for 70% of supercentenarians who have been autopsied.[4]

Usually manifesting itself between 20 and 40 years of age, it is characterized by pain, paresthesia, muscular weakness and autonomic dysfunction. In its terminal state, the kidneys and the heart are affected. FAP is characterized by the systemic deposition of amyloidogenic variants of the transthyretin protein, especially in the peripheral nervous system, causing a progressive sensory and motor polyneuropathy.

FAP is caused by a mutation of the TTR gene, located on human chromosome 18q12.1-11.2.[5] A replacement of valine by methionine at position 30 (TTR V30M) is the mutation most commonly found in FAP.[1] The variant TTR is mostly produced by the liver.[citation needed] The transthyretin protein is a tetramer. The tetramer has to dissociate into misfolded monomers to aggregate into a variety of structures including amyloid fibrils. Because most patients are heterozygotes, they deposit both mutant and wild type TTR subnits.

FAP is inherited in an autosomal dominant manner.[2] This means that the defective gene responsible for the disorder is located on anautosome (chromosome 18 is an autosome), and only one copy of the defective gene is sufficient to cause the disorder, when inherited from a parent who has the disorder.

FAP can be ameliorated by liver transplantation.  Wikipedia  https://en.wikipedia.org/wiki/Transthyretin-related_hereditary_amyloidosis


ATTRV30M Amyloidosis

Synonym(s) ATTRV30M-related amyloidosis
Familial amyloid polyneuropathy type I (Portuguese-Swedish-Japanese Type)
TTR amyloid neuropathy
Transthyretin amyloid neuropathy
Transthyretin amyloid polyneuropathy
Prevalence Unknown
Inheritance Autosomal dominant
Age of onset Adult


  • Familial amyloid polyneuropathy (FAP) or transthyretin (TTR) amyloid polyneuropathy is a progressive sensorimotor and autonomic neuropathy of adulthood onset. Weight loss and cardiac involvement are frequent; ocular or renal complications may also occur. The prevalence worldwide is unknown, but the prevalence in the general population in Japan has recently been estimated at around 1 per million.
  • FAP is clinically heterogeneous, with the clinical presentation depending on the genotype and geographic origin. FAP usually presents as a length-dependent sensory polyneuropathy with autonomic disturbances. Inaugural manifestations are paresthesiae, pain or trophic lesions of the feet, gastrointestinal disorders or weight loss. The most pronounced sensory loss involves pain and temperature sensation. Motor loss occurs later. Autonomic features include postural hypotension, and gastrointestinal and genitourinary disorders.
  • FAP is transmitted as an autosomal dominant trait and is caused by mutations in the TTR gene (18q12.1).  100 TTR mutations have been identified so far and are associated with varying patterns of organ involvement, age of onset and disease progression. The most common variant is the TTR Val30Met substitution for which several endemic foci have been identified most notably from Portugal, Japan and Sweden. However, the Val30Met phenotype varies between these countries.
  • Detection of amyloid-associated TTR mutations is required for diagnosis. However, identification of a disease-causing mutation is not considered as diagnostic because penetrance is variable. Clinical observation and tissue biopsy (from the nerve or kidney, labial salivary glands, subcutaneous fat tissue or rectal mucosa) are required for a definitive diagnosis: amyloid deposits are characterized by Congo red staining on light microscopy and green birefringence on polarized light microscopy.
  • The differential diagnosis should include diabetic neuropathy, chronic inflammatory demyelinating polyneuropathy (see this term), and light chain (AL), gelsolin and apolipoprotein A1 amyloidosis (see these terms). Antenatal diagnosis through chorionic villus sampling should be proposed to patients with early-onset (< 40 years) forms of FAP.
  • Genetic counseling should be offered to affected families and presymptomatic detection of relatives of an index case is important to allow early diagnosis.
  • Management of FAP should be multidisciplinary, involving a neurologist, geneticist, cardiologist and liver surgeon. Liver transplantation (LT) is currently the only treatment for preventing synthesis of the amyloidogenic variants of TTR. LT can stop progression of the disease during its early stages. Symptomatic treatments are essential for sensorimotor and autonomic neuropathy and visceral complications.
  • FAP is a severe and disabling disease. Severe cardiac, renal and ocular manifestations may develop. Death occurs within a mean interval of 10.8 years after onset of the inaugural symptoms and may occur suddenly or may be secondary to infections or cachexia.


Lancet Neurol. 2011 Dec;10(12):1086-97. doi: 10.1016/S1474-4422(11)70246-0.

Familial amyloid polyneuropathy

Planté-Bordeneuve V1Said GAuthor information

Familial amyloid polyneuropathies (FAPs) are a group of life-threatening multisystem disorders transmitted as an autosomal dominant trait. Nerve lesions are induced by deposits of amyloid fibrils, most commonly due to mutated transthyretin (TTR). Less often the precursor of amyloidosis is mutant apolipoprotein A-1 or gelsolin. The first identified cause of FAP-the TTR Val30Met mutation-is still the most common of more than 100 amyloidogenic point mutations identified worldwide. The penetrance and age at onset of FAP among people carrying the same mutation vary between countries. The symptomatology and clinical course of FAP can be highly variable. TTR FAP typically causes a nerve length-dependent polyneuropathy that starts in the feet with loss of temperature and pain sensations, along with life-threatening autonomic dysfunction leading to cachexia and death within 10 years on average. TTR is synthesised mainly in the liver, and liver transplantation seems to have a favourable effect on the course of neuropathy, but not on cardiac or eye lesions. Oral administration of tafamidis meglumine, which prevents misfolding and deposition of mutated TTR, is under evaluation in patients with TTR FAP. In future, patients with FAP might benefit from gene therapy; however, genetic counselling is recommended for the prevention of all types of FAP.

The course and prognostic factors of familial amyloid polyneuropathy after liver transplantation

David Adams, Didier Samuel, Catherine Goulon-Goeau, …, Henri Bismuth, Gérard Said

DOI: http://dx.doi.org/10.1093/brain/123.7.1495 1495-1504

Familial amyloid polyneuropathy (FAP) associated with mutations of the transthyretin (TTR) gene is the most common type of FAP, a devastating disease causing death within 10 years after the first symptoms. Because most of the amyloidogenic mutated TTR is secreted by the liver, transplantation is widely used to treat these patients, but long-term quantitative evaluation of the effects of liver transplantation on the progression of the neuropathy are not available. We have treated 45 patients with symptomatic TTR-FAP, including 43 with the Met30 TTR gene mutation, and report on the results of periodic evaluation of markers of neuropathy in 25 of them, who have been followed for more than 2 years after liver transplantation (mean follow-up 4 years). The overall survival rates at 1 and 5 years were 82 and 60%, respectively. Urinary incontinence and a low Norris score at liver transplantation were associated with poorer outcome. The motor score stabilized in seven of 11 patients (64%) with mild sensorimotor neuropathy (walking unaided) and in two of the eight patients (25%) with severe sensorimotor deficit (walking with aid) at liver transplantation. In five other patients, deterioration of motor deficit occurred only within the first year after liver transplantation, but was progressive after this interval in two patients. None of the six patients with pure sensory neuropathy developed motor loss and superficial sensory loss remained unchanged. Two years after liver transplantation, the rate of myelinated axon loss in nerve biopsy specimens was markedly lower in seven transplanted patients (0.9/mm2 of endoneurial area/month) than in non-transplanted patients (70/mm2 of endoneurial area/month). Symptoms of dysautonomia and quantitated cardiocirculatory autonomic tests remained unchanged. In all patients, serum mutated TTR decreased to 2.5% of pre-liver transplantation values and remained at this level during follow-up. We presently recommend liver transplantation in FAP patients at onset of first symptoms and exclusion of those with a Norris score below 55 and/or with urinary incontinence.

Familial Amyloid Polyneuropathy (Amyloidotic transthyretin related neuropathy [ATTR]

Transthyretin (TTR) amyloidosis is an autosomal dominant disorder caused by the deposition of insoluble amyloid fibrils around peripheral nerves and in various tissues, including the heart muscle. Based on the predominant organ involvement, several distinct subtypes have been reported.

Familial amyloid polyneuropathy (FAP) aka TTR amyloid neuropathy is characterized by slowly progressive, peripheral sensorimotor polyneuropathy and autonomic dysfunction. Disease onset is usually in the third to fourth decade of life. Sensory neuropathy starts in the lower extremities with paresthesia, impaired pain and temperature sensation, followed by loss of motor function. Autonomic neuropathy usually manifests with orthostatic hypotension, constipation alternating with diarrhea, vomiting, impotence or hypohidrosis. Unrelated to neuropathy, other organs manifestations may include cardiomyopathy, vitreous opacities and CNS amyloidosis.

Leptomeningeal amyloidosis aka oculoleptomeningeal amyloidosis affects predominantly the central nervous system, sometimes combined with visual impairment.

Cardiac amyloidosis usually manifests in the sixth decade of life with progressive left ventricular hypertrophy and restrictive cardiomyopathy. In a subset of families with cardiac amyloidosis, peripheral neuropathy may be completely absent or very mild.

Treatment: Currently, the only effective treatment for FAP is an orthotopic liver transplant to stop production of misfolded amyloid protein. In patients with severe amyloid cardiomyopathy, a heart transplant may be necessary. Different drugs designed to prevent or alleviate accumulation of TTR amyloid protein (transthyretin amyloidois inhibitors) are currently under investigation.

Contemporary Reviews in Cardiovascular Medicine

  • Transthyretin (TTR) Cardiac Amyloidosis

Frederick L. Ruberg, John L. Berk

Circulation.2012; 126: 1286-1300      doi: 10.1161/CIRCULATIONAHA.111.078915

The systemic amyloidoses are a family of diseases induced by misfolded or misassembled proteins. Extracellular deposition of these proteins as soluble or insoluble cross β-sheets disrupts vital organ function.1 More than 27 different precursor proteins have the propensity to form amyloid fibrils.2 The particular precursor protein that misfolds to form amyloid fibrils defines the amyloid type and predicts the patient’s clinical course. Several types of amyloid can infiltrate the heart, resulting in progressive diastolic and systolic dysfunction, congestive heart failure, and death. Treatment of cardiac amyloidosis is dictated by the amyloid type and degree of involvement. Consequently, early recognition and accurate classification are essential.3

The diagnosis of amyloidosis requires histological identification of amyloid deposits. Congo Red staining renders amyloid deposits salmon pink by light microscopy, with a characteristic apple green birefringence under polarized light conditions (Figure 1). Additional immunohistochemical staining for precursor proteins identifies the type of amyloidosis (Figure 2).4 Ultimately, immunogold electron microscopy and mass spectrometry confer the greatest sensitivity and specificity for amyloid typing.5,6


Transthyretin participates in beta-amyloid transport from the brain to the liver- involvement of the low-density lipoprotein receptor-related protein 1?

Mobina AlemiCristiana GaiteiroCarlos Alexandre RibeiroLuís Miguel Santos,João Rodrigues GomesSandra Marisa Oliveira,  ….., Maria João Saraiva & Isabel Cardoso

Scientific Reports 6, Article number: 20164 (2016)    doi:10.1038/srep20164

Transthyretin (TTR) binds Aβ peptide, preventing its deposition and toxicity. TTR is decreased in Alzheimer’s disease (AD) patients. Additionally, AD transgenic mice with only one copy of the TTR gene show increased brain and plasma Aβ levels when compared to AD mice with both copies of the gene, suggesting TTR involvement in brain Aβ efflux and/or peripheral clearance. Here we showed that TTR promotes Aβ internalization and efflux in a human cerebral microvascular endothelial cell line, hCMEC/D3. TTR also stimulated brain-to-blood but not blood-to-brain Aβ permeability in hCMEC/D3, suggesting that TTR interacts directly with Aβ at the blood-brain-barrier. We also observed that TTR crosses the monolayer of cells only in the brain-to-blood direction, as confirmed by in vivo studies, suggesting that TTR can transport Aβ from, but not into the brain. Furthermore, TTR increased Aβ internalization by SAHep cells and by primary hepatocytes from TTR+/+ mice when compared to TTR−/− animals. We propose that TTR-mediated Aβ clearance is through LRP1, as lower receptor expression was found in brains and livers of TTR−/− mice and in cells incubated without TTR. Our results suggest that TTR acts as a carrier of Aβ at the blood-brain-barrier and liver, using LRP1.

Transthyretin (Prealbumin) in Health and Disease: Nutritional Implications

Annual Review of Nutrition

Vol. 14: 495-533 (Volume publication date July 1994)

DOI: 10.1146/annurev.nu.14.070194.002431

Y Ingenbleek, and V Young


Plasma Transthyretin as a Biomarker of Lean Body Mass and Catabolic States1,2

Yves Ingenbleek3,* and Larry H Bernstein4

Adv Nutr Sep 2015; 6:572-580, 2015    doi: 10.3945/an.115.008508

Plasma transthyretin (TTR) is a plasma protein secreted by the liver that circulates bound to retinol-binding protein 4 (RBP4) and its retinol ligand. TTR is the sole plasma protein that reveals from birth to old age evolutionary patterns that are closely superimposable to those of lean body mass (LBM) and thus works as the best surrogate analyte of LBM. Any alteration in energy-to-protein balance impairs the accretion of LBM reserves and causes early depression of TTR production. In acute inflammatory states, cytokines induce urinary leakage of nitrogenous catabolites, deplete LBM stores, and cause an abrupt decrease in TTR and RBP4 concentrations. As a result, thyroxine and retinol ligands are released in free form, creating a second frontline that strengthens that primarily initiated by cytokines. Malnutrition and inflammation thus keep in check TTR and RBP4 secretion by using distinct and unrelated physiologic pathways, but they operate in concert to downregulate LBM stores. The biomarker complex integrates these opposite mechanisms at any time and thereby constitutes an ideally suited tool to determine residual LBM resources still available for metabolic responses, hence predicting outcomes of the most interwoven disease conditions.


Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma
Hans E. Purkey, Michael I. Dorrell, and Jeffery W. Kelly*
Department of Chemistry and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB12, La Jolla, CA 92037

Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembly into amyloid fibrils that putatively cause senile systemic amyloidosis and familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting amyloid fibril formation in vitro. A method is presented here to evaluate the binding selectivity of these inhibitors to TTR in human plasma, a complex biological fluid composed of more than 60 proteins and numerous small molecules. Our immunoprecipitation approach isolates TTR and bound small molecules from a biological fluid such as plasma, and quantifies the amount of small molecules bound to the protein by HPLC analysis. This approach demonstrates that only a small subset of the inhibitors that saturate the TTR binding sites in vitro do so in plasma. These selective inhibitors can now be tested in animal models of TTR amyloid disease to probe the validity of the amyloid hypothesis. This method could be easily extended to evaluate small molecule binding selectivity to any protein in a given biological fluid without the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distribution, metabolism, activity, and toxicity of potential drugs.

Amyloid diseases are characterized by the conversion of soluble proteins or peptides into insoluble b-sheet-rich amyloid fibrils. There are currently 17 different human proteins known to form amyloid fibrils in vivo (1–4). These fibrils, or their oligomeric precursors, are thought to cause pathology either through disruption of normal cellular function or by direct toxicity (5–8). X-ray fibril diffraction and electron microscopy reconstruction of amyloid fibrils reveal filaments that have a lamellar cross b-sheet structure wrapped around one another (9–13). Folded proteins form amyloid fibrils through partial unfolding triggered by a change of local environment, a mutation in the protein, or both (8, 14–20).

Transthyretin (TTR) is a tetrameric protein composed of identical 127-aa subunits that fold into a b-sandwich tertiary structure. It is found in both the plasma (3.6 mM) and cerebrospinal fluid (CSF) (0.36 mM) of humans. The TTR tetramer has two negatively cooperative C2 symmetric thyroxine (T4)-binding sites (21–23). In the CSF, it binds and transports the thyroid hormone T4 and the retinol-binding protein (RBP), which in turn transports vitamin A. In the plasma, only 10–15% of TTR has T4 bound to it, as thyroid-binding globulin has an order of magnitude higher affinity for T4 than does TTR (24). TTR fibril formation is linked to two amyloid diseases in which the fibrils are composed of full-length protein. Deposition of wild-type TTR is associated with cardiac dysfunction in the disease senile systemic amyloidosis (SSA) (25, 26). More than 70 different single-site mutants have been linked to early-onset amyloid deposition in diseases with a spectrum of clinical manifestations, collectively referred to as familial amyloid polyneuropathy (FAP) (27–35).

We have discovered compounds, through both screening and structure-based design, that dramatically inhibit TTR amyloid fibril formation in vitro (36–42). To stabilize the TTR tetramer and thus prevent amyloid fibril formation in SSA and FAP, these small molecules must be able to selectively bind to TTR in human blood plasma over all other plasma proteins. Possible competitors include thyroid-binding globulin (TBG), which has an order of magnitude higher affinity for TTR’s natural ligand, T4; and albumin, which binds numerous hydrophobic small molecules and is present at a concentration two orders of magnitude higher than TTR, as well as the other plasma proteins. Historically, one was forced to choose two or three of the most likely protein competitors and evaluate their relative affinities for the small molecule in comparison to the protein of interest. The advantage of the approach outlined within this article is that the binding selectivity of TTR amyloid inhibitors in human plasma is determined without having to make assumptions as to which proteins may competitively bind the TTR ligand. Compounds that bind to TTR selectively in plasma are the best candidates for further evaluation in animal models and, ultimately, in human clinical trials.


Analysis of Nonsteroidal Antiinflammatory Drugs (NSAIDs)

The first compounds evaluated for selective binding to TTR in plasma were the NSAIDs previously identified to be potent TTR amyloid inhibitors in vitro (37, 42). Because these compounds are already approved by the Food and Drug Administration, they could easily be evaluated in human clinical trials for another indication if they proved to be selective TTR binders in human plasma. However, none of the NSAIDs exhibited significant selectivity for binding TTR in human plasma at a concentration of 10.8 mM (Table 1), although most exhibit a submicromolar Kd (37, 50). The most selective NSAIDs, flufenamic acid (1) and mefenamic acid (2), only had 0.2 eq of a maximum of 2 molar eq bound to TTR. However, fenoprofen (7), flurbiprofen (6), flufenamic acid (1), mefenamic acid (2), and diflunisal (4) all have maximal therapeutic plasma concentrations exceeding 20 mM (51, 52). When these compounds were incubated with plasma at their maximal therapeutic concentrations, flufenamic acid and diflunisal showed increased binding selectivity (stoichiometry) to TTR (Table 1). Diflunisal (4) is notable in that its 224 mM maximal therapeutic concentration leads to 0.85 eq of drug bound to TTR. Increasing the concentration of all other NSAIDs to their maximal therapeutic dose did not result in dramatic increases in binding selectivity, likely because of binding to other plasma proteins.

Analysis of the Remaining Lead Compounds. Approximately 40 additional small molecule amyloid inhibitors were evaluated for their ability to bind to TTR in plasma by using our immunoprecipitationyHPLC approach (Fig. 2). These compounds were derived from screening or structure-based design and identified as promising by an in vitro fibril formation assay (refs. 40–42; V. H. Oza, H. M. Petrassi, and J.W.K., unpublished data). Lead compounds having diverse structures including biaryls, biarylamines, stilbenes, and dibenzofurans showed promising selectivity (Table 2). Three compounds from this group (9-11) possess excellent TTR-binding selectivity in plasma. At a concentration of 10.8 mM, they exhibit saturation of .1 of the two possible binding sites in the TTR tetramer. Determination of the TTR-binding affinities of these three compounds in buffer shows that the Kd values do not correlate with the plasma binding selectivity (Table 2). For example, inhibitors 9 and 10 have greater than an order of magnitude difference in Kd but nearly identical binding selectivity (stoichiometry) to TTR in plasma. Moreover, compounds with modest binding selectivity, such as flufenamic acid (1), have been previously determined to have Kd values very similar to those exhibiting excellent selectivities (37). Mass spectrometry confirmed the identity of inhibitors 9-12 isolated by immunoprecipitation HPLC as the compounds that were initially incubated with plasma.

Binding to plasma proteins is an important factor in determining the overall distribution, metabolism, activity, and toxicity of a drug (55). In this particular case, we desire small molecules that bind to the plasma protein TTR over protein competitors whose identities are not known and likely change with the small molecule under evaluation. This binding is known to stabilize TTR’s normally folded state, thus preventing the conformational changes required for amyloidogenicity (14, 36). The immunoprecipitationyHPLCbased binding selectivity method outlined above allows quantification of the stoichiometry of small molecule binding to TTR in human plasma in the presence of all other plasma proteins and numerous competing small molecules without the use of tags, such as radiolabels, on the small molecule. Immunoprecipitation has been used previously to determine the stoichiometry of metal ion binding to specific plasma proteins (56, 57). However, to our knowledge, this is the first use of the technique to determine the binding selectivity of a small molecule to a single protein in human plasma. In principle, this approach is applicable to evaluate the binding selectivity of small molecules to any plasma protein, provided that a highly selective antibody for the protein can be generated, binding does not block the epitope or destroy it by conformational change, and appropriate wash steps can be introduced to avoid nonspecific binding of the small molecule and other proteins to the resin.


The Kelly Group Research

Transthyretin amyloid diseases: understanding the mechanism of proteotoxicity and inhibition of amyloid fibril formation

Figure 1: Three dimensional structure of the flufenamic acid-transthyretin tetramer complex. The small organic molecule, flufenamic acid, inhibits the conformational changes of transthyretin associated with amyloid fibril formation. Courtesy of Steven Johnson.

Transthyretin is a 55 kDa homotetrameric protein (Figure 1) that transports L-thyroxine and holo-retinol binding protein in the serum and cerebrospinal fluid of humans. We discovered that conformational changes alone enable transthyretin aggregation. Transthyretin amyloid formation in vivo is initiated by dissociation of the native tetramer under a denaturation stress of unknown origin. Subsequently, the resulting monomers can partially denature and misassemble into amyloid fibrils and other amorphous aggregates. Aggregation by transthyretin monomers under acidic conditions (conditions that render transthyretin amyloidogenesis fast on a laboratory time scale) occurs via a downhill polymerization mechanism, which means that every step along the amyloid formation pathway is energetically favorable and fast relative to tetramer dissociation. Thus, tetramer dissociation is rate limiting for transthyretin amyloid formation in the case of the wild type protein and for the vast majority of mutants.

Stabilizing the native tetrameric state of transthyretin should ameliorate transthyretin amyloid diseases. This hypothesis is supported by the observation of trans-suppression, in which compound heterozygotes expressing both a disease-associated mutation (e.g., V30M) and a trans-suppressor mutation (e.g., T119M) do not develop transthyretin amyloid disease. We have shown that the trans-suppressor mutation T119M inhibits amyloid formation by kinetically stabilizing (i.e., dramatically slowing the dissociation of) mixed transthyretin tetramers.

We have designed numerous small molecules based on the crystal structures of transthyretin that are now established to avidly bind to the unoccupied L-thyroxine binding sites within transthyretin. We have shown that small molecule binding to these sites inhibits amyloid formation in vitro by selectively stabilizing the native tetrameric state over the dissociative transition state, thus raising the energetic barrier for tetramer dissociation, dramatically slowing the rate-limiting step in the aggregation pathway. Over the past decade, we have identified and synthesized over 1000 small molecule inhibitors of transthyretin amyloid formation that group into half a dozen distinct families. The inhibitors are typically composed of two differentially-substituted aromatic rings connected by linkers of variable chemical composition.

These small molecule kinetic stabilizers either just bind to transthyretin or bind and then react chemoselectively with only one of eight lysine ε-amino groups within transthyretin. In collaboration with Dr. Ian Wilson (The Scripps Research Institute, Department of Molecular Biology), we systematically ranked a myriad of possibilities for the three substructures composing a typical transthyretin kinetic stabilizer. We used these data in a substructure combination strategy to develop very potent and selective transthyretin kinetic stabilizers. In collaboration with Dr. Joel Buxbaum (The Scripps Research Institute, Department of Molecular and Experimental Medicine), these and other compounds are being tested in cell and mouse models.

These data should allow us to be able to predict the structures of potent and selective transthyretin amyloidogenesis inhibitors that are largely devoid of characteristics undesirable for a clinical candidate.

In a recently completed placebo-controlled, double-blind clinical trial carried out by FoldRx Pharmaceuticals (a company that Kelly cofounded), benzoxazoles discovered by the Kelly Laboratory and developed by FoldRx Pharmaceuticals proved safe and effective at halting the progression of familial amyloid polyneuropathy by a myriad of metrics. This transthyretin kinetic stabilizer, now named Tafamidis, provides the first pharmacological evidence that the process of amyloid fibril formation causes the transthyretin amyloid diseases—reinvigorating efforts of other investigators and companies to do the same in other amyloid diseases. In collaboration with Dr. Martha Skinner and Dr. John Berk at Boston University, another kinetic stabilizer discovered by the Kelly Laboratory, diflunisal, is being tested in a second placebo-controlled human clinical trial for familial amyloid polyneuropathy that is currently enrolling patients.

In collaboration with Dr. Bill Balch (The Scripps Research Institute, Department of Cell Biology) and Dr. Luke Wiseman (The Scripps Research Institute, Department of Molecular and Experimental Medicine), we investigated the relationship between the secretion of destabilized transthyretin variants and the pathology of the disease they cause. The earliest onset (onset at 20-30 years of age) and most severe transthyretin amyloid diseases are generally associated with mutations that strongly destabilize the transthyretin tetramer, resulting in facile transthyretin dissociation and misfolded monomer misassembly into aggregates in the peripheral nerves. However, the most destabilized variants characterized to date, A25T and D18G transthyretin, do not cause an early onset systemic amyloid disease because they are intercepted by the degradation component of the proteostasis network within liver cells—the liver is where most of the transthyretin in the blood plasma is produced. Because of endoplasmic reticulum-associated degradation of these highly destabilized transthyretin variants within the secretory pathway of liver cells, the concentration of the destabilized mutant transthyretin in blood plasma would not be high enough to enable the amyloidogenesis responsible for pathology. Interestingly, these mutants lead to a very rare brain disease, with an intermediate age of onset (40-50 years old), because the choroid plexus is more permissive in its ability to secrete highly destabilized transthyretin variants for reasons that we are seeking to understand. These results suggest that endoplasmic reticulum-assisted folding mediated by the proteostasis network determines protein secretion in a tissue-specific manner, and we propose that its competition with endoplasmic reticulum-associated degradation may explain the appearance of tissue-selective amyloid diseases.

Is there a CRISPR alternative?

CRISPR biotech Intellia strikes licensing deal with Regeneron, readies IPO


Dive Brief:

  • Intellia Therapeutics, one of several companies working to develop CRISPR/Cas 9 technology commercially, on Monday filed to go public in an initially priced $120 million IPO. Concurrently, the biotech announced a collaboration and licensing deal with Regeneron Pharmaceuticals aimed at advancing up to 10 CRISPR-based programs, focusing primarily on liver diseases. 
  • Regeneron will pay Intellia $75 million upfront, as well as investing $50 million in Intellia’s forthcoming IPO. Additionally, the agreement includes as much as $320 million in milestone payments, according to SEC filing documents
  • The first program to be co-developed with Regeneron will target a rare genetic disorder known as ATTR, which can causes severely impaired nerve or cardiac function. 

Intellia hopes CRISPR gene-editing can cure the genetic disorder ATTR, which is caused by a buildup of the transthyretin (TTR) protein in tissue. By “knocking out” TTR expression in the liver, Intellia could reduce or eliminate the disease-causing buildup.

A view by Larry H. Bernstein, MD, FCAP

I can only wish them success in a project that may well be a search for the Trojan Horse.  TTR is also synthesized in the choroid plexus.  In addition, knocking out TTR expression in the liver is not likely to fit a mechanism leading to TTR buildup in tissue.  The TTR is a plasma tetrameric transport protein released into the circulation for transport of TH with a bound retinol-binding protein carrying one retinol per tetrameric protein.  Other considerations are the nuclear release and the cell binding that have been extensively studied.  The buildup in tissues is a misfolded protein – amyloid, which has extensively studied fibrils.  Another issue is the actual functional transport of hormone and delivery of retinoid to cross the blood brain barrier.


Transthyretin participates in betaamyloid transport from the brain to the liver- involvement of the lowdensity lipoprotein receptor-related protein 1?

Mobina Alemi1,2,3, Cristiana Gaiteiro1,2, Carlos Alexandre Ribeiro1,2, Luís Miguel Santos1,2, João Rodrigues Gomes1,2, Sandra Marisa Oliveira1,2,3, Pierre-Olivier Couraud4, Babette Weksler5, Ignacio Romero6, Maria João Saraiva1,2 & Isabel Cardoso1


Distinctive binding and structural properties of piscine transthyretin

Claudia Follia, Nicola Pasquatob, Ileana Ramazzinaa, Roberto Battistuttab;c, Giuseppe Zanottib;c, Rodolfo Bernia;
FEBS Letters 555 (2003) 279-284

The thyroid hormone binding protein transthyretin (TTR) forms a macromolecular complex with the retinol-specific carrier retinol binding protein (RBP) in the blood of higher vertebrates. Piscine TTR is shown here to exhibit high binding affnity for L-thyroxine and negligible affnity for RBP. The 1.56 A % resolution X-ray structure of sea bream TTR, compared with that of human TTR, reveals a high degree of conservation of the thyroid hormone binding sites. In contrast, some amino acid differences in discrete regions of sea bream TTR appear to be responsible for the lack of protein-protein recognition, providing evidence for the crucial role played by a limited number of residues in the interaction between RBP and TTR. Overall, this study makes it possible to draw conclusions on evolutionary relationships for RBPs and TTRs of phylogenetically distant vertebrates.

Jeffery Kelly, Ph.D., Lita Annenberg Hazen Professor of Chemistry
Chairman, Department of Molecular and Experimental Medicine
California Campus, Scripps Institute
The Skaggs Institute for Chemical Biology
San Diego, CA

Studies directed at understanding the principles of b-sheet folding utilizing rapid kinetic and thermodynamic measurements are ongoing and focused on the WW domain, a 34-residue 3-stranded b-sheet. Chemical synthesis of the WW domain allows us to incorporate unique amino acids into the fold to probe structural determinants of transition state and ground state structure.

Awards & Professional Activities

  • Searle Scholar Award in Biomedical Sciences, 1991-1994
  • Camille and Henry Dreyfus Teacher Scholar Award, 1994
  • Texas A&M University Teacher Scholar Award, 1994-1995
  • The Biophysical Society National Lecturer, 1999
  • The Protein Society – Dupont Young Investigator Award, 1999
  • Alumni Distinguished Achievement Award, State University of New York at Fredonia, 2000
  • Arthur C. Cope Scholar Award, American Chemical Society, 2001

Selected References

Deechongkit, S.; Nguyen, H.; Dawson, P.E.; Gruebele, M.; Kelly, J.W. “Context Dependent Contributions of Backbone H-Bonding to b-Sheet Folding Energetics” Nature 2004, 430, 101-105.

Cohen,F.: Kelly, J.W. “Therapeutic Approaches to Protein Folding Diseases” Nature, 2003, 426, 905-910.

Hammarstrom, P.; Wiseman, R.L.; Powers, E.T.; Kelly, J.W., “Prevention of Transthyretin Amyloid Disease by Changing Protein Misfolding Energetics” Science 2003, 299, 713-716.

Sawkar, A.; Cheng, W-C.; Beutler, E.; Wong, C.-H.; Balch, W.E.; Kelly, J.W. “Chemical Chaperones Increase the Cellular Activity of N370S β glucosidase; A Therapeutic Strategy for Gaucher’s Disease” Proc.Natl.Acad.Sci., 2002, 99, 15428-15433.


The Skaggs Institute for Chemical Biology

TSRI Scientists Discover Therapeutic Strategy for “Misfolding Diseases” Analogous to Alzheimer’s Disease

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Beyond tau and amyloid

Larry H. Bernstein, MD, FCAP, Curator






Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders.

Berislav V. Zlokovic

Nature Reviews Neuroscience 12, 723-738 (December 2011) |   http:dx.doi.org:/10.1038/nrn3114

The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood–brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal ‘milieu’, which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer’s disease, and highlights therapeutic opportunities relating to these neurovascular deficits.



The neurovascular unit comprises vascular cells (endothelial cells, pericytes and vascular smooth muscle cells (VSMCs)), glial cells (astrocytes, microglia and oliogodendroglia) and neurons.
Neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) are associated with microvascular dysfunction and/or degeneration in the brain, neurovascular disintegration, defective blood–brain barrier (BBB) function and/or vascular factors.
The interactions between endothelial cells and pericytes are crucial for the formation and maintenance of the BBB. Indeed, pericyte deficiency leads to BBB breakdown and extravasation of multiple vasculotoxic and neurotoxic circulating macromolecules, which can contribute to neuronal dysfunction, cognitive decline and neurodegenerative changes.
Alterations in cerebrovascular metabolic functions can also lead to the secretion of multiple neurotoxic and inflammatory factors.
BBB dysfunction and/or breakdown and cerebral blood flow (CBF) reductions and/or dysregulation may occur in sporadic Alzheimer’s disease and experimental models of this disease before cognitive decline, amyloid-β deposition and brain atrophy. In patients with ALS and in some experimental models of ALS, CBF dysregulation, blood–spinal cord barrier breakdown and spinal cord hypoperfusion have been reported prior to motor neuron cell death.
Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder have shown diminished amyloid-β clearance from brain tissue. The recognition of amyloid-β clearance pathways opens exciting new therapeutic opportunities for this disease.
‘Multiple-target, multiple-action’ agents will stand a better chance of controlling the complex disease mechanisms that mediate neurodegeneration in disorders such as Alzheimer’s disease than will agents that have only one target. According to the vasculo-neuronal-inflammatory triad model of neurodegenerative disorders, in addition to neurons, brain endothelium, VSMCs, pericytes, astrocytes and activated microglia all represent important therapeutic targets.


Neurons depend on blood vessels for their oxygen and nutrient supplies, and for the removal of carbon dioxide and other potentially toxic metabolites from the brain’s interstitial fluid (ISF). The importance of the circulatory system to the human brain is highlighted by the fact that although the brain comprises ~2% of total body mass, it receives up to 20% of cardiac output and is responsible for ~20% and ~25% of the body’s oxygen consumption and glucose consumption, respectively1. To underline this point, when cerebral blood flow (CBF) stops, brain functions end within seconds and damage to neurons occurs within minutes2.

Neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) are associated with microvascular dysfunction and/or degeneration in the brain, neurovascular disintegration, defective blood–brain barrier (BBB) function and/or vascular factors1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Microvascular deficits diminish CBF and, consequently, the brain’s supply of oxygen, energy substrates and nutrients. Moreover, such deficits impair the clearance of neurotoxic molecules that accumulate and/or are deposited in the ISF, non-neuronal cells and neurons. Recent evidence suggests that vascular dysfunction leads to neuronal dysfunction and neurodegeneration, and that it might contribute to the development of proteinaceous brain and cerebrovascular ‘storage’ disorders. Such disorders include cerebral β-amyloidosis and cerebral amyloid angiopathy (CAA), which are caused by accumulation of the peptide amyloid-β in the brain and the vessel wall, respectively, and are features of Alzheimer’s disease1.

In this Review, I will discuss neurovascular pathways to neurodegeneration, placing a focus on Alzheimer’s disease because more is known about neurovascular dysfunction in this disease than in other neurodegenerative disorders. The article first examines transport mechanisms for molecules to cross the BBB, before exploring the processes that are involved in BBB breakdown at the molecular and cellular levels, and the consequences of BBB breakdown, hypoperfusion, and hypoxia and endothelial metabolic dysfunction for neuronal function. Next, the article reviews evidence for neurovascular changes during normal ageing and neurovascular BBB dysfunction in various neurodegenerative diseases, including evidence suggesting that vascular defects precede neuronal changes. Finally, the article considers specific mechanisms that are associated with BBB dysfunction in Alzheimer’s disease and ALS, and therapeutic opportunities relating to these neurovascular deficits.

The neurovascular unit

The neurovascular unit (NVU) comprises vascular cells (that is, endothelium, pericytes and vascular smooth muscle cells (VSMCs)), glial cells (that is, astrocytes, microglia and oliogodendroglia) and neurons1,2, 13 (Fig. 1). In the NVU, the endothelial cells together form a highly specialized membrane around blood vessels. This membrane underlies the BBB and limits the entry of plasma components, red blood cells (RBCs) and leukocytes into the brain. The BBB also regulates the delivery into the CNS of circulating energy metabolites and essential nutrients that are required for proper neuronal and synaptic function. Non-neuronal cells and neurons act in concert to control BBB permeability and CBF. Vascular cells and glia are primarily responsible for maintenance of the constant ‘chemical’ composition of the ISF, and the BBB and the blood–spinal cord barrier (BSCB) work together with pericytes to prevent various potentially neurotoxic and vasculotoxic macromolecules in the blood from entering the CNS, and to promote clearance of these substances from the CNS1.

In the brain, pial arteries run through the subarachnoid space (SAS), which contains the cerebrospinal fluid (CSF). These vessels give rise to intracerebral arteries, which penetrate into brain parenchyma. Intracerebral arteries are separated from brain parenchyma by a single, interrupted layer of elongated fibroblast-like cells of the pia and the astrocyte-derived glia limitans membrane that forms the outer wall of the perivascular Virchow–Robin space. These arteries branch into smaller arteries and subsequently arterioles, which lose support from the glia limitans and give rise to pre-capillary arterioles and brain capillaries. In an intracerebral artery, the vascular smooth muscle cell (VSMC) layer occupies most of the vessel wall. At the brain capillary level, vascular endothelial cells and pericytes are attached to the basement membrane. Pericyte processes encase most of the capillary wall, and they communicate with endothelial cells directly through synapse-like contacts containing connexins and N-cadherin. Astrocyte end-foot processes encase the capillary wall, which is composed of endothelium and pericytes. Resting microglia have a ‘ramified’ shape and can sense neuronal injury.

Figure 2 | Blood–brain barrier transport mechanisms.

Small lipophilic drugs, oxygen and carbon dioxide diffuse across the blood–brain barrier (BBB), whereas ions require ATP-dependent transporters such as the (Na++K+)ATPase. Transporters for nutrients include the glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)), the lactate transporter monocarboxylate transporter 1 (MCT1) and the L1 and y+ transporters for large neutral and cationic essential amino acids, respectively. These four transporters are expressed at both the luminal and albuminal membranes. Non-essential amino acid transporters (the alanine, serine and cysteine preferring system (ASC), and the alanine preferring system (A)) and excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are located at the abluminal side. The ATP-binding cassette (ABC) efflux transporters that are found in the endothelial cells include multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) and solute carrier organic anion transporter family member 1C1 (OATP1C1). Finally, transporters for peptides or proteins include the endothelial protein C receptor (EPCR) for activated protein C (APC); the insulin receptors (IRs) and the transferrin receptors (TFRs), which are associated with caveolin 1 (CAV1); low-density lipoprotein receptor-related protein 1 (LRP1) for amyloid-β, peptide transport system 1 (PTS1) for encephalins; and the PTS2 and PTS4–vasopressin V1a receptor (V1AR) for arginine vasopressin.


Transport across the blood–brain barrier. The endothelial cells that form the BBB are connected by tight and adherens junctions, and it is the tight junctions that confer the low paracellular permeability of the BBB1. Small lipophilic molecules, oxygen and carbon dioxide diffuse freely across the endothelial cells, and hence the BBB, but normal brain endothelium lacks fenestrae and has limited vesicular transport.

The high number of mitochondria in endothelial cells reflects a high energy demand for active ATP-dependent transport, conferred by transporters such as the sodium pump ((Na++K+)ATPase) and the ATP-binding cassette (ABC) efflux transporters. Sodium influx and potassium efflux across the abluminal side of the BBB is controlled by (Na++K+)ATPase (Fig. 2). Changes in sodium and potassium levels in the ISF influence the generation of action potentials in neurons and thus directly affect neuronal and synaptic functions1, 12.

Brain endothelial cells express transporters that facilitate the transport of nutrients down their concentration gradients, as described in detail elsewhere1, 14 (Fig. 2). Glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)) — the BBB-specific glucose transporter — is of special importance because glucose is a key energy source for the brain.

Monocarboxylate transporter 1 (MCT1), which transports lactate, and the L1 and y+ amino acid transporters are expressed at the luminal and abluminal membranes12, 14. Sodium-dependent excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are expressed at the abluminal side of the BBB15 and enable removal of glutamate, an excitatory neurotransmitter, from the brain (Fig. 2). Glutamate clearance at the BBB is essential for protecting neurons from overstimulation of glutaminergic receptors, which is neurotoxic16.

ABC transporters limit the penetration of many drugs into the brain17. For example, multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) controls the rapid removal of ingested toxic lipophilic metabolites17 (Fig. 2). Some ABC transporters also mediate the efflux of nutrients from the endothelium into the ISF. For example, solute carrier organic anion transporter family member 1C1 (OATP1C1) transports thyroid hormones into the brain. MCT8 mediates influx of thyroid hormones from blood into the endothelium18 (Fig. 2).

The transport of circulating peptides across the BBB into the brain is restricted or slow compared with the transport of nutrients19. Carrier-mediated transport of neuroactive peptides controls their low levels in the ISF20, 21, 22, 23, 24 (Fig. 2). Some proteins, including transferrin, insulin, insulin-like growth factor 1 (IGF1), leptin25, 26, 27 and activatedprotein C (APC)28, cross the BBB by receptor-mediated transcytosis (Fig. 2).

Circumventricular organs. Several small neuronal structures that surround brain ventricles lack the BBB and sense chemical changes in blood or the cerebrospinal fluid (CSF) directly. These brain areas are known as circumventricular organs (CVOs). CVOs have important roles in multiple endocrine and autonomic functions, including the control of feeding behaviour as well as regulation of water and salt metabolism29. For example, the subfornical organ is one of the CVOs that are capable of sensing extracellular sodium using astrocyte-derived lactate as a signal for local neurons to initiate neural, hormonal and behavioural responses underlying sodium homeostasis30. Excessive sodium accumulation is detrimental, and increases in plasma sodium above a narrow range are incompatible with life, leading to cerebral oedema (swelling), seizures and death29.

Vascular-mediated pathophysiology

The key pathways of vascular dysfunction that are linked to neurodegenerative diseases include BBB breakdown, hypoperfusion–hypoxia and endothelial metabolic dysfunction (Fig. 3). This section examines processes that are involved in BBB breakdown at the molecular and cellular levels, and explores the consequences of all three pathways for neuronal function and viability.

Figure 3 | Vascular-mediated neuronal damage and neurodegeneration.

a | Blood–brain barrier (BBB) breakdown that is caused by pericyte detachment leads to leakage of serum proteins and focal microhaemorrhages, with extravasation of red blood cells (RBCs). RBCs release haemoglobin, which is a source of iron. In turn, this metal catalyses the formation of toxic reactive oxygen species (ROS) that mediate neuronal injury. Albumin promotes the development of vasogenic oedema, contributing to hypoperfusion and hypoxia of the nervous tissue, which aggravates neuronal injury. A defective BBB allows several potentially vasculotoxic and neurotoxic proteins (for example, thrombin, fibrin and plasmin) to enter the brain. b | Progressive reductions in cerebral blood flow (CBF) lead to increasing neuronal dysfunction. Mild hypoperfusion, oligaemia, leads to a decrease in protein synthesis, whereas more-severe reductions in CBF, leading to hypoxia, cause an array of detrimental effects.

Blood–brain barrier breakdown. Disruption to tight and adherens junctions, an increase in bulk-flow fluid transcytosis, and/or enzymatic degradation of the capillary basement membrane cause physical breakdown of the BBB.

The levels of many tight junction proteins, their adaptor molecules and adherens junction proteins decrease in Alzheimer’s disease and other diseases that cause dementia1, 9, ALS31, multiple sclerosis32 and various animal models of neurological disease8, 33. These decreases might be partly explained by the fact that vascular-associated matrix metalloproteinase (MMP) activity rises in many neurodegenerative disorders and after ischaemic CNS injury34, 35; tight junction proteins and basement membrane extracellular matrix proteins are substrates for these enzymes34. Lowered expression of messenger RNAs that encode several key tight junction proteins, however, has also been reported in some neurodegenerative disorders, such as ALS31.

Endothelial cell–pericyte interactions are crucial for the formation36, 37and maintenance of the BBB33, 38. Pericyte deficiency can lead to a reduction in expression of certain tight junction proteins, including occludin, claudin 5 and ZO1 (Ref. 33), and to an increase in bulk-flow transcytosis across the BBB, causing BBB breakdown38. Both processes can lead to extravasation of multiple small and large circulating macromolecules (up to 500 kDa) into the brain parenchyma33, 38. Moreover, in mice, an age-dependent progressive loss of pericytes can lead to BBB disruption and microvasular degeneration and, subsequently, neuronal dysfunction, cognitive decline and neurodegenerative changes33. In their lysosomes, pericytes concentrate and degrade multiple circulating exogenous39 and endogenous proteins, including serum immunoglobulins and fibrin33, which amplify BBB breakdown in cases of pericyte deficiency.

BBB breakdown typically leads to an accumulation of various molecules in the brain. The build up of serum proteins such as immunoglobulins and albumin can cause brain oedema and suppression of capillary blood flow8, 33, whereas high concentrations of thrombin lead to neurotoxicity and memory impairment40, and accelerate vascular damage and BBB disruption41. The accumulation of plasmin (derived from circulating plasminogen) can catalyse the degradation of neuronal laminin and, hence, promote neuronal injury42, and high fibrin levels accelerate neurovascular damage6. Finally, an increase in the number of RBCs causes deposition of haemoglobin-derived neurotoxic products including iron, which generates neurotoxic reactive oxygen species (ROS)8, 43(Fig. 3a). In addition to protein-mediated vasogenic oedema, local tissue ischaemia–hypoxia depletes ATP stores, causing (Na++K+)ATPase pumps and Na+-dependent ion channels to stop working and, consequently, the endothelium and astrocytes to swell (known as cytotoxic oedema)44. Upregulation of aquaporin 4 water channels in response to ischaemia facilitates the development of cytotoxic oedema in astrocytes45.

Hypoperfusion and hypoxia. CBF is regulated by local neuronal activity and metabolism, known as neurovascular coupling46. The pial and intracerebral arteries control the local increase in CBF that occurs during brain activation, which is termed ‘functional hyperaemia’. Neurovascular coupling requires intact pial circulation, and for VSMCs and pericytes to respond normally to vasoactive stimuli33, 46, 47. In addition to VSMC-mediated constriction and vasodilation of cerebral arteries, recent studies have shown that pericytes modulate brain capillary diameter through constriction of the vessel wall47, which obstructs capillary flow during ischaemia48. Astrocytes regulate the contractility of intracerebral arteries49, 50.

Progressive CBF reductions have increasingly serious consequences for neurons (Fig. 3b). Briefly, mild hypoperfusion — termed oligaemia — affects protein synthesis, which is required for the synaptic plasticity mediating learning and memory46. Moderate to severe CBF reductions and hypoxia affect ATP synthesis, diminishing (Na++K+)ATPase activity and the ability of neurons to generate action potentials9. In addition, such reductions can lower or increase pH, and alter electrolyte balances and water gradients, leading to the development of oedema and white matter lesions, and the accumulation of glutamate and proteinaceous toxins (for example, amyloid-β and hyperphopshorylated tau) in the brain. A reduction of greater than 80% in CBF results in neuronal death2.

The effect of CBF reductions has been extensively studied at the molecular and cellular levels in relation to Alzheimer’s disease. Reduced CBF and/or CBF dysregulation occurs in elderly individuals at high risk of Alzheimer’s disease before cognitive decline, brain atrophy and amyloid-β accumulation10, 46, 51, 52, 53, 54. In animal models, hypoperfusion can induce or amplify Alzheimer’s disease-like neuronal dysfunction and/or neuropathological changes. For example, bilateral carotid occlusion in rats causes memory impairment, neuronal dysfunction, synaptic changes and amyloid-β oligomerization55, leading to accumulation of neurotoxic amyloid-β oligomers56. In a mouse model of Alzheimer’s disease, oligaemia increases neuronal amyloid-β levels and neuronal tau phosphophorylation at an epitope that is associated with Alzheimer’s disease-type paired helical filaments57. In rodents, ischaemia leads to the accumulation of hyperphosphorylated tau in neurons and the formation of filaments that resemble those present in human neurodegenerative tauopathies and Alzheimer’s disease58. Mice expressing amyloid-β precursor protein (APP) and transforming growth factor β1 (TGFβ1) develop deficient neurovascular coupling, cholinergic denervation, enhanced cerebral and cerebrovascular amyloid-β deposition, and age-dependent cognitive decline59.

Recent studies have shown that ischaemia–hypoxia influences amyloidogenic APP processing through mechanisms that increase the activity of two key enzymes that are necessary for amyloid-β production; that is, β-secretase and γ-secretase60, 61, 62, 63. Hypoxia-inducible factor 1α (HIF1α) mediates transcriptional increase in β-secretase expression61. Hypoxia also promotes phosphorylation of tau through the mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase (ERK)) pathway64, downregulates neprilysin — an amyloid-β-degrading enzyme65 — and leads to alterations in the expression of vascular-specific genes, including a reduction in the expression of the homeobox protein MOX2 gene mesenchyme homeobox 2 (MEOX2) in brain endothelial cells5 and an increase in the expression of the myocardin gene (MYOCD) in VSMCs66. In patients with Alzheimer’s disease and in models of this disorder, these changes cause vessel regression, hypoperfusion and amyloid-β accumulation resulting from the loss of the key amyloid-β clearance lipoprotein receptor (see below). In addition, hypoxia facilitates alternative splicing of Eaat2 mRNA in Alzheimer’s disease transgenic mice before amyloid-β deposition67 and suppresses glutamate reuptake by astrocytes independently of amyloid formation68, resulting in glutamate-mediated neuronal injury that is independent of amyloid-β.

In response to hypoxia, mitochondria release ROS that mediate oxidative damage to the vascular endothelium and to the selective population of neurons that has high metabolic activity. Such damage has been suggested to occur before neuronal degeneration and amyloid-β deposition in Alzheimer’s disease69, 70. Although the exact triggers of hypoxia-mediated neurodegeneration and the role of HIF1α in neurodegeneration versus preconditioning-mediated neuroprotection remain topics of debate, mitochondria-generated ROS seem to have a primary role in the regulation of the HIF1α-mediated transcriptional switch that can activate an array of responses, ranging from mechanisms that increase cell survival and adaptation to mechanisms inducing cell cycle arrest and death71. Whether inhibition of hypoxia-mediated pathogenic pathways will delay onset and/or control progression in neurodegenerative conditions such as Alzheimer’s disease remains to be determined.

When comparing the contributions of BBB breakdown and hypoperfusion to neuronal injury, it is interesting to consider Meox2+/− mice. Such animals have normal pericyte coverage and an intact BBB but a substantial perfusion deficit5 that is comparable to that found in pericyte-deficient mice that develop BBB breakdown33 Notably, however, Meox2+/− mice show less pronounced neurodegenerative changes than pericyte-deficient mice, indicating that chronic hypoperfusion–hypoxia alone can cause neuronal injury, but not to the same extent as hypoperfusion–hypoxia combined with BBB breakdown.

Endothelial neurotoxic and inflammatory factors. Alterations in cerebrovascular metabolic functions can lead to the secretion of multiple neurotoxic and inflammatory factors72, 73. For example, brain microvessels that have been isolated from individuals with Alzheimer’s disease (but not from neurologically normal age-matched and young individuals) and brain microvessels that have been treated with inflammatory proteins release neurotoxic factors that kill neurons74, 75. These factors include thrombin, the levels of which increase with the onset of Alzheimer’s disease76. Thrombin can injure neurons directly40and indirectly by activating microglia and astrocytes73. Compared with those from age-matched controls, brain microvessels from individuals with Alzheimer’s disease secrete increased levels of multiple inflammatory mediators, such as nitric oxide, cytokines (for example, tumour necrosis factor (TNF), TGFβ1, interleukin-1β (IL-1β) and IL-6), chemokines (for example, CC-chemokine ligand 2 (CCL2; also known as monocyte chemoattractant protein 1 (MCP1)) and IL-8), prostaglandins, MMPs and leukocyte adhesion molecules73. Endothelium-derived neurotoxic and inflammatory factors together provide a molecular link between vascular metabolic dysfunction, neuronal injury and inflammation in Alzheimer’s disease and, possibly, in other neurodegenerative disorders.

Neurovascular changes

This section examines evidence for neurovascular changes during normal ageing and for neurovascular and/or BBB dysfunction in various neurodegenerative diseases, as well as the possibility that vascular defects can precede neuronal changes.

Age-associated neurovascular changes. Normal ageing diminishes brain circulatory functions, including a detectable decay of CBF in the limbic and association cortices that has been suggested to underlie age-related cognitive changes77. Alterations in the cerebral microvasculature, but not changes in neural activity, have been shown to lead to age-dependent reductions in functional hyperaemia in the visual system in cats78 and in the sensorimotor cortex in pericyte-deficient mice33. Importantly, a recent longitudinal CBF study in neurologically normal individuals revealed that people bearing the apolipoprotein E (APOE) ɛ4allele — the major genetic risk factor for late-onset Alzheimer’s disease79, 80, 81 — showed greater regional CBF decline in brain regions that are particularly vulnerable to pathological changes in Alzheimer’s disease than did people without this allele82.

A meta-analysis of BBB permeability in 1,953 individuals showed that neurologically healthy humans had an age-dependent increase in vascular permeability83. Moreover, patients with vascular or Alzheimer’s disease-type dementia and leucoaraiosis — a small-vessel disease of the cerebral white matter — had an even greater age-dependent increase in vascular permeability83. Interestingly, an increase in BBB permeability in brain areas with normal white matter in patients with leukoaraiosis has been suggested to play a causal part in disease and the development of lacunar strokes84. Age-related changes in the permeability of the blood–CSF barrier and the choroid plexus have been reported in sheep85.

Vascular pathology. Patients with Alzheimer’s disease or other dementia-causing diseases frequently show focal changes in brain microcirculation. These changes include the appearance of string vessels (collapsed and acellular membrane tubes), a reduction in capillary density, a rise in endothelial pinocytosis, a decrease in mitochondrial content, accumulation of collagen and perlecans in the basement membrane, loss of tight junctions and/or adherens junctions3, 4, 5, 6, 9,46, 86, and BBB breakdown with leakage of blood-borne molecules4, 6,7, 9. The time course of these vascular alterations and how they relate to dementia and Alzheimer’s disease pathology remain unclear, as no protocol that allows the development of the diverse brain vascular pathology to be scored, and hence to be tracked with ageing, has so far been developed and widely validated87. Interestingly, a recent study involving 500 individuals who died between the ages of 69 and 103 years showed that small-vessel disease, infarcts and the presence of more than one vascular pathological change were associated with Alzheimer’s disease-type pathological lesions and dementia in people aged 75 years of age87. These associations were, however, less pronounced in individuals aged 95 years of age, mainly because of a marked ageing-related reduction in Alzheimer’s disease neuropathology relative to a moderate but insignificant ageing-related reduction in vascular pathology87.

Accumulation of amyloid-β and amyloid deposition in pial and intracerebral arteries results in CAA, which is present in over 80% of Alzheimer’s disease cases88. In patients who have Alzheimer’s disease with established CAA in small arteries and arterioles, the VSMC layer frequently shows atrophy, which causes a rupture of the vessel wall and intracerebral bleeding in about 30% of these patients89, 90. These intracerebral bleedings contribute to, and aggravate, dementia. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type have accelerated VSMC degeneration resulting in haemorrhagic strokes and dementia91. Duplication of the gene encoding APP causes early-onset Alzheimer’s disease dementia with CAA and intracerebral haemorrhage92.

Early studies of serum immunoglobulin leakage reported that patients with ALS had BSCB breakdown and BBB breakdown in the motor cortex93. Microhaemorrhages and BSCB breakdown have been shown in the spinal cord of transgenic mice expressing mutant variants of human superoxide dismutase 1 (SOD1), which in mice cause an ALS-like disease8, 94, 95. In mice with ALS-like disease and in patients with ALS, BSCB breakdown has been shown to occur before motor neuron degeneration or brain atrophy8, 11, 95.

BBB breakdown in the substantia nigra and the striatum has been detected in murine models of Parkinson’s disease that are induced by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)96, 97, 98. However, the temporal relationship between BBB breakdown and neurodegeneration in Parkinson’s disease is currently unknown. Notably, the prevalence of CAA and vascular lesions increases in Parkinson’s disease99, 100. Vascular lesions in the striatum and lacunar infarcts can cause vascular parkinsonism syndrome101. A recent study reported BBB breakdown in a rat model of Huntington’s disease that is induced with the toxin 3-nitropropionic acid102.

Several studies have established disruption of BBB with a loss of tight junction proteins during neuroinflammatory conditions such as multiple sclerosis and its murine model, experimental allergic encephalitis. Such disruption facilitates leukocyte infiltration, leading to oliogodendrocyte death, axonal damage, demyelination and lesion development32.

Functional changes in the vasculature. In individuals with Alzheimer’s disease, GLUT1 expression at the BBB decreases103, suggesting a shortage in necessary metabolic substrates. Studies using18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) have identified reductions in glucose uptake in asymptomatic individuals with a high risk of dementia104, 105. Several studies have suggested that reduced glucose uptake across the BBB, as seen by FDG PET, precedes brain atrophy104, 105, 106, 107, 108.

Amyloid-β constricts cerebral arteries109. In a mouse model of Alzheimer’s disease, impairment of endothelium-dependent regulation of neocortical microcirculation110, 111 occurs before amyloid-β accumulation. Recent studies have shown that CD36, a scavenger receptor that binds amyloid-β, is essential for the vascular oxidative stress and diminished functional hyperaemia that occurs in response to amyloid-β exposure112. Neuroimaging studies in patients with Alzheimer’s disease have shown that neurovascular uncoupling occurs before neurodegenerative changes10, 51, 52, 53. Moreover, cognitively normal APOE ɛ4 carriers at risk of Alzheimer’s disease show impaired CBF responses to brain activation in the absence of neurodegenerative changes or amyloid-β accumulation54. Recently, patients with Alzheimer’s disease as well as mouse models of this disease with high cerebrovascular levels of serum response factor (SRF) and MYOCD, the two transcription factors that control VSMC differentiation, have been shown to develop a hypercontractile arterial phenotype resulting in brain hypoperfusion, diminished functional hyperaemia and CAA66, 113. More work is needed to establish the exact role of SRF and MYOCD in the vascular dysfunction that results in the Alzheimer’s disease phenotype and CAA.

PET studies with 11C-verapamil, an ABCB1 substrate, have indicated that the function of ABCB1, which removes multiple drugs and toxins from the brain, decreases with ageing114 and is particularly compromised in the midbrain of patients with Parkinson’s disease, progressive supranuclear palsy or multiple system atrophy115. More work is needed to establish the exact roles of ABC BBB transporters in neurodegeneration and whether their failure precedes the loss of dopaminergic neurons that occurs in Parkinson’s disease.

In mice with ALS-like disease and in patients with ALS, hypoperfusion and/or dysregulated CBF have been shown to occur before motor neuron degeneration or brain atrophy8, 116. Reduced regional CBF in basal ganglia and reduced blood volume have been reported in pre-symptomatic gene-tested individuals at risk for Huntington’s disease117. Patients with Huntington’s disease display a reduction in vasomotor activity in the cerebral anterior artery during motor activation118.

Vascular and neuronal common growth factors. Blood vessels and neurons share common growth factors and molecular pathways that regulate their development and maintenance119, 120. Angioneurins are growth factors that exert both vasculotrophic and neurotrophic activities121. The best studied angioneurin is vascular endothelial growth factor (VEGF). VEGF regulates vessel formation, axonal growth and neuronal survival120. Ephrins, semaphorins, slits and netrins are axon guidance factors that also regulate the development of the vascular system121. During embryonic development of the neural tube, blood vessels and choroid plexus secrete IGF2 into the CSF, which regulates the proliferation of neuronal progenitor cells122. Genetic and pharmacological manipulations of angioneurin activity yielded various vascular and cerebral phenotypes121. Given the dual nature of angioneurin action, these studies have not been able to address whether neuronal dysfunction results from a primary insult to neurons and/or whether it is secondary to vascular dysfunction.

Increased levels of VEGF, a hypoxia-inducible angiogenic factor, were found in the walls of intraparenchymal vessels, perivascular deposits, astrocytes and intrathecal space of patients with Alzheimer’s disease, and were consistent with the chronic cerebral hypoperfusion and hypoxia that were observed in these individuals73. In addition to VEGF, brain microvessels in Alzheimer’s disease release several molecules that can influence angiogenesis, including IL-1β, IL-6, IL-8, TNF, TGFβ, MCP1, thrombin, angiopoietin 2, αVβ3 and αVβ5 integrins, and HIF1α73. However, evidence for increased vascularity in Alzheimer’s disease is lacking. On the contrary, several studies have reported that focal vascular regression and diminished microvascular density occur in Alzheimer’s disease4, 5, 73 and in Alzheimer’s disease transgenic mice123. The reason for this discrepancy is not clear. The anti-angiogenic activity of amyloid-β, which accumulates in the brains of individuals with Alzheimer’s disease and Alzheimer’s disease models, may contribute to hypovascularity123. Conversely, genome-wide transcriptional profiling of brain endothelial cells from patients with Alzheimer’s disease revealed that extremely low expression of vascular-restricted MEOX2 mediates aberrant angiogenic responses to VEGF and hypoxia, leading to capillary death5. This finding raises the interesting question of whether capillary degeneration in Alzheimer’s disease results from unsuccessful vascular repair and/or remodelling. Moreover, mice that lack one Meox2 allele have been shown to develop a primary cerebral endothelial hypoplasia with chronic brain hypoperfusion5, resulting in secondary neurodegenerative changes33.

Does vascular dysfunction cause neuronal dysfunction? In summary, the evidence that is discussed above clearly indicates that vascular dysfunction is tightly linked to neuronal dysfunction. There are many examples to illustrate that primary vascular deficits lead to secondary neurodegeneration, including CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts), an hereditary small-vessel brain disease resulting in multiple small ischaemic lesions, neurodegeneration and dementia124; mutations in SLC2A1 that cause dysfunction of the BBB-specific GLUT1 transporter in humans resulting in seizures; cognitive impairment and microcephaly125; microcephaly and epileptiform discharges in mice with genetic deletion of a single Slc2a1allele126; and neurodegeneration mediated by a single Meox2 homebox gene deletion restricted to the vascular system33. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type provide another example showing that primary vascular dysfunction — which in this case is caused by deposition of vasculotropic amyloid-β mutants in the arterial vessel wall — leads to dementia and intracerebral bleeding. Moreover, as reviewed in the previous sections, recent evidence suggests that BBB dysfunction and/or breakdown, and CBF reductions and/or dysregulation may occur in sporadic Alzheimer’s disease and experimental models of this disease before cognitive decline, amyloid-β deposition and brain atrophy. In patients with ALS and in some experimental models of ALS, CBF dysregulation, BSCB breakdown and spinal cord hypoperfusion have been reported to occur before motor neuron cell death. Whether neurological changes follow or precede vascular dysfunction in Parkinson’s disease, Huntington’s disease and multiple sclerosis remains less clear. However, there is little doubt that vascular injury mediates, amplifies and/or lowers the threshold for neuronal dysfunction and loss in several neurological disorders.

Disease-specific considerations

This section examines how amyloid-β levels are kept low in the brain, a process in which the BBB has a central role, and how faulty BBB-mediated clearance mechanisms go awry in Alzheimer’s disease. On the basis of this evidence and the findings discussed elsewhere in the Review, a new hypothesis for the pathogenesis of Alzheimer’s disease that incorporates the vascular evidence is presented. ALS-specific disease mechanisms relating to the BBB are then examined.

Alzheimer’s disease. Amyloid-β clearance from the brain by the BBB is the best studied example of clearance of a proteinaceous toxin from the CNS. Multiple pathways regulate brain amyloid-β levels, including its production and clearance (Fig. 4). Recent studies127, 128, 129 have confirmed earlier findings in multiple rodent and non-human primate models demonstrating that peripheral amyloid-β is an important precursor of brain amyloid-β130, 131, 132, 133, 134, 135, 136. Moreover, peripheral amyloid-β sequestering agents such as soluble LRP1 (ref.137), anti-amyloid-β antibodies138, 139, 140, gelsolin and the ganglioside GM1 (Ref. 141), or systemic expression of neprilysin142, 143have been shown to reduce the amyloid burden in Alzheimer’s disease mice by eliminating contributions of the peripheral amyloid-β pool to the total brain pool of this peptide.

Figure 4 | The role of blood–brain barrier transport in brain homeostasis of amyloid-β.

Amyloid-β (Aβ) is produced from the amyloid-β precursor protein (APP), both in the brain and in peripheral tissues. Clearance of amyloid-β from the brain normally maintains its low levels in the brain. This peptide is cleared across the blood–brain barrier (BBB) by the low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 mediates rapid efflux of a free, unbound form of amyloid-β and of amyloid-β bound to apolipoprotein E2 (APOE2), APOE3 or α2-macroglobulin (not shown) from the brain’s interstitial fluid into the blood, and APOE4 inhibits such transport. LRP2 eliminates amyloid-β that is bound to clusterin (CLU; also known as apolipoprotein J (APOJ)) by transport across the BBB, and shows a preference for the 42-amino-acid form of this peptide. ATP-binding cassette subfamily A member 1 (ABCA1; also known as cholesterol efflux regulatory protein) mediates amyloid-β efflux from the brain endothelium to blood across the luminal side of the BBB (not shown). Cerebral endothelial cells, pericytes, vascular smooth muscle cells, astrocytes, microglia and neurons express different amyloid-β-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme (IDE), tissue plasminogen activator (tPA) and matrix metalloproteinases (MMPs), which contribute to amyloid-β clearance. In the circulation, amyloid-β is bound mainly to soluble LRP1 (sLRP1), which normally prevents its entry into the brain. Systemic clearance of amyloid-β is mediated by its removal by the liver and kidneys. The receptor for advanced glycation end products (RAGE) provides the key mechanism for influx of peripheral amyloid-β into the brain across the BBB either as a free, unbound plasma-derived peptide and/or by amyloid-β-laden monocytes. Faulty vascular clearance of amyloid-β from the brain and/or an increased re-entry of peripheral amyloid-β across the blood vessels into the brain can elevate amyloid-β levels in the brain parenchyma and around cerebral blood vessels. At pathophysiological concentrations, amyloid-β forms neurotoxic oligomers and also self-aggregates, which leads to the development of cerebral β-amyloidosis and cerebral amyloid angiopathy.

The receptor for advanced glycation end products (RAGE) mediates amyloid-β transport in brain and the propagation of its toxicity. RAGE expression in brain endothelium provides a mechanism for influx of amyloid-β144, 145 and amyloid-β-laden monocytes146 across the BBB, as shown in Alzheimer’s disease models (Fig. 4). The amyloid-β-rich environment in Alzheimer’s disease and models of this disorder increases RAGE expression at the BBB and in neurons147, 148, amplifying amyloid-β-mediated pathogenic responses. Blockade of amyloid-β–RAGE signalling in Alzheimer’s disease is a promising strategy to control self-propagation of amyloid-β-mediated injury.

Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder149 have shown that diminished amyloid-β clearance occurs in brain tissue in this disease. LRP1 plays an important part in the three-step serial clearance of this peptide from brain and the rest of the body150 (Fig. 4). In step one, LRP1 in brain endothelium binds brain-derived amyloid-β at the abluminal side of the BBB, initiating its clearance to blood, as shown in many animal models151, 152, 153, 154, 155, 156 and BBB models in vitro151, 157,158. The vasculotropic mutants of amyloid-β that have low binding affinity for LRP1 are poorly cleared from the brain or CSF151, 159, 160. APOE4, but not APOE3 or APOE2, blocks LRP1-mediated amyloid-β clearance from the brain and, hence, promotes its retention161, whereas clusterin (also known as apolipoprotein J (APOJ)) mediates amyloid-β clearance across the BBB via LRP2 (Ref. 153). APOE and clusterin influence amyloid-β aggregation162, 163. Reduced LRP1 levels in brain microvessels, perhaps in addition to altered levels of ABCB1, are associated with amyloid-β cerebrovascular and brain accumulation during ageing in rodents, non-human primates, humans, Alzheimer’s disease mice and patients with Alzheimer’s disease66, 151, 152, 164, 165, 166. Moreover, recent work has shown that brain LRP1 is oxidized in Alzheimer’s disease167, and may contribute to amyloid-β retention in brain because the oxidized form cannot bind and/or transport amyloid-β137. LRP1 also mediates the removal of amyloid-β from the choroid plexus168.

In step two, circulating soluble LRP1 binds more than 70% of plasma amyloid-β in neurologically normal humans137. In patients with Alzheimer’s disease or mild cognitive impairment (MCI), and in Alzheimer’s disease mice, amyloid-β binding to soluble LRP1 is compromised due to oxidative changes137, 169, resulting in elevated plasma levels of free amyloid-β isoforms comprising 40 or 42 amino acids (amyloid-β1–40 and amyloid-β1–42). These peptides can then re-enter the brain, as has been shown in a mouse model of Alzheimer’s disease137. Rapid systemic removal of amyloid-β by the liver is also mediated by LRP1 and comprises step three of the clearance process170.

In brain, amyloid-β is enzymatically degraded by neprilysin171, insulin-degrading enzyme172, tissue plasminogen activator173 and MMPs173,174 in various cell types, including endothelial cells, pericytes, astrocytes, neurons and microglia. Cellular clearance of this peptide by astrocytes and VSMCs is mediated by LRP1 and/or another lipoprotein receptor66, 175. Clearance of amyloid-β aggregates by microglia has an important role in amyloid-β-directed immunotherapy176 and reduction of the amyloid load in brain177. Passive ISF–CSF bulk flow and subsequent clearance through the CSF might contribute to 10–15% of total amyloid-β removal152, 153, 178. In the injured human brain, increasing soluble amyloid-β concentrations in the ISF correlated with improvements in neurological status, suggesting that neuronal activity might regulate extracellular amyloid-β levels179.

The role of BBB dysfunction in amyloid-β accumulation, as discussed above, underlies the contribution of vascular dysfunction to Alzheimer’s disease (see Fig. 5 for a model of vascular damage in Alzheimer’s disease). The amyloid hypothesis for the pathogenesis of Alzheimer’s disease maintains that this peptide initiates a cascade of events leading to neuronal injury and loss and, eventually, dementia180, 181. Here, I present an alternative hypothesis — the two-hit vascular hypothesis of Alzheimer’s disease — that incorporates the vascular contribution to this disease, as discussed in this Review (Box 1). This hypothesis states that primary damage to brain microcirculation (hit one) initiates a non-amyloidogenic pathway of vascular-mediated neuronal dysfunction and injury, which is mediated by BBB dysfunction and is associated with leakage and secretion of multiple neurotoxic molecules and/or diminished brain capillary flow that causes multiple focal ischaemic or hypoxic microinjuries. BBB dysfunction also leads to impairment of amyloid-β clearance, and oligaemia leads to increased amyloid-β generation. Both processes contribute to accumulation of amyloid-β species in the brain (hit two), where these peptides exert vasculotoxic and neurotoxic effects. According to the two-hit vascular hypothesis of Alzheimer’s disease, tau pathology develops secondary to vascular and/or amyloid-β injury.

Figure 5 | A model of vascular damage in Alzheimer’s disease.

a | In the early stages of Alzheimer’s disease, small pial and intracerebral arteries develop a hypercontractile phenotype that underlies dysregulated cerebral blood flow (CBF). This phenotype is accompanied by diminished amyloid-β clearance by the vascular smooth muscle cells (VSMCs). In the later phases of Alzheimer’s disease, amyloid deposition in the walls of intracerebral arteries leads to cerebral amyloid angiopathy (CAA), pronounced reductions in CBF, atrophy of the VSMC layer and rupture of the vessels causing microbleeds. b | At the level of capillaries in the early stages of Alzheimer’s disease, blood–brain barrier (BBB) dysfunction leads to a faulty amyloid-β clearance and accumulation of neurotoxic amyloid-β oligomers in the interstitial fluid (ISF), microhaemorrhages and accumulation of toxic blood-derived molecules (that is, thrombin and fibrin), which affect synaptic and neuronal function. Hyperphosphorylated tau (p-tau) accumulates in neurons in response to hypoperfusion and/or rising amyloid-β levels. At this point, microglia begin to sense neuronal injury. In the later stages of the disease in brain capillaries, microvascular degeneration leads to increased deposition of basement membrane proteins and perivascular amyloid. The deposited proteins and amyloid obstruct capillary blood flow, resulting in failure of the efflux pumps, accumulation of metabolic waste products, changes in pH and electrolyte composition and, subsequently, synaptic and neuronal dysfunction. Neurofibrillary tangles (NFTs) accumulate in response to ischaemic injury and rising amyloid-β levels. Activation of microglia and astrocytes is associated with a pronounced inflammatory response. ROS, reactive oxygen species.

Amyotrophic lateral sclerosis. The cause of sporadic ALS, a fatal adult-onset motor neuron neurodegenerative disease, is not known182. In a relatively small number of patients with inherited SOD1 mutations, the disease is caused by toxic properties of mutant SOD1 (Ref. 183). Mutations in the genes encoding ataxin 2 and TAR DNA-binding protein 43 (TDP43) that cause these proteins to aggregate have been associated with ALS182, 184. Some studies have suggested that abnormal SOD1 species accumulate in sporadic ALS185. Interestingly, studies in ALS transgenic mice expressing a mutant version of human SOD1 in neurons, and in non-neuronal cells neighbouring these neurons, have shown that deletion of this gene from neurons does not influence disease progression186, whereas deletion of this gene from microglia186 or astrocytes187 substantially increases an animal’s lifespan. According to an emerging hypothesis of ALS that is based on studies in SOD1 mutant mice, the toxicity that is derived from non-neuronal neighbouring cells, particularly microglia and astrocytes, contributes to disease progression and motor neuron degeneration186, 187, 188, 189, 190, whereas BBB dysfunction might be critical for disease initiation8, 43, 94, 95. More work is needed to determine whether this concept of disease initiation and progression may also apply to cases of sporadic ALS.

Human data support a role for angiogenic factors and vessels in the pathogenesis of ALS. For example, the presence of VEGF variations (which were identified in large meta-analysis studies) has been linked to ALS191. Angiogenin is another pro-angiogenic gene that is implicated in ALS because heterozygous missense mutations in angiogenin cause familial and sporadic ALS192. Moreover, mice with a mutation that eliminates hypoxia-responsive induction of the Vegf gene (Vegfδ/δ mice) develop late-onset motor neuron degeneration193. Spinal cord ischaemia worsens motor neuron degeneration and functional outcome in Vegfδ/δmice, whereas the absence of hypoxic induction of VEGF in mice that develop motor neuron disease from expression of ALS-linked mutant SOD1G93A results in substantially reduced survival191.

Therapeutic opportunities

Many investigators believe that primary neuronal dysfunction resulting from an intrinsic neuronal disorder is the key underlying event in human neurodegenerative diseases. Thus, most therapeutic efforts for neurodegenerative diseases have so far been directed at the development of so-called ‘single-target, single-action’ agents to target neuronal cells directly and reverse neuronal dysfunction and/or protect neurons from injurious insults. However, most preclinical and clinical studies have shown that such drugs are unable to cure or control human neurological disorders2, 181, 183, 194, 195. For example, although pathological overstimulation of glutaminergic NMDA receptors (NMDARs) has been shown to lead to neuronal injury and death in several disorders, including stroke, Alzheimer’s disease, ALS and Huntington’s disease16, NMDAR antagonists have failed to show a therapeutic benefit in the above-mentioned human neurological disorders.

Recently, my colleagues and I coined the term vasculo-neuronal-inflammatory triad195 to indicate that vascular damage, neuronal injury and/or neurodegeneration, and neuroinflammation comprise a common pathological triad that occurs in multiple neurological disorders. In line with this idea, it is conceivable that ‘multiple-target, multiple-action’ agents (that is, drugs that have more than one target and thus have more than one action) will have a better chance of controlling the complex disease mechanisms that mediate neurodegeneration than agents that have only one target (for example, neurons). According to the vasculo-neuronal-inflammatory triad model, in addition to neurons, brain endothelium, VSMCs, pericytes, astrocytes and activated microglia are all important therapeutic targets.

Here, I will briefly discuss a few therapeutic strategies based on the vasculo-neuronal-inflammatory triad model. VEGF and other angioneurins may have multiple targets, and thus multiple actions, in the CNS120. For example, preclinical studies have shown that treatment of SOD1G93A rats with intracerebroventricular VEGF196 or intramuscular administration of a VEGF-expressing lentiviral vector that is transported retrogradely to motor neurons in SOD1G93A mice197 reduced pathology and extended survival, probably by promoting angiogenesis and increasing the blood flow through the spinal cord as well as through direct neuronal protective effects of VEGF on motor neurons. On the basis of these and other studies, a phase I–II clinical trial has been initiated to evaluate the safety of intracerebroventricular infusion of VEGF in patients with ALS198. Treatment with angiogenin also slowed down disease progression in a mouse model of ALS199.

IGF1 delivery has been shown to promote amyloid-β vascular clearance and to improve learning and memory in a mouse model of Alzheimer’s disease200. Local intracerebral implantation of VEGF-secreting cells in a mouse model of Alzheimer’s disease has been shown to enhance vascular repair, reduce amyloid burden and improve learning and memory201. In contrast to VEGF, which can increase BBB permeability, TGFβ, hepatocyte growth factor and fibroblast growth factor 2 promote BBB integrity by upregulating the expression of endothelial junction proteins121 in a similar way to APC43. However, VEGF and most growth factors do not cross the BBB, so the development of delivery strategies such as Trojan horses is required for their systemic use25.

A recent experimental approach with APC provides an example of a neurovascular medicine that has been shown to favourably regulate multiple pathways in non-neuronal cells and neurons, resulting in vasculoprotection, stabilization of the BBB, neuroprotection and anti-inflammation in several acute and chronic models of the CNS disorders195 (Box 2).

The recognition of amyloid-β clearance pathways (Fig. 4), as discussed above, opens exciting new therapeutic opportunities for Alzheimer’s disease. Amyloid-β clearance pathways are promising therapeutic targets for the future development of neurovascular medicines because it has been shown both in animal models of Alzheimer’s disease1 and in patients with sporadic Alzheimer’s disease149 that faulty clearance from brain and across the BBB primarily determines amyloid-β retention in brain, causing the formation of neurotoxic amyloid-β oligomers56 and the promotion of brain and cerebrovascular amyloidosis3. The targeting of clearance mechanisms might also be beneficial in other diseases; for example, the clearance of extracellular mutant SOD1 in familial ALS, the prion protein in prion disorders and α-synuclein in Parkinson’s disease might all prove beneficial. However, the clearance mechanisms for these proteins in these diseases are not yet understood.

Conclusions and perspectives

Currently, no effective disease-modifying drugs are available to treat the major neurodegenerative disorders202, 203, 204. This fact leads to a question: are we close to solving the mystery of neurodegeneration? The probable answer is yes, because the field has recently begun to recognize that, first, damage to neuronal cells is not the sole contributor to disease initiation and progression, and that, second, correcting disease pathways in vascular and glial cells may offer an array of new approaches to control neuronal degeneration that do not involve targeting neurons directly. These realizations constitute an important shift in paradigm that should bring us closer to a cure for neurodegenerative diseases. Here, I raise some issues concerning the existing models of neurodegeneration and the new neurovascular paradigm.

The discovery of genetic abnormalities and associations by linkage analysis or DNA sequencing has broadened our understanding of neurodegeneration204. However, insufficient effort has been made to link genetic findings with disease biology. Another concern for neurodegenerative research is how we should interpret findings from animal models202. Genetically engineered models of human neurodegenerative disorders in Drosophila melanogaster andCaenorhabditis elegans have been useful for dissecting basic disease mechanisms and screening compounds. However, in addition to having much simpler nervous systems, insects and avascular species do not have cerebrovascular and immune systems that are comparable to humans and, therefore, are unlikely to replicate the complex disease pathology that is found in people.

For most neurodegenerative disorders, early steps in the disease processes remain unclear, and biomarkers for these stages have yet to be identified. Thus, it is difficult to predict whether mammalian models expressing human genes and proteins that we know are implicated in the intermediate or later stages of disease pathophysiology, such as amyloid-β or tau in Alzheimer’s disease7, 181, will help us to discover therapies for the early stages of disease and for disease prevention, because the exact role of these pathological accumulations during disease onset remains uncertain. According to the two-hit vascular hypothesis of Alzheimer’s disease, incorporating vascular factors that are associated with Alzheimer’s disease into current models of this disease may more faithfully replicate dementia events in humans. Alternatively, by focusing on the comorbidities and the initial cellular and molecular mechanisms underlying early neurovascular dysfunction that are associated with Alzheimer’s disease, new models of dementia and neurodegeneration may be developed that do not require the genetic manipulation of amyloid-β or tau expression.

The proposed neurovascular triad model of neurodegenerative diseases challenges the traditional neurocentric view of such disorders. At the same time, this model raises a set of new important issues that require further study. For example, the molecular basis of the neurovascular link with neurodegenerative disorders is poorly understood, in terms of the adhesion molecules that keep the physical association of various cell types together, the molecular crosstalk between different cell types (including endothelial cells, pericytes and astrocytes) and how these cellular interactions influence neuronal activity. Addressing these issues promises to create new opportunities not only to better understand the molecular basis of the neurovascular link with neurodegeneration but also to develop novel neurovascular-based medicines.

The construction of a human BBB molecular atlas will be an important step towards understanding the role of the BBB and neurovascular interactions in health and disease. Achievement of this goal will require identifying new BBB transporters by using genomic and proteomic tools, and by cloning some of the transporters that are already known. Better knowledge of transporters at the human BBB will help us to better understand their potential as therapeutic targets for disease.

Development of higher-resolution imaging methods to evaluate BBB integrity, key transporters’ functions and CBF responses in the microregions of interest (for example, in the entorhinal region of the hippocampus) will help us to understand how BBB dysfunction correlates with cognitive outcomes and neurodegenerative processes in MCI, Alzheimer’s disease and related disorders.

The question persists: are we missing important therapeutic targets by studying the nervous system in isolation from the influence of the vascular system? The probable answer is yes. However, the current exciting and novel research that is based on the neurovascular model has already begun to change the way that we think about neurodegeneration, and will continue to provide further insights in the future, leading to the development of new neurovascular therapies.


  1. Zlokovic, B. V. The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201 (2008).

  2. Moskowitz, M. A., Lo, E. H. & Iadecola, C. The science of stroke: mechanisms in search of treatments. Neuron 67, 181–198 (2010).
    A comprehensive review describing mechanisms of ischaemic injury to the neurovascular unit.

  3. Zlokovic, B. V. Neurovascular mechanisms of Alzheimer’s neurodegeneration. Trends Neurosci. 28, 202–208 (2005).

  4. Brown, W. R. & Thore, C. R. Review: cerebral microvascular pathology in ageing and neurodegeneration. Neuropathol. Appl. Neurobiol. 37, 56–74 (2011).

  5. Wu, Z. et al. Role of the MEOX2 homeobox gene in neurovascular dysfunction in Alzheimer disease. Nature Med. 11, 959–965 (2005).
    A study demonstrating that low expression of MEOX2 in brain endothelium leads to aberrant angiogenesis and vascular regression in Alzheimer’s disease.

  6. Paul, J., Strickland, S. & Melchor, J. P. Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer’s disease. J. Exp. Med. 204, 1999–2008 (2007).
    A study showing BBB breakdown in models of Alzheimer’s disease.

  7. Zipser, B. D. et al. Microvascular injury and blood–brain barrier leakage in Alzheimer’s disease. Neurobiol. Aging 28, 977–986 (2007).

  8. Zhong, Z. et al. ALS-causing SOD1 mutants generate vascular changes prior to motor neuron degeneration. Nature Neurosci. 11, 420–422 (2008).
    A study demonstrating that BSCB defects precede motor neuron degeneration in mice that develop an ALS-like disease.

  9. Kalaria, R. N. Vascular basis for brain degeneration: faltering controls and risk factors for dementia. Nutr. Rev. 68, S74–S87 (2010).

  10. Knopman, D. S. & Roberts, R. Vascular risk factors: imaging and neuropathologic correlates. J. Alzheimers Dis. 20, 699–709 (2010).

  11. Miyazaki, K. et al. Disruption of neurovascular unit prior to motor neuron degeneration in amyotrophic lateral sclerosis. J. Neurosci. Res. 89, 718–728 (2011).

  12. Neuwelt, E. A. et al. Engaging neuroscience to advance translational research in brain barrier biology. Nature Rev. Neurosci. 12, 169–182 (2011).

  13. Guo, S. & Lo, E. H. Dysfunctional cell–cell signaling in the neurovascular unit as a paradigm for central nervous system disease.Stroke 40, S4–S7 (2009).

  14. Redzic, Z. Molecular biology of the blood–brain and the blood–cerebrospinal fluid barriers: similarities and differences. Fluids Barriers CNS 8, 3 (2011).

  15. O’Kane, R. L., Martinez-Lopez, I., DeJoseph, M. R., Vina, J. R. & Hawkins, R. A. Na+-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood–brain barrier. A mechanism for glutamate removal. J. Biol. Chem. 274, 31891–31895 (1999).

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Author affiliations

  1. Department of Physiology and Biophysics, and Center for Neurodegeneration and Regeneration at the Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, 1501 San Pablo Street, Los Angeles, California 90089, USA.
    Email: bzlokovi@usc.edu


Retromer in Alzheimer disease, Parkinson disease and other neurological disorders.

Scott A. Small and Gregory A. Petsko

Nature Reviews Neuroscience  2015; 16:126-132.   http://dx.doi.org:/10.1038/nrn3896


Retromer is a protein assembly that has a central role in endosomal trafficking, and retromer dysfunction has been linked to a growing number of neurological disorders. First linked to Alzheimer disease, retromer dysfunction causes a range of pathophysiological consequences that have been shown to contribute to the core pathological features of the disease. Genetic studies have established that retromer dysfunction is also pathogenically linked to Parkinson disease, although the biological mechanisms that mediate this link are only now being elucidated. Most recently, studies have shown that retromer is a tractable target in drug discovery for these and other disorders of the nervous system.

Yeast has proved to be an informative model organism in cell biology and has provided early insight into much of the molecular machinery that mediates the intracellular transport of proteins1,2. Indeed, the term ‘retromer’ was first introduced in a yeast study in 1998 (Ref. 3). In this study, retromer was referred to as a complex of proteins that was dedicated to transporting cargo in a retrograde direction, from the yeast endosome back to the Golgi.

By 2004, a handful of studies had identified the molecular4 and the functional5, 6 homologies of the mammalian retromer, and in 2005 retromer was linked to its first human disorder, Alzheimer disease (AD)7. At the time, the available evidence suggested that the mammalian retromer might match the simplicity of its yeast homologue. Since then, a dramatic and exponential rise in research focusing on retromer has led to more than 300 publications. These studies have revealed the complexity of the mammalian retromer and its functional diversity in endosomal transport, and have implicated retromer in a growing number of neurological disorders.

New evidence indicates that retromer is a ‘master conductor’ of endosomal sorting and trafficking8. Synaptic function heavily depends on endosomal trafficking, as it contributes to the presynaptic release of neurotransmitters and regulates receptor density in the postsynaptic membrane, a process that is crucial for neuronal plasticity9. Therefore, it is not surprising that a growing number of studies are showing that retromer has an important role in synaptic biology10, 11, 12, 13. These observations may account for why the nervous system seems particularly sensitive to genetic and other defects in retromer. In this Progress article, we briefly review the molecular organization and the functional role of retromer, before discussing studies that have linked retromer dysfunction to several neurological diseases — notably, AD and Parkinson disease (PD).

Function and organization

The endosome is considered a hub for intracellular transport. From the endosome, transmembrane proteins can be actively sorted and trafficked to various intracellular sites via distinct transport routes (Fig. 1a). Studies have shown that the mammalian retromer mediates two of the three transport routes out of endosomes. First, retromer is involved in the retrieval of cargos from endosomes and in their delivery, in a retrograde direction, to the trans-Golgi network (TGN)5,6. Retrograde transport has many cellular functions but, as we describe, it is particularly important for the normal delivery of hydrolases and proteases to the endosomal–lysosomal system. The second transport route in which retromer functions is the recycling of cargos from endosomes back to the cell surface14, 15 (Fig. 1a). It is this transport route that is particularly important for neurons, as it mediates the normal delivery of glutamate and other receptors to the plasma membrane during synaptic remodelling and plasticity10, 11, 12, 13.

Figure 1: Retromer’s endosomal transport function and molecular organization.
Retromer's endosomal transport function and molecular organization.

a | Retromer mediates two transport routes out of endosomes via tubules that extend out of endosomal membranes. The first is the retrograde pathway in which cargo is retrieved from the endosome and trafficked to the trans-Golgi network (TGN). The second is the recycling pathway in which cargo is trafficked back from the endosome to the cell surface. The degradation pathway, which is not mediated by retromer, involves the trafficking of cargo from endosomes to lysosomes for degradation. b | The retromer assembly of proteins can be organized into distinct functional modules, all of which work together as part of retromer’s transport role. The ‘cargo-recognition core’ is the central module of the retromer assembly and comprises a trimer of proteins, in which vacuolar protein sorting-associated protein 26 (VPS26) and VPS29 bind VPS35. The ‘tubulation’ module includes protein complexes that bind the cargo-recognition core and aid in the formation and stabilization of tubules that extend out of endosomes, directing the transport of cargos towards their final destinations. The ‘membrane-recruiting’ proteins recruit the cargo-recognition core to the endosomal membrane. The WAS protein family homologue (WASH) complex of proteins also binds the cargo-recognition core and is involved in endosomal ‘actin remodelling’ to form actin patches, which are important for directing cargos towards retromer’s transport pathways. Retromer cargos includes a range of receptors — which bind the cargo-recognition core — and their ligands. PtdIns3P, phosphatidylinositol-3-phosphate.

As well as extending the endosomal transport routes, recent studies have considerably expanded the number of molecular constituents and what is known about the functional organization of the mammalian retromer. Following this expansion in knowledge of the molecular diversity and organizational complexity, retromer might be best described as a multimodular protein assembly. The protein or group of proteins that make up each module can vary, but each module is defined by its distinct function, and the modules work in unison in support of retromer’s transport role.

Two modules are considered central to the retromer assembly. First and foremost is a trimeric complex that functions as a ‘cargo-recognition core’, which selects and binds to the transmembrane proteins that need to be transported and that reside in endosomal membranes5, 6. This trimeric core comprises vacuolar protein sorting-associated protein 26 (VPS26), VPS29 and VPS35; VPS35 functions as the core’s backbone to which the other two proteins bind16. VPS26 is the only member of the core that has been found to have two paralogues, VPS26a and VPS26b17,18, and studies suggest that VPS26b might be differentially expressed in the brain19, 20. Some studies suggest that VPS26a and VPS26b are functionally redundant21, whereas others suggest that they might form distinct cargo-recognition cores20, 22.

The second central module of the retromer assembly is the ‘tubulation’ module, which is made up of proteins that work together in the formation and the stabilization of tubules that extend out of endosomes and that direct the transport of cargo towards its final destination (Fig. 1b). The proteins in this module, which directly binds the cargo-recognition core, are members of the subgroup of the sorting nexin (SNX) family that are characterized by the inclusion of a carboxy-terminal BIN–amphiphysin–RVS (BAR) domain23. These members include SNX1, SNX2, SNX5 and SNX6 (Refs 24,25). As part of the tubulation module, these SNX-BAR proteins exist in different dimeric combinations, but typically SNX1 interacts with SNX5 or SNX6, and SNX2 interacts with SNX5 or SNX6 (Refs 26,27). The EPS15-homology domain 1 (EHD1) protein can be included in this module, as it is involved in stabilizing the tubules formed by the SNX-BAR proteins28.

A third module of the retromer assembly functions to recruit the cargo-recognition core to endosomal membranes and to stabilize the core once it is there (Fig. 1b). Proteins that are part of this ‘membrane-recruiting’ module include SNX3 (Ref. 29), the RAS-related protein RAB7A30, 31,32 and TBC1 domain family member 5 (TBC1D5), which is a member of the TRE2–BUB2–CDC16 (TBC) family of RAB GTPase-activating proteins (GAPs)28. In addition, the lipid phosphatidylinositol-3-phosphate (PtdIns3P), which is found on endosomal membranes, contributes to recruiting most of the retromer-related SNXs through their phox homology domains33. Interestingly, another SNX with a phox homology domain, SNX27, was recently linked to retromer and its function15, 34. SNX27 functions as an adaptor for binding to PDZ ligand-containing cargos that are destined for transport to the cell surface via the recycling pathway. Thus, according to the functional organization of the retromer assembly, SNX27 belongs to the module that engages in cargo recognition and selection.

Recent studies have identified a fourth module of the retromer assembly. The five proteins in this module — WAS protein family homologue 1 (WASH1), FAM21, strumpellin, coiled-coil domain-containing protein 53 (CCDC53) and KIAA1033 (also known as WASH complex subunit 7) — form the WASH complex and function as an ‘actin-remodelling’ module28, 35, 36 (Fig. 1b). Specifically, the WASH complex functions in the rapid polymerization of actin to create patches of actin filaments on endosomal membranes. The complex is recruited to endosomal membranes by binding VPS35 (Ref. 28), and together they divert cargo towards retromer transport pathways and away from the degradation pathway.

The cargos that are transported by retromer include the receptors that directly bind the cargo-recognition core and the ligands of these receptors that are co-transported with the receptors. The receptors that are transported by retromer that have so far been identified to be the most relevant to neurological diseases are the family of VPS10 domain-containing receptors (including sortilin-related receptor 1 (SORL1; also known as SORLA), sortilin, and SORCS1, SORCS2 and SORCS3)7; the cation-independent mannose-6-phosphate receptor (CIM6PR)6, 5; glutamate receptors10; and phagocytic receptors that mediate the clearing function of microglia37. The most disease-relevant ligand to be identified that is trafficked as retromer cargo is the β-amyloid precursor protein (APP)7, 38, 39, 40, 41, which binds SORL1 and perhaps other VPS10 domain-containing receptors42 at the endosomal membrane.

Retromer dysfunction

Guided by retromer’s established function, and on the basis of empirical evidence, there are three well-defined pathophysiological consequences of retromer dysfunction that have proven to be relevant to AD and nervous system disorders. First, retromer dysfunction can cause cargos that typically transit rapidly through the endosome to reside in the endosome for longer than normal durations, such that they can be pathogenically processed into neurotoxic fragments (for example, APP, when stalled in the endosome, is more likely to be processed into amyloid-β, which is implicated in AD43 (Fig. 2a)). Second, by reducing endosomal outflow via impairment of the recycling pathway, retromer dysfunction can lead to a reduction in the number of cell surface receptors that are important for brain health (for example, microglia phagocytic receptors37 (Fig. 2b)).

Figure 2: The pathophysiology of retromer dysfunction.
The pathophysiology of retromer dysfunction.

Retromer dysfunction has three established pathophysiological consequences. In the examples shown, the left graphic represents a cell with normal retromer function and the right graphic represents a cell with a deficit in retromer function. a | Retromer dysfunction causes increased levels of cargo to reside in endosomes. For example, in primary neurons, retromer transports the β-amyloid precursor protein (APP) out of endosomes. Accordingly, retromer dysfunction increases APP levels in endosomes, leading to accelerated APP processing, resulting in an accumulation of neurotoxic fragments of APP (namely, β-carboxy-terminal fragment (βCTF) and amyloid-β) that are pathogenic in Alzheimer disease. b | Retromer dysfunction causes decreased cargo levels at the cell surface. For example, in microglia, retromer mediates the transport of phagocytic receptors to the cell surface and retromer dysfunction results in a decrease in the delivery of these receptors. Studies suggest that this cellular phenotype might have a pathogenic role in Alzheimer disease. c | Retromer dysfunction causes decreased delivery of proteases to the endosome. Retromer is required for the normal retrograde transport of the cation-independent mannose-6-phosphate receptor (CIM6PR) from the endosome back to the trans-Golgi network (TGN). It is in the TGN that this receptor binds cathepsin D and other proteases, and transports them to the endosome, to support the normal function of the endosomal–lysosomal system. By impairing the retrograde transport of the receptor, retromer dysfunction ultimately leads to reduced delivery of cathepsin D to this system. Cathepsin D deficiency has been shown to disrupt the endosomal–lysosomal system and to trigger tau pathology either within endosomes or secondarily in the cytosol.

The third consequence (Fig. 2c) is a result of the established role that retromer has in the retrograde transport of receptors, such as CIM6PR5, 6 or sortilin44, after these receptors transport proteases from the TGN to the endosome. Once at the endosome, the proteases disengage from the receptors, are released into endosomes and migrate to lysosomes. These proteases function in the endosomal–lysosomal system to degrade proteins, protein oligomers and aggregates45. Retromer functions to transfer the ‘naked’ receptor from the endosome back to the TGN via the retrograde pathway5, 6, allowing the receptors to continue in additional rounds of protease delivery. Accordingly, by reducing the normal retrograde transport of these receptors, retromer dysfunction has been shown to reduce the proper delivery of proteases to the endosomal–lysosomal system5,6, which, as discussed below, is a pathophysiological state linked to several brain disorders.

Although requiring further validation, recent studies suggest that retromer dysfunction might be involved in two other mechanisms that have a role in neurological disease. One study suggested that retromer might be involved in trafficking the transmembrane protein autophagy-related protein 9A (ATG9A) to recycling endosomes, from where it can then be trafficked to autophagosome precursors — a trafficking step that is crucial in the formation and the function of autophagosomes46. Autophagy is an important mechanism by which neurons clear neurotoxic aggregates that accumulate in numerous neurodegenerative diseases47. A second study has suggested that retromer dysfunction might enhance the seeding and the cell-to-cell spread of intracellular neurotoxic aggregates48, which have emerged as novel pathophysiological mechanisms that are relevant to AD49, PD50 and other neurodegenerative diseases.

Alzheimer disease

Retromer was first implicated in AD in a molecular profiling study that relied on functional imaging observations in patients and animal models to guide its molecular analysis7. Collectively, neuroimaging studies confirmed that the entorhinal cortex is the region of the hippocampal circuit that is affected first in AD, even in preclinical stages, and suggested that this effect was independent of ageing (as reviewed in Ref. 51). At the same time, neuroimaging studies identified a neighbouring hippocampal region, the dentate gyrus, that is relatively unaffected in AD52. Guided by this information, a study was carried out in which the two regions of the brain were harvested post mortem from patients with AD and from healthy individuals, intentionally covering a broad range of ages. A statistical analysis was applied to the determined molecular profiles of the regions that was designed to address the following question: among the thousands of profiled molecules, which are the ones that are differentially affected in the entorhinal cortex versus the dentate gyrus, in patients versus controls, but that are not affected by age? The final results led to the determination that the brains of patients with AD are deficient in two core retromer proteins — VPS26 and VPS35 (Ref. 7).

Little was known about the receptors of the neuronal retromer, so to understand how retromer deficiency might be mechanistically linked to AD, an analysis was carried out on the molecular data set that looked for transmembrane molecules for which expression levels correlated with VPS35 expression. The top ‘hit’ was the transcript encoding the transmembrane protein SORL1 (Ref. 43). As SORL1 belongs to the family of VPS10-containing receptors and as VPS10 is the main retromer receptor in yeast3, it was postulated that SORL1 and the family of other VPS10-containing proteins (sortillin, SORCS1, SORCS2 and SORCS3) might function as retromer receptors in neurons7. In addition, SORL1 had recently been reported to bind APP53, so if SORL1 was assumed to be a receptor that is trafficked by retromer, then APP might be the cargo that is co-trafficked by retromer. This led to a model in which retromer traffics APP out of endosomes7, which are the organelles in which APP is most likely to be cleaved by βAPP-cleaving enzyme 1 (BACE1; also known as β-secretase 1)43; this is the initial enzymatic step in the pathogenic processing of APP.

Subsequent studies were required to further establish the pathogenic link between retromer and AD, and to test the proposed model. The pathogenic link was further supported by human genetic studies. First, a genetic study investigating the association between AD, the genes encoding the components of the retromer cargo-recognition core and the family of VPS10-containing receptors found that variants of SORL1 increase the risk of developing AD38. This finding was confirmed by numerous studies, including a recent large-scale AD genome-wide association study54. Other genetic studies identified AD-associated variants in genes encoding proteins that are linked to nearly all modules of the retromer assembly55, including genes encoding proteins of the retromer tubulation module (SNX1), genes encoding proteins of the retromer membrane-recruiting module (SNX3 and RAB7A) and genes encoding proteins of the retromer actin-remodelling module (KIAA1033). In addition, nearly all of the genes encoding the family of VPS10-containing retromer receptors have been found to have variants that associate with AD56. Finally, a study found that brain regions that are differentially affected in AD are deficient in PtdIns3P, which is the phospholipid required for recruiting many sorting nexins to endosomal membranes57. Thus, together with the observation that the brains of patients with AD are deficient in VPS26a and VPS35 (Refs 7,37), all modules in the retromer assembly are implicated in AD.

Studies in mice39, 58, 59, flies39 and cells in culture34, 40, 41, 60, 61 have investigated how retromer dysfunction leads to the pathogenic processing of APP. Although rare discrepancies have been observed among these studies62, when viewed in total, the most consistent findings are that retromer dysfunction causes increased pathogenic processing of APP by increasing the time that APP resides in endosomes. Moreover, these studies have confirmed that SORL1 and other VPS10-containing proteins function as APP receptors that mediate APP trafficking out of endosomes.

Retromer has unexpectedly been linked to microglial abnormalities37 — another core feature of AD — which, on the basis of recent genetic findings, seem to have an upstream role in disease pathogenesis54, 63. A recent study found that microglia harvested from the brains of individuals with AD are deficient in VPS35 and provided evidence suggesting that retromer’s recycling pathway regulates the normal delivery of various phagocytic receptors to the cell surface of microglia37, including the phagocytic receptor triggering receptor expressed on myeloid cells 2 (TREM2) (Fig. 2b). Mutations in TREM2 have been linked to AD63, and a recent study indicates that these mutations cause a reduction in its cell surface delivery and accelerate TREM2 degradation, which suggests that the mutations are linked to a recycling defect64. While they are located at the microglial cell surface, these phagocytic receptors function in the clearance of extracellular proteins and other molecules from the extracellular space65. Taken together, these recent studies suggest that defects in the retromer’s recycling pathway can, at least in part, account for the microglial defects observed in the disease.

The microtubule-associated protein tau is the key element of neurofibrillary tangles, which are the other hallmark histological features of AD. Although a firm link between retromer dysfunction and tau toxicity remains to be established, recent insight into tau biology suggests several plausible mechanisms that are worth considering. Tau is a cytosolic protein, but nonetheless, through mechanisms that are still undetermined, it is released into the extracellular space from where it gains access to neuronal endosomes via endocytosis66, 67. In fact, recent studies suggest that the pathogenic processing of tau is triggered after it is endocytosed into neurons and while it resides in endosomes67. Of note, it still remains unknown which specific tau processing step — its phosphorylation, cleavage or aggregation — is an obligate step towards tau-related neurotoxicity. Accordingly, if defects in microglia or in other phagocytic cells reduce their capacity to clear extracellular tau, this would accelerate tau endocytosis in neurons and its pathogenic processing.

A second possibility comes from the established role retromer has in the proper delivery of cathepsin D and other proteases to the endosomal–lysosomal system via CIM6PR or sortilin (Fig. 2c). Studies in sheep, mice and flies68 have shown that cathepsin D deficiency can enhance tau toxicity and that this is mediated by a defective endosomal–lysosomal system68. Whether this mechanism leads to abnormal processing of tau within endosomes or in the cytosol via caspase activation68 remains unclear. As discussed above, retromer dysfunction will lead to a decrease in the normal delivery of cathepsin D to the endosome and will result in endosomal–lysosomal system defects. Retromer dysfunction can therefore be considered as a functional phenocopy of cathepsin D deficiency, which suggests a plausible link between retromer dysfunction and tau toxicity. Nevertheless, although these recent insights establish plausibility and support further investigation into the link between retromer and tau toxicity, whether this link exists and how it may be mediated remain open and outstanding questions.

Parkinson disease

The pathogenic link between retromer and PD is singular and straightforward: exome sequencing has identified autosomal-dominant mutations in VPS35 that cause late-onset PD69, 70, one of a handful of genetic causes of late-onset disease. However, the precise mechanism by which these mutations cause the disease is less clear.

Among a group of recent studies, all46, 48, 71, 72, 73, 74, 75, 76 but one77 strongly suggest that these mutations cause a loss of retromer function. At the molecular level, the mutations do not seem to disrupt mutant VPS35 from interacting normally with VPS26 and VPS29, and from forming the cargo-recognition core. Rather, two studies suggest that the mutations have a restricted effect on the retromer assembly but reduce the ability of VPS35 to associate with the WASH complex46, 75. Studies disagree about the pathophysiological consequences of the mutations. Four studies suggest that the mutations affect the normal retrograde transport of CIM6PR71, 73, 75, 76 from the endosome back to the TGN (Fig. 2c). In this scenario, the normal delivery of cathepsin D to the endosomal–lysosomal system should be reduced and this has been empirically shown73. Cathepsin D has been shown to be the dominant endosomal–lysosomal protease for the normal processing of α-synuclein76, and mutations could therefore lead to abnormal α-synuclein processing and to the formation of α-synuclein aggregates, which are thought to have a key pathogenic role in PD.

A separate study suggested that the mutation might cause a mistrafficking of ATG9, and thereby, as discussed above, reduce the formation and the function of autophagosomes46. Autophagosomes have also been implicated as an intracellular site in which α-synuclein aggregates are cleared. Thus, although future studies are needed to resolve these discrepant findings (which may in fact not be mutually exclusive), these studies are generally in agreement that retromer defects will probably increase the neurotoxic levels of α-synuclein aggregates48.

Several studies in flies71, 74 and in rat neuronal cultures71 provide strong evidence that increasing retromer function by overexpressing VPS35 rescues the neurotoxic effects of the most common PD-causing mutations in leucine-rich repeat kinase 2 (LRRK2). Moreover, a separate study has shown that increasing retromer levels rescues the neurotoxic effect of α-synuclein aggregates in a mouse model48. These findings have immediate therapeutic implications for drugs that increase VPS35 and retromer function, as discussed in the next section, but they also offer mechanistic insight. LRRK2 mutations were found to phenocopy the transport defects caused either by theVPS35 mutations or by knocking down VPS35 (Ref. 71). Together, this and other studies78suggest that LRRK2 might have a role in retromer-dependent transport, but future studies are required to clarify this role.

Other neurological disorders

Besides AD and PD, in which a convergence of findings has established a strong pathogenic link, retromer is being implicated in an increasing number of other neurological disorders. Below, we briefly review three disorders for which the evidence of the involvement of retromer in their pathophysiology is currently the most compelling.

The first of these disorders is Down syndrome (DS), which is caused by an additional copy of chromosome 21. Given the hundreds of genes that are duplicated in DS, it has been difficult to identify which ones drive the intellectual impairments that characterize this condition. A recent elegant study provides strong evidence that a deficiency in the retromer cargo-selection protein SNX27 might be a primary driver for some of these impairments79. This study found that the brains of individuals with DS were deficient in SNX27 and that this deficiency may be caused by an extra copy of a microRNA (miRNA) encoded by human chromosome 21 (the miRNA is produced at elevated levels and thereby decreases SNX27 expression). Consistent with the known role of SNX27 in retromer function, decreased expression of this protein in mice disrupted glutamate receptor recycling in the hippocampus and led to dendritic dysfunction. Importantly, overexpression of SNX27 rescued cognitive and other defects in animal models79, which not only strengthens the causal link between retromer dysfunction and cognitive impairment in DS but also has important therapeutic implications.

Hereditary spastic paraplegia (HSP) is another disorder linked to retromer. HSP is caused by genetic mutations that affect upper motor neurons and is characterized by progressive lower limb spasticity and weakness. Although there are numerous mutations that cause HSP, most are unified by their effects on intracellular transport80. One HSP-associated gene in particular encodes strumpellin81, which is a member of the WASH complex.

The third disorder linked to retromer is neuronal ceroid lipofuscinosis (NCL). NCL is a young-onset neurodegenerative disorder that is part of a larger family of lysosomal storage diseases and is caused by mutations in one of ten identified genes — nine neuronal ceroid lipofuscinosis (CLN) genes and the gene encoding cathepsin D82. Besides cathepsin D, for which the link to retromer has been discussed above, CLN3 seems to function in the normal trafficking of CIM6PR83. However, the most direct link to retromer has been recently described for CLN5, which seems to function, at least in part, as a retromer membrane-recruiting protein84.

Retromer as a therapeutic target

As suggested by the first study implicating retromer in AD7, and in several subsequent studies71,85, increasing the levels of retromer’s cargo-recognition core enhances retromer’s transport function. Motivated by this observation and after a decade-long search86, we identified a novel class of ‘retromer pharmacological chaperones’ that can bind and stabilize retromer’s cargo-recognition core and increase retromer levels in neurons61.

Validating the motivating hypothesis, the chaperones were found to enhance retromer function, as shown by the increased transport of APP out of endosomes and a reduction in the accumulation of APP-derived neurotoxic fragments61. Although there are numerous other pharmacological approaches for enhancing retromer function, this success provides the proof-of-principle that retromer is a tractable therapeutic target.

As retromer functions in all cells, a general concern is whether enhancing its function will have toxic adverse effects. However, studies have found that in stark contrast to even mild retromer deficiencies, increasing retromer levels has no obvious negative consequences in yeast, neuronal cultures, flies or mice40, 48, 61, 71. This might make sense because unlike drugs that, for example, function as inhibitors, simply increasing the normal flow of transport through the endosome might not be cytotoxic.

If retromer drugs are safe and can effectively enhance retromer function in the nervous system — which are still outstanding issues — there are two general indications for considering their clinical application. One rests on the idea that these agents will only be efficacious in patients who have predetermined evidence of retromer dysfunction. The most immediate example is that of individuals with PD that is caused by LRRK2 mutations. As discussed above, several ‘preclinical’ studies in flies and neuronal cultures have already established that increasing retromer levels71, 74can reverse the neurotoxic effects of such mutations and, thus, if this approach is proven to be safe, LRRK2-linked PD might be an appropriate indication for clinical trials.

Alternatively, the pathophysiology of a disease might be such that retromer-enhancing drugs would be efficacious regardless of whether there is documented evidence of retromer dysfunction. AD illustrates this point. As reviewed above, current evidence suggests that retromer-enhancing drugs will, at the very least, decrease pathogenic processing of APP in neurons and enhance microglial function, even if there are no pre-existing defects in retromer.

More generally, histological studies comparing the entorhinal cortex of patients with sporadic AD to age-matched controls have documented that enlarged endosomes are a defining cellular abnormality in AD87, 88. Importantly, enlarged endosomes are uniformly observed in a broad range of patients with sporadic AD, which suggests that enlarged endosomes reflect an intracellular site at which molecular aetiologies converge87. In addition, because they are observed in early stages of the disease in regions of the brain without evidence of amyloid pathology87, enlarged endosomes are thought to be an upstream event. Mechanistically, the most likely cause of enlarged endosomes is either too much cargo flowing into endosomes — as occurs, for example, with apolipoprotein E4 (APOE4), which has been shown to accelerate endocytosis89, 90 — or too little cargo flowing out, as observed in retromer dysfunction40, 61 and related transport defects57. By any mechanism, retromer-enhancing drugs might correct this unifying cellular defect and might be expected to be beneficial regardless of the specific aetiology.


The fact that retromer defects, including those derived from bona fide genetic mutations, seem to differentially target the nervous system suggests that the nervous system is differentially dependent on retromer for its normal function. We think that this reflects the unique cellular properties of neurons and how synaptic biology heavily depends on endosomal transport and trafficking. Although plausible, future studies are required to confirm and to test the details of this hypothesis.

However, currently, it is the clinical rather than the basic neuroscience of retromer that is much better understood, with the established pathophysiological consequences of retromer dysfunction providing a mechanistic link to the disorders in which retromer has been implicated. Nevertheless, many questions remain. The two most interesting questions, which are in fact inversions of each other, relate to regional vulnerability in the nervous system. First, why does retromer dysfunction target specific neuronal populations? Second, how can retromer dysfunction cause diseases that target different regions of the nervous system? Recent evidence hints at answers to both questions, which must somehow be rooted in the functional and molecular diversity of retromer.

The type and the extent of retromer defects linked to different disorders might provide pathophysiological clues as well as reasons for differential vulnerability. As discussed, in AD there seem to be across-the-board defects in retromer, such that each module of the retromer assembly as well as multiple retromer cargos have been pathogenically implicated. By contrast, the profile of retromer defects in PD seems to be more circumscribed, involving selective disruption of the interaction between VPS35 and the WASH complex. These insights might agree with histological87, 88 and large-scale genetic studies54 that suggest that endosomal dysfunction is a unifying focal point in the cellular pathogenesis of AD. In contrast, genetics and other studies91suggest that the cellular pathobiology of PD is more distributed, implicating the endosome but other organelles as well, in particular the mitochondria.

Interestingly, studies suggest that the entorhinal cortex — a region that is differentially vulnerable to AD — has unique dendritic structure and function92, which are highly dependent on endosomal transport. We speculate that it is the unique synaptic biology of the entorhinal cortex that can account for why it might be particularly sensitive to defects in endosomal transport in general and retromer dysfunction in particular, and for why this region is the early site of disease. Future studies are required to investigate this hypothesis, as well as to understand why the substantia nigra or other regions that are differentially vulnerable to PD would be particularly sensitive to the more circumscribed defect in retromer.

Perhaps the most important observation for clinical neuroscience is the now well-established fact that increasing levels of retromer proteins enhances retromer function and has already proved capable of reversing defects associated with AD, PD and DS in either cell culture or in animal models. The relationships between protein levels and function are not always simple, but emerging pharmaceutical technologies that selectively and safely increase protein levels are now a tractable goal in drug discovery93. With the evidence mounting that retromer has a pathogenic role in two of the most common neurodegenerative diseases, we think that targeting retromer to increase its functional activity is an important goal that has strong therapeutic promise.


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……. 93


Taub Institute for Research on Alzheimer’s Disease and the Ageing Brain, Departments of Neurology, Radiology, and Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.

Scott A. Small

Helen and Robert Appel Alzheimer’s Disease Research Institute, Department of Neurology and Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, New York 10065, USA.

Gregory A. Petsko


See also:

Neurobiol Aging. 2011 Nov;32(11):2109.e1-14. doi: 10.1016/j.neurobiolaging.2011.05.025.
Altered intrinsic neuronal excitability and reduced Na+ currents in a mouse model of Alzheimer’s disease.
Brown JT, Chin J, Leiser SC, Pangalos MN, Randall AD.

Trends Neurosci. 2013 Jun;36(6):325-35. doi: 10.1016/j.tins.2013.03.002.
Why size matters – balancing mitochondrial dynamics in Alzheimer’s disease.
DuBoff B, Feany M, Götz J.

Neuron. 2014 Dec 3;84(5):1023-33. doi: 10.1016/j.neuron.2014.10.024.
Dendritic structural degeneration is functionally linked to cellular hyperexcitability in a mouse model of Alzheimer’s disease.
Šišková Z, Justus D, Kaneko H, Friedrichs D, Henneberg N, Beutel T, Pitsch J, Schoch S, Becker A, von der Kammer H, Remy S.



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Protein misfolding and prions

Larry H/ Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 4.9

Revised 9/30/2015

Susan L. Lindquist, Stanley B. Prusiner

Whitehead Member Susan Lindquist is a pioneer in the study of protein folding. She has shown that changes in protein folding can have profound and unexpected influences in fields as wide-ranging as human disease, evolution and nanotechnology.

Protein misfolding has been implicated as a major mechanism in many severe neurological disorders including Parkinson’s and Huntington’s diseases. Lindquist and colleagues have developed yeast strains that serve as living test tubes in which to study these disorders, unraveling how protein folding contributes to them.

Prions are proteins that can change into a self-perpetuating form. They have only been discovered recently, but one of them is already well known as the cause of mad cow disease. The Lindquist lab investigates both how prions form and the diseases they cause. In addition, Lindquist is convinced that other prion proteins play many important and positive roles in biological processes. The first evidence for this was shown in her work with Nobel Laureate Eric Kandel, which demonstrated that prions may be integral to memory storage in the brain.

Heat shock proteins are a group of molecular chaperone proteins that, as their name might suggest, guide other proteins to fold and mature correctly. Lindquist has established that heat shock protein 90 (Hsp90) can reveal hidden genetic variation in fruit flies and in cress plants (Arabidopsis) under certain environmental conditions.

Lindquist is a Member and former Director (2001-2004) of Whitehead Institute, a Professor of Biology at MIT, and a Howard Hughes Medical Institute investigator. Previously she was the Albert D. Lasker Professor of Medical Sciences from 1999-2001, and a Professor in the Department of Molecular Biology, University of Chicago, since 1978. She received a PhD in Biology from Harvard University in 1976, and was elected to the American Academy of Arts and Sciences in 1997, the National Academy of Sciences in 1997 and the Institute of Medicine in 2006.

QnAs with Susan L. Lindquist

http://www.pnas.org/content/108/50/19861.full  Dec 13, 2011  Susan L. Lindquist. PNAS: How did you suspect that prions might have a positive role in cells?
Lindquist: We were working on a heat shock …

Dr. Susan Lindquist – “Alzheimer’s Disease: An Entirely New Point of 

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Whitehead Institute Member Susan Lindquist’s keynote from the 2011 Whitehead Colloquium, November 5, 2011.

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Sue Lindquist Plenary Lecture at AAAS Annual Meeting 2014

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From Yeast Cells to Patient Neurons: A Powerful Discovery Platform for Parkinson’s and Alzheimer’s Disease

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Susan Lindquist ISSCR 6 21 14

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Susan Lindquist Keynote Lecture at ISSCR in Vancouver, June 2014

Stanley B. Prusiner, MD

Director, Institute for Neurodegenerative Diseases
Professor, Department of Neurology

Prusiner discovered an unprecedented class of pathogens that he named prions. Prions are proteins that acquire an alternative shape that becomes self-propagating. As prions accumulate, they cause neurodegenerative diseases in animals and humans. Prusiner’s discovery lead him to develop a novel disease paradigm: prions cause disorders such as Creutzfeldt-Jakob disease (CJD) in humans that manifest as (1) sporadic, (2) inherited and (3) infectious illnesses.  Based on his seminal discovery that prions can assemble into amyloid fibrils, Prusiner proposed that the more common neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases may be caused by prions.

Prusiner’s contributions to scientific research have been internationally recognized: He is a member of the National Academy of Sciences, the Institute of Medicine, the American Academy of Arts and Sciences and the American Philosophical Society, and a foreign member of the Royal Society, London. He is the recipient of numerous prizes, including the Potamkin Prize for Alzheimer’s Disease Research from the American Academy of Neurology (1991); the Richard Lounsbery Award for Extraordinary Scientific Research in Biology and Medicine from the National Academy of Sciences (1993); the Gairdner Foundation International Award (1993); the Albert Lasker Award for Basic Medical Research (1994); the Wolf Prize in Medicine from the State of Israel (1996); the Nobel Prize in Physiology or Medicine (1997); and the United States Presidential National Medal of Science (2009).

Stanley Prusiner – National Medal of Science – YouTube

Nov 29, 2010  2009 Medal of Science Laureate for his discovery of prions — a new class of infectious agents comprised only of proteins. Produced by Evolving ..

2011 Bay Area Council Outlook Conference – Dr. Stanley Prusiner 

Apr 27, 2011  2011 Bay Area Council Outlook Conference – Dr. Stanley Prusiner …. President Obama Awards National Medal of Scienceand Medal of …

By Jennifer O’Brien on October 15, 2010

UCSF Nobel laureate Stanley B. Prusiner, MD, UCSF professor of neurology and director of the Institute for Neurodegenerative Diseases, today (Oct. 15, 2010) was named to receive the National Medal of Science, the nation’s highest honor for science and technology.

Prusiner received the medal for his discovery of and ongoing research on a novel infectious agent, which he named the prion (PREE-on). The prion, composed solely of protein, causes bovine spongiform encephalopathy, or “mad cow” disease, and other related fatal neurodegenerative diseases in animals and humans.


Stanley B. Prusiner

PNAS Nov 10, 1998; 95(23):13363–13383,    http://dx.doi.org:/10.1073/pnas.95.23.13363

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high β-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc.

The torturous path of the scientific investigation that led to an understanding of familial Creutzfeldt–Jakob disease (CJD) chronicles a remarkable scientific odyssey. By 1930, the high incidence of familial (f) CJD in some families was known (12). Almost 60 years were to pass before the significance of this finding could be appreciated (35). CJD remained a curious, rare neurodegenerative disease of unknown etiology throughout this period of three score years (6)(7).

Once CJD was shown to be an infectious disease, relatively little attention was paid to the familial form of the disease since most cases were not found in families(812). Libyan Jews living in Israel developed CJD about 30 times more frequently than other Israelis (13). This finding prompted some investigators to propose that the Libyan Jews had contracted CJD by eating lightly cooked brain from scrapie-infected sheep when they lived in Tripoli prior to emigration. Subsequently, the Libyan Jewish patients were all found to carry a mutation at codon 200 in their prion protein (PrP) gene (1416).

Slow Viruses.

The term “slow virus” had been coined by Bjorn Sigurdsson in 1954 while he was working in Iceland on scrapie and visna of sheep (17). Five years later, William Hadlow had suggested that kuru, a disease of New Guinea highlanders, was similar to scrapie and thus, it, too, was caused by a slow virus (18). Seven more years were to pass before the transmissibility of kuru was established by passaging the disease to chimpanzees inoculated intracerebrally (19). Just as Hadlow had made the intellectual leap between scrapie and kuru, Igor Klatzo made a similar connection between kuru and CJD (20). Neuropathologists were struck by the similarities in light microscopic pathology of the central nervous system (CNS) that kuru exhibited with scrapie or CJD. In 1968, the transmission of CJD to chimpanzees after intracerebral inoculation was reported (7).

In scrapie, kuru, CJD, and all of the other disorders now referred to as prion diseases (Table 1), spongiform degeneration and astrocytic gliosis is found upon microscopic examination of the CNS (Fig. 1) (2122).

Table 1

The prion disease

Figure 1


Neuropathologic changes in Swiss mice after inoculation with RML scrapie prions. (a) Hematoxylin and eosin stain of a serial section of the hippocampus shows spongiform degeneration of the neuropil, with vacuoles 10–30 μm in diameter.

Prions: A Brief Overview.

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases mediated by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP), a constituent of normal mammalian cells (23). CJD generally presents as progressive dementia, whereas scrapie of sheep and bovine spongiform encephalopathy (BSE) are generally manifest as ataxic illnesses (Table 1) (24).

Prions are devoid of nucleic acid and seem to be composed exclusively of a modified isoform of PrP designated PrPSc. The normal, cellular PrP, denoted PrPC, is converted into PrPSc through a process whereby a portion of its α-helical and coil structure is refolded into β-sheet (25). This structural transition is accompanied by profound changes in the physicochemical properties of the PrP. The amino acid sequence of PrPSc corresponds to that encoded by the PrP gene of the mammalian host in which it last replicated. In contrast to pathogens with a nucleic acid genome that encode strain-specific properties in genes, prions encipher these properties in the tertiary structure of PrPSc (2628). Transgenetic studies argue that PrPScacts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein.

More than 20 mutations of the PrP gene are now known to cause the inherited human prion diseases, and significant genetic linkage has been established for five of these mutations (4162931). The prion concept readily explains how a disease can be manifest as a heritable as well as an infectious illness.

Resistance of Scrapie Agent to Radiation.

My fascination with CJD quickly shifted to scrapie once I learned of the remarkable radiobiological data that Tikvah Alper and her colleagues had collected on the scrapie agent (3234). The scrapie agent had been found to be extremely resistant to inactivation by UV and ionizing radiation, as was later shown for the CJD agent (35). It seemed to me that the most intriguing question was the chemical nature of the scrapie agent. Suggestions as to the nature of the scrapie agent  (3642).

Scrapie of sheep and goats possesses a history no less fascinating than that of CJD. The resistance of the scrapie agent to inactivation by formalin and heat treatments (43), suggested that the scrapie agent might be different from viruses. British scientists had argued for many years about whether natural scrapie was a genetic or an infectious disease (4446). Scrapie, like kuru and CJD, produced death of the host without any sign of an immune response to a “foreign infectious agent.”

From studies on the sedimentation properties of scrapie infectivity in mouse spleens and brains  I concluded that hydrophobic interactions were responsible for the nonideal physical behavior of the scrapie particle (4748). The scrapie agent demanded new approaches and better assays (49).


In 1972, when I became fascinated by the enigmatic nature of the scrapie agent, I thought that the most direct path to determining the molecular structure of the scrapie agent was purification. Few systematic investigations had been performed on the fundamental characteristics of the infectious scrapie particle (42). Knowledge of the sedimentation properties of the scrapie agent under defined conditions seemed mandatory.

Incubation time assays in hamsters.

In view of these daunting logistical problems, the identification of an inoculum that produced scrapie in the golden Syrian hamster (SHa) in ≈70 days after intracerebral inoculation proved to be an important advance (5354) once an incubation time assay was developed (55,56). In earlier studies, SHa had been inoculated with prions, but serial passage with short incubation times was not reported (57). Development of the incubation time bioassay reduced the time required to measure prions in samples with high titers by a factor of 5: only 70 days were required instead of the 360 days previously needed. Equally important, 4 animals could be used in place of the 60 that were required for endpoint titrations, making possible a large number of parallel experiments. With this bioassay, research on the nature of the scrapie agent was accelerated nearly 100-fold and the hamster with high prion titers in its brain became the experimental animal of choice for biochemical studies.

The incubation time assay enabled development of effective purification schemes for enriching fractions for scrapie infectivity. It provided a means to assess quantitatively those fractions that were enriched for infectivity and those that were not.  With a ≈100-fold purification of infectivity relative to protein, >98% of the proteins and polynucleotides were eliminated.

The Prion Concept.

As reproducible data began to accumulate indicating that scrapie infectivity could be reduced by procedures that hydrolyze or modify proteins but was resistant to procedures that alter nucleic acids, a family of hypotheses about the molecular architecture of the scrapie agent began to emerge (58). These data established, for the first time, that a particular macromolecule was required for infectivity and that this macromolecule was a protein. The experimental findings extended earlier observations on resistance of scrapie infectivity to UV irradiation at 250 nm (33) in that the four different procedures used to probe for a nucleic acid are based on physical principles that are independent of UV radiation damage.

Once the requirement of protein for infectivity was established, I thought that it was appropriate to give the infectious pathogen of scrapie a provisional name that would distinguish it from both viruses and viroids. After some contemplation, I suggested the term “prion,” derived from proteinaceous and infectious (58). At that time, I defined prions as proteinaceous infectious particles that resist inactivation by procedures that modify nucleic acids. I never imagined the irate reaction of some scientists to the word “prion”—it was truly remarkable!

Current definitions.

Perhaps the best current working definition of a prion is a proteinaceous infectious particle that lacks nucleic acid (28). Because a wealth of data supports the contention that scrapie prions are composed entirely of a protein that adopts an abnormal conformation, it is not unreasonable to define prions as infectious proteins (25275960). But I hasten to add that we still cannot eliminate a small ligand bound to PrPSc as an essential component of the infectious prion particle.  (61). From a broader perspective, prions are elements that impart and propagate conformational variability.

Although PrPSc is the only known component of the infectious prion particles, these unique pathogens share several phenotypic traits with other infectious entities such as viruses. (6267).

Families of hypotheses.

Once the requirement for a protein was established, it was possible to revisit the long list of hypothetical structures that had been proposed for the scrapie agent and to eliminate carbohydrates, lipids, and nucleic acids as the infective elements within a scrapie agent devoid of protein (58) (5868).

The family of hypotheses that remained after identifying a protein component was still large and required a continued consideration of all possibilities in which a protein was a critical element (49). The prion concept evolved from a family of hypotheses in which an infectious protein was only one of several possibilities. With the accumulation of experimental data on the molecular properties of the prion, it became possible to discard an increasing number of hypothetical structures. (69).

Genes and DNA.

In some respects, the early development of the prion concept mirrors the story of DNA (7075). Those who attacked the hypothesis that the prion is composed only of protein had more than 30 years of cumulative evidence showing that genetic information in all organisms on our planet is encoded in DNA and that biological diversity resides in DNA. Studies of viruses and eventually viroids extended this concept to these small infectious pathogens (76) and showed that genes could also be composed of RNA (7778).

Discovery of the Prion Protein.

The discovery of the prion protein transformed research on scrapie and related diseases (7980). It provided a molecular marker that was subsequently shown to be specific for these illnesses as well as the major, and very likely the only, constituent of the infectious prion.

PrP 27–30 was discovered by enriching fractions from SHa brain for scrapie infectivity (7980). This protein is the protease-resistant core of PrPSc and has an apparent molecular mass of 27–30 kDa (8182). Although resistance to limited proteolysis proved to be a convenient tool for many but not all studies, use of proteases to enrich fractions for scrapie infectivity created a problem when the NH2-terminal sequence of PrP 27–30 was determined (81). The ragged NH2 terminus of PrP 27–30 yielded three sets of signals in almost every cycle of the Edman degradation. Only after these signals were properly interpreted and placed in correct register could a unique sequence be assigned for the NH2 terminus of PrP 27–30. The determination of the amino acid sequence of the NH2 terminus of PrP 27–30 made subsequent molecular cloning studies of the PrP gene possible (8384).

The finding that PrP mRNA levels were similar in normal uninfected and scrapie-infected tissues caused some investigators to argue that PrP 27–30 was not related to the infectious prion particle (83). An alternate interpretation prompted a search for a prion protein in uninfected animals that was found to be protease sensitive and soluble in nondenaturing detergents, unlike PrP 27–30. This isoform was designated PrPC(Fig. 2) (8485). Deduced amino acid sequences from PrP cDNA as well as immunoblotting studies revealed that PrP 27–30 was NH2-terminally truncated and was derived from a larger molecule, designated PrPSc, that was unique to infected animals (81828486).

Figure 2


With the discovery of PrP 27–30 and the production of antiserum (87), brains from humans and animals with putative prion diseases were examined for the presence of this protein. In each case, PrP 27–30 was found, and it was absent in other neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (8891). The extreme specificity of PrPSc for prion disease is an important feature of the protein and is consistent with the postulated role of PrPSc in both the transmission and pathogenesis of these illnesses (Table 2) (92).

Table 2

Arguments for prions being composed largely, if not entirely, of PrPSc molecules and devoid of nucleic acid

The accumulation of PrPSc contrasts markedly with that of glial fibrillary acidic protein (GFAP) in prion disease. In scrapie, GFAP mRNA and protein levels rise as the disease progresses (93), but the accumulation of GFAP is neither specific nor necessary for either the transmission or the pathogenesis of disease. Mice deficient for GFAP show no alteration in their incubation times (9495).

Except for PrPSc, no macromolecule has been found in tissues of patients dying of the prion diseases that is specific for these encephalopathies. In searches for a scrapie-specific nucleic acid, cDNAs have been identified that are complementary to mRNAs encoding other proteins with increased expression in prion disease (9698). Yet none of the proteins has been found to be specific for prion disease.

Attempts to Falsify the Prion Hypothesis.

Numerous attempts to disprove the prion hypothesis over the past 15 years have failed (99), and no replication of prions in PrP-deficient (Prnp0/0) mice was found (100104).

Copurification of PrP 27–30 and scrapie infectivity demands that the physicochemical properties as well as antigenicity of these two entities be similar (105) (Table 2). The results of a wide array of inactivation experiments demonstrated the similarities in the properties of PrP 27–30 and scrapie infectivity (61,106109). To explain these findings in terms of the virus hypothesis, the PrP-like coat protein or the PrPSc/virus complex must display properties indistinguishable from PrPSc alone. With each species that the putative virus invades, it must incorporate a new PrP sequence during replication.

Search for a scrapie-specific nucleic acid.

The inability to inactivate preparations highly enriched for scrapie infectivity by procedures that modify nucleic acids militates against the existence of a scrapie-specific nucleic acid (58110111). Both UV and ionizing radiation inactivation studies as well as physical studies have eliminated the possibility of a large nucleic acid hiding within purified preparations of prions (11011263115).

PrP amyloid.

In preparations highly enriched for scrapie infectivity and containing only PrP 27–30 by silver staining of gels after SDS/PAGE, numerous rod-shaped particles were seen by electron microscopy after negative staining (Fig. 3) (107),(116). These irregular rods, composed largely, if not entirely, of PrP 27–30, were indistinguishable morphologically from many other purified amyloids (117). Studies of the prion rods with Congo red dye demonstrated that the rods also fulfilled the tinctorial criteria for amyloid (107), and immunostaining later showed that PrP is a major component of amyloid plaques in some animals and humans with prion disease (118120). Subsequently, it was recognized that the prion rods were not required for scrapie infectivity (121); (Fig. 3) (122). 

Figure 3


The idea that scrapie prions were composed of an amyloidogenic protein was truly heretical when it was introduced (107). Since the prevailing view at the time was that scrapie is caused by an atypical virus, many argued that amyloid proteins are mammalian polypeptides and not viral proteins!

Some investigators have argued that the prion rods are synonymous with scrapie-associated fibrils (123125) even though morphologic and tinctorial features of these fibrils clearly differentiated them from amyloid and as such from the prion rods (126127). The scrapie-associated fibrils were identified by their unique ultrastructure in which two or four subfilaments were helically wound around each other (126) and were proposed to represent the first example of a filamentous animal virus (128).

Search for the ubiquitous “scrapie virus.”

When PrP gene mutations were discovered to cause familial prion diseases (4), it was postulated that PrPC is a receptor for the ubiquitous scrapie virus that binds more tightly to mutant than to wt PrPC (131). A similar hypothesis was proposed to explain why the length of the scrapie incubation time was found to be inversely proportional to the level of PrP expression in transgenic (Tg) mice and why Prnp0/0 mice are resistant to scrapie (132). The higher the level of PrP expression, the faster the spread of the putative virus, which results in shorter incubation times; conversely, mice deficient for PrP lack the receptor required for spread of the virus (63). The inability to find virus-like particles in purified preparations of PrPSc was attributed to these particles being hidden (115) even though tobacco mosaic viruses could be detected when one virion was added per ID50 unit of scrapie prions (121).

Recent studies on the transmission of mutant prions from FFI and fCJD(E200K) to Tg(MHu2M) mice, which results in the formation of two different PrPSc molecules (27), has forced a corollary to the ubiquitous virus postulate. To accommodate this result, at least two different viruses must reside worldwide, each of which binds to a different mutant HuPrPC and each of which induces a different MHu2M PrPSc conformer when transferred to Tg mice. Even more difficult to imagine is how one ubiquitous virus might acquire different mutant PrPSc molecules corresponding to FFI or fCJD(E200K) and then induce different MHu2M PrPScconformers upon transmission to Tg mice.

Artificial prions.

To explain the production of artificial prions from chimeric or mutant PrP transgenes in terms of a virus (133135), mutated PrPSc molecules must be incorporated into the virus. In the case of mice expressing chimeric PrP transgenes, artificial prions are produced with host ranges not previously found in nature. Similarly, deleting specific regions of PrP resulted in the formation of “miniprions” with a unique host range and neuropathology as described below. The production of artificial prions that were generated by modifying the PrP gene sequence and exhibit unique biological properties is another compelling argument against the proposition that scrapie and CJD are caused by viruses.

Prions Defy Rules of Protein Structure.

Once cDNA probes for PrP became available, the PrP gene was found to be constitutively expressed in adult, uninfected brain (8384). This finding eliminated the possibility that PrPSc stimulated production of more of itself by initiating transcription of the PrP gene as proposed nearly two decades earlier (37). Determination of the structure of the PrP gene eliminated a second possible mechanism that might explain the appearance of PrPSc in brains already synthesizing PrPC. Since the entire protein coding region was contained within a single exon, there was no possibility for the two PrP isoforms to be the products of alternatively spliced mRNAs (82). Next, a posttranslational chemical modification that distinguishes PrPScfrom PrPC was considered, but none was found in an exhaustive study (59) and we considered it likely that PrPC and PrPSc differed only in their conformation, a hypothesis also proposed earlier (37). However, this idea was no less heretical than that of an infectious protein.

For more than 25 years, it had been widely accepted that the amino acid sequence specifies one biologically active conformation of a protein (136). Yet in scrapie we were faced with the possibility that one primary structure for PrP might adopt at least two different conformations to explain the existence of both PrPC and PrPSc. When the secondary structures of the PrP isoforms were compared by optical spectroscopy, they were found to be markedly different (25). Fourier-transform infrared (FTIR) and circular dichroism (CD) studies showed that PrPC contains about 40% α-helix and little β-sheet, whereas PrPSc is composed of about 30% α-helix and 45% β-sheet (25137). Nevertheless, these two proteins have the same amino acid sequence!

Prior to comparative studies on the structures of PrPC and PrPSc, we found by metabolic labeling studies that the acquisition of PrPSc protease resistance is a posttranslational process (138). In our quest for a chemical difference that would distinguish PrPSc from PrPC, we found ethanolamine in hydrolysates of PrP 27–30, which signaled the possibility that PrP might contain a GPI anchor (139). Both PrP isoforms were found to carry GPI anchors, and PrPC was found on the surface of cells where it could be released by cleavage of the anchor. Subsequent studies showed that PrPSc formation occurs after PrPC reaches the cell surface (140141) and is localized to caveolae-like domains (142145).

Modeling PrP structures.

Molecular modeling studies predicted that PrPC is a four-helix bundle protein containing four regions of secondary structure denoted H1, H2, H3, and H4 (Fig. 4) (146147). Subsequent NMR studies of a synthetic PrP peptide containing residues 90–145 provided good evidence for H1 (148). This peptide contains the residues 113–128, which are most highly conserved among all species studied (Fig. 4A) (147149150) and correspond to a transmembrane region of PrP that was delineated in cell-free translation studies (151152). Recent studies show that a transmembrane form of PrP accumulates in GSS caused by the A117V mutation and in Tg mice overexpressing either mutant or wild-type (wt)PrP (153). The paradoxical lack of evidence for an α-helix in this region from NMR studies of recombinant PrP in aqueous buffers (154156) could be explained if the recombinant PrPs correspond to the secreted form of PrP that was also identified in cell-free translation studies. This contention is supported by studies with recombinant antibody fragments (Fabs) showing the GPI-anchored PrPC on the surface of cells exhibits an immunoreactivity similar to that of recombinant PrP that was prepared with an α-helical conformation (157,158). GPI-anchored PrPC is synthesized within the secretory pathway and transported to the surface of the cell (139159).

Figure 4


Species variations and mutations of the prion protein gene. (A) Species variations. The x-axis represents the human PrP sequence, with the five octarepeats and H1–H4 regions of putative secondary structure shown as well as the three α-helices A, B, and C and the two β-strands S1 and S2 as determined by NMR. The precise residues corresponding to each region of secondary structure are given in Fig. 5. Vertical bars above the axis indicate the number of species that differ from the human sequence at each position. Below the axis, the length of the bars indicates the number of alternative amino acids at each position in the alignment. (B) Mutations causing inherited human prion disease and polymorphisms in human, mouse, and sheep. Above the line of the human sequence are mutations that cause prion disease. Below the lines are polymorphisms, some but not all of which are known to influence the onset as well as the phenotype of disease. Data were compiled by Paul Bamborough and Fred E. Cohen.

Optical spectroscopic measurements of PrPC provided the necessary background for more detailed structural studies (25). Unable to produce crystals of PrP, we and others utilized NMR to determine the structure of an α-helical form of a recombinant PrP. The NMR structure of a COOH-terminal fragment of MoPrP consisting of 111 residues showed three helices, two of which corresponded to H3 and H4 in the PrPC model, and two small β-strands each consisting of three residues (154). How the structure of this MoPrP(121–231) fragment differs from PrPC is of interest because this fragment is lethal when expressed in Tg mice (160). Subsequently, structural studies were performed on a longer fragment of PrP containing residues 90–231 and corresponding to SHaPrP 27–30 (155161162). Expression of PrP(90–231) in Tg mice did not produce spontaneous disease (163164). More recently, NMR structures of recombinant full-length PrP have been reported (156165).

Models of PrPSc suggested that formation of the disease-causing isoform involves refolding of residues within the region between residues 90 and 140 into β-sheets (166); the single disulfide bond joining COOH-terminal helices would remain intact because the disulfide is required for PrPSc formation (Fig. 5E) (167,168). The high β-sheet content of PrPSc was predicted from the ability of PrP 27–30 to polymerize into amyloid fibrils (107). Subsequent optical spectroscopy confirmed the presence of β-sheet in both PrPSc and PrP 27–30 (25169171). Deletion of each of the regions of putative secondary structure in PrP, except for the NH2-terminal 66 amino acids (residues 23–88) (163172) and a 36-amino acid region (mouse residues 141–176) prevented formation of PrPSc as measured in scrapie–infected cultured neuroblastoma cells (168). With anti-PrP Fabs selected from phage display libraries (157) and two monoclonal antibodies (mAbs) derived from hybridomas (173175), the major conformational change that occurs during conversion of PrPC into PrPSc has been localized largely, but not entirely, to a region bounded by residues 90 and 112 (158). Similar conclusions were drawn from studies with an anti-PrP IgM mAb (176). While these results indicate that PrPSc formation involves primarily a conformational change in the domain composed of residues 90–112, mutations causing inherited prion diseases have been found throughout the protein (Fig. 4B). Interestingly, all of the known point mutations in PrP with biological significance occur either within or adjacent to regions of putative secondary structure in PrP and as such, appear to destabilize the structure of PrP (147148154). 

Figure 5


Structures of prion proteins. (A) NMR structure of SHa recombinant (r) PrP(90–231). Presumably, the structure of the α-helical form of rPrP(90–231) resembles that of PrPC. rPrP(90–231) is viewed from the interface where PrPSc is thought to bind to PrPC. The color scheme is as follows: α-helices A (residues 144–157), B (172193), and C (200227) in pink; disulfide between Cys-179 and Cys-214 in yellow; conserved hydrophobic region composed of residues 113–126 in red; loops in gray; residues 129–134 in green encompassing strand S1 and residues 159–165 in blue encompassing strand S2; the arrows span residues 129–131 and 161–163, as these show a closer resemblance to β-sheet (155). (B) NMR structure of rPrP(90–231) is viewed from the interface where protein X is thought to bind to PrPC. Protein X appears to bind to the side chains of residues that form a discontinuous epitope: some amino acids are in the loop composed of residues 165–171 and at the end of helix B (Gln-168 and Gln-172 with a low-density van der Waals rendering), whereas others are on the surface of helix C (Thr-215 and Gln-219 with a high-density van der Waals rendering) (178). (C) PrP residues governing the transmission of prions (180). NMR structure of recombinant SHaPrP region 121–231 (155) shown with the putative epitope formed by residues 184, 186, 203, and 205 highlighted in red. Residue numbers correspond to SHaPrP. Additional residues (138, 139, 143, 145, 148, and 155) that might participate in controlling the transmission of prions across species are depicted in green. Residues 168, 172, 215, and 219 that form the epitope for the binding of protein X are shown in blue. The three helices (A, B, and C) are highlighted in pink. (D) Schematic diagram showing the flexibility of the polypeptide chain for PrP(29–231) (156). The structure of the portion of the protein representing residues 90–231 was taken from the coordinates of PrP(90–231) (155). The remainder of the sequence was hand-built for illustration purposes only. The color scale corresponds to the heteronuclear {1H}-15N nuclear Overhauser enhancement data: red for the lowest (most negative) values, where the polypeptide is most flexible, to blue for the highest (most positive) values in the most structured and rigid regions of the protein. (E) Plausible model for the tertiary structure of HuPrPSc(166). Color scheme is as follows: S1 β-strands are 108–113 and 116–122 in red; S2 β-strands are 128–135 and 138–144 in green; α-helices H3 (residues 178–191) and H4 (residues 202–218) in gray; loop (residues 142–177) in yellow. Four residues implicated in the species barrier are shown in ball-and-stick form (Asn-108, Met-112, Met-129, Ala-133).

NMR structure of recombinant PrP.

The NMR structure of recombinant (r) SHaPrP(90–231) derived from Escherichia coli was determined after the protein was purified and refolded (Fig. 5A). Residues 90–112 are not shown because marked conformational heterogeneity was found in this region, while residues 113–126 constitute the conserved hydrophobic region, which also displays some structural plasticity (162). Although some features of the structure of rPrP(90–231) are similar to those reported earlier for the smaller recombinant MoPrP(121–231) fragment (154177), substantial differences were found. For example, the loop at the NH2 terminus of helix B is well defined in rPrP(90–231) but is disordered in MoPrP(121–231); in addition, helix C is composed of residues 200–227 in rPrP(90–231) but extends only from 200–217 in MoPrP(121–231). The loop and the COOH-terminal portion of helix C are particularly important because they form the site to which protein X binds as described below (Fig. 5B) (178). Whether the differences between the two recombinant PrP fragments are because of (i) their different lengths, (ii) species-specific differences in sequences, or (iii) the conditions used for solving the structures remains to be determined.

Studies of chimeric SHa/Mo and Hu/Mo PrP transgenes identified a domain composed of residues 95–170, where PrPC binds to PrPSc (133179). When chimeric bovine (Bo)/Mo PrP transgenes failed to render mice sensitive to BSE prions, we examined the differences among the sequences in the chimeric and parent PrP genes (180). The findings identified a second domain in PrP composed of residues 180–205 that seems to modulate the interaction between PrPC and PrPSc (Fig. 5C).

Recent NMR studies of full-length MoPrP(23–231) and SHaPrP(29–231) have shown that the NH2 termini are highly flexible and lack identifiable secondary structure under the experimental conditions employed (Fig. 5D) (156165). Studies of SHaPrP(29–231) indicate transient interactions between the COOH terminus of helix B and the highly flexible, NH2-terminal random-coil containing the octareapeats (residues 29–125) (156); such interactions were not reported for MoPrP(23–231) (165).

PrP appears to bind copper.

The highly flexible NH2 terminus of recombinant PrP may be more structured in the presence of copper. Each SHaPrP(29–231) molecule was found to bind two atoms of Cu2+; other divalent cations did not bind to PrP (181). Earlier studies with synthetic peptides corresponding to the octarepeat sequence demonstrated the binding of Cu2+ ions (182183), and optical spectroscopy showed that Cu2+ induced an α-helix formation in these peptides (184). More recently, PrP-deficient (Prnp0/0) mice were found to have lower levels of Zn/Cu superoxide dismutase (SOD) activity than do controls (185); SOD activity has been shown to mirror the state of copper metabolism (186). Measurements of membrane extracts from brains of Prnp0/0 mice showed low levels of Cu, whereas Fe and Zn were unchanged suggesting PrPC might function as a Cu2+-binding protein (187).

Disturbances in Cu2+ homeostasis leading to dysfunction of the CNS are well documented in humans and animals but are not known to be due to abnormalities in PrP metabolism: Menkes disease is manifest at birth and is due to a mutation of the MNK gene on the X chromosome, whereas Wilson’s disease appears in childhood and is due to a mutation of the WD gene on chromosome 13 (188191). Both the MNK and WDgenes encode copper-transporting ATPases. While both Menkes and Wilson’s diseases are recessive disorders, only Wilson’s disease can be treated with copper-chelating reagents. Interestingly, cuprizone, a Cu2+-chelating reagent, has been used in mice to induce neuropathological changes similar to those found in the prion diseases (192193).

PrP Gene Structure and Expression.

The entire open reading frame (ORF) of all known mammalian and avian PrP genes resides within a single exon (482194195). The mouse, sheep, cattle, and rat PrP genes contain three exons with the ORFs in exon 3 (196200) which is analogous to exon 2 of the SHa gene (82). The two exons of the SHaPrP gene are separated by a 10-kb intron: exon 1 includes a portion of the 5′ untranslated leader sequence, while exon 2 includes the ORF and 3′ untranslated region (82). Recently, a low abundance SHaPrP mRNA containing an additional small exon in the 5′ untranslated region was discovered that is encoded by the SHaPrP gene (201). Comparative sequencing of sheep and Hu cosmid clones containing PrP genes revealed an additional putative, small untranslated 5′ exon in the HuPrP gene (202). The promoters of both the SHa- and MoPrP genes contain multiple copies of G+C-rich repeats and are devoid of TATA boxes. These G+C nonamers represent a motif that may function as a canonical binding site for the transcription factor Sp1 (203). Mapping of PrP genes to the short arm of Hu chromosome 20 and to the homologous region of Mo chromosome 2 argues for the existence of PrP genes prior to the speciation of mammals (204,205).

Although PrP mRNA is constitutively expressed in the brains of adult animals (8384), it is highly regulated during development. In the septum, levels of PrP mRNA and choline acetyltransferase were found to increase in parallel during development (206). In other brain regions, PrP gene expression occurred at an earlier age. In situ hybridization studies show that the highest levels of PrP mRNA are found in neurons (207).

PrPC expression in brain was defined by standard immunohistochemistry (208) and by histoblotting in the brains of uninfected controls (209). Immunostaining of PrPC in the SHa brain was most intense in the stratum radiatum and stratum oriens of the CA1 region of the hippocampus and was virtually absent from the granule cell layer of the dentate gyrus and the pyramidal cell layer throughout Ammon’s horn. PrPScstaining was minimal in those regions that were intensely stained for PrPC. A similar relationship between PrPC and PrPSc was found in the amygdala. In contrast, PrPSc accumulated in the medial habenular nucleus, the medial septal nuclei, and the diagonal band of Broca; in contrast, these areas were virtually devoid of PrPC. In the white matter, bundles of myelinated axons contained PrPSc but were devoid of PrPC. These findings suggest that prions are transported along axons and are in agreement with earlier findings in which scrapie infectivity was found to migrate in a pattern consistent with retrograde transport (210212).

Molecular Genetics of Prion Diseases.

Independent of enriching brain fractions for scrapie infectivity that led to the discovery of PrPSc, the PrP gene was shown to be genetically linked to a locus controlling scrapie incubation times (213). Subsequently, mutation of the PrP gene was shown to be genetically linked to the development of familial prion disease (4). At the same time, expression of a SHaPrP transgene in mice was shown to render the animals highly susceptible to SHa prions, demonstrating that expression of a foreign PrP gene could abrogate the species barrier (214). Later, PrP-deficient (Prnp0/0) mice were found to be resistant to prion infection and failed to replicate prions, as expected (100101). The results of these studies indicated PrP must play a central role in the transmission and pathogenesis of prion disease, but equally important, they argued that the abnormal isoform is an essential component of the prion particle (23).

PrP gene dosage controls length of incubation time.

Scrapie incubation times in mice were used to distinguish prion strains and to identify a gene controlling its length (135215).  With a PrP cDNA probe, we demonstrated genetic linkage between the PrP gene and a locus controlling the incubation time in crosses between NZW/LacJ and I/Ln mice (213). We provisionally designated these genes as components of the Prn complex but eventually found that the incubation time gene, Prn-i, is either congruent with or closely linked to the PrP gene, Prnp (195).

Although the amino acid substitutions in PrP that distinguish NZW (Prnpa ) from I/Ln (Prnpb) mice argued for congruency of Prnp and Prn-i, experiments with Prnpa mice expressing Prnpb transgenes demonstrated a “paradoxical” shortening of incubation times (196). We had predicted that these Tg mice would exhibit a prolongation of the incubation time after inoculation with RML prions on the basis of (Prnpa × Prnpb)F1mice, which do exhibit long incubation times. We described those findings as “paradoxical shortening” because we and others had believed for many years that long incubation times are dominant traits (213,215). From studies of congenic and transgenic mice expressing different numbers of the a and b alleles ofPrnp, we learned that these findings were not paradoxical; indeed, they resulted from increased PrP gene dosage (218). We concluded that both Sincand Prn-i are congruent with PrP (218), and recent gene targeting studies have confirmed this view (219).

Overexpression of wtPrP transgenes.

Mice were constructed expressing different levels of the wt SHaPrP transgene (214). Inoculation of these Tg(SHaPrP) mice with SHa prions demonstrated abrogation of the species barrier, resulting in abbreviated incubation times (220). The length of the incubation time after inoculation with SHa prions was inversely proportional to the level of SHaPrPC in the brains of Tg(SHaPrP) mice (220). Bioassays of brain extracts from clinically ill Tg(SHaPrP) mice inoculated with Mo prions revealed that only Mo prions but no SHa prions were produced. Conversely, inoculation of Tg(SHaPrP) mice with SHa prions led only to the synthesis of SHa prions. Although the rate of PrPScsynthesis appears to be a function of the level of PrPC expression in Tg mice, the level to which PrPSc finally accumulates seems to be independent of PrPC concentration (220).

During transgenetic studies, we discovered that uninoculated older mice harboring numerous copies of wtPrP transgenes derived from Syrian hamsters, sheep, and Prnpb mice spontaneously developed truncal ataxia, hind-limb paralysis, and tremors (198). These Tg mice exhibited a profound necrotizing myopathy involving skeletal muscle, a demyelinating polyneuropathy, and focal vacuolation of the CNS. Development of disease was dependent on transgene dosage. For example, Tg(SHaPrP+/+)7 mice homozygous for the SHaPrP transgene array regularly developed disease between 400 and 600 days of age, whereas hemizygous Tg(SHaPrP+/0)7 mice also developed disease, but only after >650 days.

PrP-deficient mice.

The development and lifespan of two lines of PrP-deficient (Prnp0/0) mice were indistinguishable from those of controls (221222), whereas two other lines exhibited ataxia and Purkinje cell degeneration at ≈70 weeks of age (223) (R. Moore and D. Melton, personal communication). In the former two lines with normal development, altered sleep–wake cycles have been reported (224), and altered synaptic behavior in brain slices was reported (225226) but could not be confirmed by others (227,228).

Prnp0/0 mice are resistant to prions (100101). Prnp0/0 mice were sacrificed 5, 60, 120, and 315 days after inoculation with RML prions, and brain extracts were bioassayed in CD-1 Swiss mice. Except for residual infectivity from the inoculum detected at 5 days after inoculation, no infectivity was detected in the brains ofPrnp0/0 mice (101). One group of investigators found that Prnp0/0 mice inoculated with RML prions and sacrificed 20 weeks later had 103.6 ID50 units/ml of homogenate by bioassay (100). Others have used this report to argue that prion infectivity replicates in the absence of PrP (67132). Neither we nor the authors of the initial report could confirm the finding of prion replication in Prnp0/0 mice (101103).

Prion Species Barrier and Protein X.

The passage of prions between species is often a stochastic process, almost always characterized by prolonged incubation times during the first passage in the new host (36). This prolongation is often referred to as the “species barrier” (36229). Prions synthesized de novo reflect the sequence of the host PrP gene and not that of the PrPSc molecules in the inoculum derived from the donor (90). On subsequent passage in a homologous host, the incubation time shortens to that recorded for all subsequent passages. From studies with Tg mice, three factors have been identified that contribute to the species barrier: (i) the difference in PrP sequences between the prion donor and recipient, (ii) the strain of prion, and (iii) the species specificity of protein X, a factor defined by molecular genetic studies that binds to PrPC and facilitates PrPSc formation. This factor is likely to be a protein, hence the provisional designation protein X (134178). The prion donor is the last mammal in which the prion was passaged and its PrP sequence represents the “species” of the prion. The strain of prion, which seems to be enciphered in the conformation of PrPSc, conspires with the PrP sequence, which is specified by the recipient, to determine the tertiary structure of nascent PrPSc. These principles are demonstrated by studies on the transmission of SHa prions to mice showing that expression of a SHaPrP transgene in mice abrogated the species barrier (Table 3) (214). Besides the PrP sequence, the strain of prion modified transmission of SHa prions to mice (Table 3) (135230231).

Table 3

Influence of prion species and strains on transmission across a species barrier

Transmission of Hu prions.

Protein X was postulated to explain the results on the transmission of Hu prions to Tg mice (Table 4) (134179). Mice expressing both Mo and HuPrP were resistant to Hu prions, whereas those expressing only HuPrP were susceptible. These results argue that MoPrPC inhibited transmission of Hu prions—i.e., the formation of nascent HuPrPSc. In contrast to the foregoing studies, mice expressing both MoPrP and chimeric MHu2MPrP were susceptible to Hu prions and mice expressing MHu2MPrP alone were only slightly more susceptible. These findings contend that MoPrPC has only a minimal effect on the formation of chimeric MHu2MPrPSc.

Table 4

Evidence for protein X from transmission studies of human prions

Genetic evidence for protein X.

When the data on Hu prion transmission to Tg mice were considered together, they suggested that MoPrPC prevented the conversion of HuPrPC into PrPSc but had little effect on the conversion of MHu2M into PrPSc by binding to another Mo protein. We interpreted these results in terms of MoPrPC binding to this Mo protein with a higher affinity than does HuPrPC. We postulated that MoPrPC had little effect on the formation of PrPSc from MHu2M (Table 4) because MoPrP and MHu2M share the same amino acid sequence at the COOH terminus. We hypothesized that MoPrPC only weakly inhibited transmission of SHa prions to Tg(SHaPrP) mice (Table 3) because SHaPrP is more closely related to MoPrP than is HuPrP.

Using scrapie-infected Mo neuroblastoma cells transfected with chimeric Hu/Mo PrP genes, we extended our studies of protein X. Substitution of a Hu residue at position 214 or 218 prevented PrPSc formation (Fig.5B) (178). The side chains of these residues protrude from the same surface of the COOH-terminal α-helix, forming a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at position 167, 171, or 218 prevented PrPSc formation; these mutant PrPs appear to act as “dominant negatives” by binding protein X and rendering it unavailable for prion propagation. Our findings within the context of protein X explain the protective effects of basic polymorphic residues in PrP of humans and sheep (199232233).

Is protein X a molecular chaperone?

Since PrP undergoes a profound structural transition during prion propagation, it seems likely that other proteins such as chaperones participate in this process. Whether protein X functions as a molecular chaperone is unknown. Interestingly, scrapie-infected cells in culture display marked differences in the induction of heat-shock proteins (234235), and Hsp70 mRNA has been reported to increase in scrapie of mice (236). While attempts to isolate specific proteins that bind to PrP have been disappointing (237), PrP has been shown to interact with Bcl-2 and Hsp60 by two-hybrid analysis in yeast (238239). Although these studies are suggestive, no molecular chaperone involved in prion formation in mammalian cells has been identified.


By using the four-helix bundle model of PrPC (Fig. 4A) (147), each region of proposed secondary structure was systematically deleted and the mutant constructs were expressed in scrapie-infected neuroblastoma (ScN2a) cells and Tg mice (164168). Deletion of any of the four putative helical regions prevented PrPScformation, whereas deletion of the NH2-terminal region containing residues 23–89 did not affect the yield of PrPSc. In addition to the 67 residues at the NH2 terminus, 36 residues from positions 141–176 could be deleted without altering PrPSc formation (Figs. 6 and 7). The resulting PrP molecule of 106 amino acids was designated PrP106. In this mutant PrP, helix A as well as the S2 β-strand were removed. When PrP106 was expressed in ScN2a cells, PrPSc106 was soluble in 1% Sarkosyl. Whether the structure of PrPSc106 can be more readily determined than that of full-length PrPSc remains uncertain. 

Figure 6


Miniprions produced by deleting PrP residues 23–89 and 141–176. The deletion of residues 141–176 (green) containing helix A and the S2 β-strand is shown. Side chains of residues 168, 172, 215, and 219, which are thought to bind protein X, are shown in cyan. 

Figure 7


Transgene-specified susceptibility.

Tg(PrP106)Prnp0/0 mice that expressed PrP106 developed neurological dysfunction ≈300 days after inoculation with RML prions previously passaged in CD-1 Swiss mice (S. Supattapone, T. Muramoto, D. Peretz, S. J. DeArmond, A. Wallace, F. E. Cohen, S.B.P., and M. R. Scott, unpublished results). The resulting prions containing PrPSc106 produced CNS disease in ≈66 days upon subsequent passage in Tg(PrP106)Prnp0/0 mice (Table 5). Besides widespread spongiform degeneration and PrP deposits, the pyramidal cells of the hippocampus constituting the CA-1, CA-2, and CA-3 fields disappeared in Tg(PrP106)Prnp0/0 mice inoculated with prions containing PrPSc106 (Fig. 7). In no previous study of Tg mice have we seen similar neuropathological lesions. The Tg(MoPrP-A) mice overexpressing MoPrP are resistant to RML106 miniprions but are highly susceptible to RML prions. These mice require more than 250 days to produce illness after inoculation with miniprions but develop disease in ≈50 days when inoculated with RML prions containing full-length MoPrPSc.

Table 5

Susceptibility and resistance of Tg mice to artificial miniprions

Smaller prions and mythical viruses.

The unique incubation times and neuropathology in Tg mice caused by miniprions are difficult to reconcile with the notion that scrapie is caused by an as-yet-unidentified virus. When the mutant or wt PrPC of the host matched PrPSc in the inoculum, the mice were highly susceptible (Table 5). However, when there was a mismatch between PrPC and PrPSc, the mice were resistant to the prions. This principle of homologous PrP interactions, which underlies the species barrier (Table 3), is recapitulated in studies of PrP106 where the amino acid sequence has been drastically changed by deleting nearly 50% of the residues. Indeed, the unique properties of the miniprions provide another persuasive argument supporting the contention that prions are infectious proteins.

Human Prion Diseases.

Most humans afflicted with prion disease present with rapidly progressive dementia, but some manifest cerebellar ataxia. Although the brains of patients appear grossly normal upon postmortem examination, they usually show spongiform degeneration and astrocytic gliosis under the microscope. The human prion diseases can present as sporadic, genetic, or infectious disorders (5) (Table 1).

Sporadic CJD.

Sporadic forms of prion disease constitute most cases of CJD and possibly a few cases of Gerstmann–Sträussler–Scheinker disease (GSS) (Table 1) (4240241). In these patients, mutations of the PrP gene are not found. How prions causing disease arise in patients with sporadic forms is unknown; hypotheses include horizontal transmission of prions from humans or animals (242), somatic mutation of the PrP gene, and spontaneous conversion of PrPC into PrPSc (515). Because numerous attempts to establish an infectious link between sporadic CJD and a preexisting prion disease in animals or humans have been unrewarding, it seems unlikely that transmission features in the pathogenesis of sporadic prion disease (912243).

Inherited prion diseases.

To date, 20 different mutations in the human PrP gene resulting in nonconservative substitutions have been found that segregate with the inherited prion diseases (Fig. 4B). Familial CJD cases suggested that genetic factors might influence pathogenesis (12244), but this was difficult to reconcile with the transmissibility of fCJD and GSS (3). The discovery of genetic linkage between the PrP gene and scrapie incubation times in mice (213) raised the possibility that mutation might feature in the hereditary human prion diseases. The P102L mutation was the first PrP mutation to be genetically linked to CNS dysfunction in GSS (Fig. 4B) (4) and has since been found in many GSS families throughout the world (245247). Indeed, a mutation in the protein coding region of the PrP gene has been found in all reported kindred with familial human prion disease; besides the P102L mutation, genetic linkage has been established for four other mutations (162931).

Tg mouse studies confirmed that mutations of the PrP gene can cause neurodegeneration. The P102L mutation of GSS was introduced into the MoPrP transgene, and five lines of Tg(MoPrP-P101L) mice expressing high levels of mutant PrP developed spontaneous CNS degeneration consisting of widespread vacuolation of the neuropil, astrocytic gliosis, and numerous PrP amyloid plaques similar to those seen in the brains of humans who die from GSS(P102L) (248250). Brain extracts prepared from spontaneously ill Tg(MoPrP-P101L) mice transmitted CNS degeneration to Tg196 mice but contained no protease-resistant PrP (249250). The Tg196 mice do not develop spontaneous disease but express low levels of the mutant transgene MoPrP-P101L and are deficient for mouse PrP (Prnp0/0) (221). These studies, combined with the transmission of prions from patients who died of GSS to apes and monkeys (3) or to Tg(MHu2M-P101L) mice (134), demonstrate that prions are generated de novo by mutations in PrP. Additionally, brain extracts from patients with some other inherited prion diseases, fCJD(E200K) or FFI, transmit disease to Tg(MHu2M) mice (27), (153251).

Infectious prion diseases.

The infectious prion diseases include kuru of the Fore people in New Guinea, where prions were transmitted by ritualistic cannibalism (242252253). With the cessation of cannibalism at the urging of missionaries, kuru began to decline long before it was known to be transmissible (Fig. 8). Sources of prions causing infectious CJD on several different continents include improperly sterilized depth electrodes, transplanted corneas, human growth hormone (HGH) and gonadotropin derived from cadaveric pituitaries, and dura mater grafts (254). Over 90 young adults have developed CJD after treatment with cadaveric HGH; the incubation periods range from 3 to more than 20 years (255256). Dura mater grafts implanted during neurosurgical procedures seem to have caused more than 60 cases of CJD; these incubation periods range from 1 to more than 14 years (257259). 

Figure 8


Disappearance of the kuru and BSE epidemics. (A) Annual number of cases of BSE in cattle in Great Britain. (B) Biannual number of cases of kuru in Papua New Guinea. Data were compiled for BSE by John Wilesmith and for kuru by Michael Alpers.

Prion Diversity.

The existence of prion strains raises the question of how heritable biological information can be enciphered in a molecule other than nucleic acid (131215260265, 231266267). The typing of prion strains in C57BL, VM, and (C57BL × VM)F1 inbred mice began with isolates from sheep with scrapie. The prototypic strains called Me7 and 22A gave incubation times of ≈150 and ≈400 days in C57BL mice, respectively (215268269). The PrP genes of C57BL and I/Ln (and later VM) mice encode proteins differing at two residues and control scrapie incubation times (195213217219270).

Until recently, support for the hypothesis that the tertiary structure of PrPSc enciphers strain-specific information (23) was minimal except for the DY strain isolated from mink with transmissible encephalopathy by passage in Syrian hamsters (26271272).  Also notable was the generation of new strains during passage of prions through animals with different PrP genes (135230).

PrPSc conformation enciphers diversity.

Persuasive evidence that strain-specific information is enciphered in the tertiary structure of PrPSc comes from transmission of two different inherited human prion diseases to mice expressing a chimeric MHu2M PrP transgene (27). In FFI, the protease-resistant fragment of PrPSc after deglycosylation has a mass of 19 kDa, whereas in fCJD(E200K) and most sporadic prion diseases it is 21 kDa (Table 6) (273274). This difference in molecular size was shown to be due to different sites of proteolytic cleavage at the NH2 termini of the two human PrPSc molecules reflecting different tertiary structures (273).

Table 6

Distinct prion strains generated in humans with inherited prion diseases and transmitted to Tg mice

Extracts from the brains of FFI patients transmitted disease to mice expressing a chimeric MHu2M PrP gene about 200 days after inoculation and induced formation of the 19-kDa PrPSc, whereas fCJD(E200K) and sCJD produced the 21-kDa PrPSc in mice expressing the same transgene (27). On second passage, Tg(MHu2M) mice inoculated with FFI prions showed an incubation time of ≈130 days and a 19-kDa PrPSc, whereas those inoculated with fCJD(E200K) prions exhibited an incubation time of ≈170 days and a 21-kDa PrPSc (28). The experimental data demonstrate that MHu2MPrPSc can exist in two different conformations based on the sizes of the protease-resistant fragments, yet the amino acid sequence of MHu2MPrPSc is invariant.

The results of our studies argue that PrPSc acts as a template for the conversion of PrPC into nascent PrPSc. Imparting the size of the protease-resistant fragment of PrPSc through conformational templating provides a mechanism for both the generation and propagation of prion strains.

Interestingly, the protease-resistant fragment of PrPSc after deglycosylation with a mass of 19 kDa has been found in a patient who developed a sporadic case of prion disease similar to FFI but with no family history. Because both PrP alleles encoded the wt sequence and a Met at position 129, we labeled this case fatal sporadic insomnia (FSI). At autopsy, the spongiform degeneration, reactive astrogliosis, and PrPScdeposition were confined to the thalamus (275). These findings argue that the clinicopathologic phenotype is determined by the conformation of PrPSc in accord with the results of the transmission of human prions from patients with FFI to Tg mice (27).

Selective neuronal targeting.

Besides incubation times, profiles of spongiform change (Fig. 1) have been used to characterize prion strains (276), but recent studies argue that such profiles are not an intrinsic feature of strains (277278). The mechanism by which prion strains modify the pattern of spongiform degeneration was perplexing, since earlier investigations had shown that PrPSc deposition precedes neuronal vacuolation and reactive gliosis (212231). When FFI prions were inoculated into Tg(MHu2M) mice, PrPSc was confined largely to the thalamus (Fig. 9A) as is the case for FFI in humans (27279). In contrast, fCJD(E200K) prions inoculated into Tg(MHu2M) mice produced widespread deposition of PrPScthroughout the cortical mantle and many of the deep structures of the CNS (Fig. 9B) as is seen in fCJD(E200K) of humans. To examine whether the diverse patterns of PrPSc deposition are influenced by Asn-linked glycosylation of PrPC, we constructed Tg mice expressing PrPs mutated at one or both of the Asn-linked glycosylation consensus sites (278). These mutations resulted in aberrant neuroanatomic topologies of PrPC within the CNS, whereas pathologic point mutations adjacent to the consensus sites did not alter the distribution of PrPC. Tg mice with mutation of the second PrP glycosylation site exhibited prion incubation times of >500 days and unusual patterns of PrPSc deposition. These findings raise two possible scenarios. First, glycosylation can modify the conformation of PrPC and affect its affinity for a particular conformer of PrPSc, which results in specific patterns of PrPSc deposition; such interactions between PrPScand PrPC are likely to determine the rate of nascent PrPSc formation. Second, glycosylation modifies the stability of PrPSc and, hence, the rate of PrPSc clearance. This latter explanation is consistent with the proposal that the binding of PrPC to protein X is the rate-limiting step in PrPSc formation under most circumstances (280345).

Figure 9



Prion strains and the species barrier are of paramount importance in understanding the BSE epidemic in Great Britain, in which it is estimated that almost one million cattle were infected with prions (281282). The mean incubation time for BSE is about 5 years. Most cattle therefore did not manifest disease because they were slaughtered between 2 and 3 years of age (283). Nevertheless, more than 160,000 cattle, primarily dairy cows, have died of BSE over the past decade (Fig. 8A) (281). BSE is a massive common-source epidemic caused by meat and bone meal (MBM) fed primarily to dairy cows (282284). The MBM was prepared from the offal of sheep, cattle, pigs, and chickens as a high-protein nutritional supplement. In the late 1970s, the hydrocarbon-solvent extraction method used in the rendering of offal began to be abandoned, resulting in MBM with a much higher fat content (284). It is now thought that this change in the rendering process allowed scrapie prions from sheep to survive rendering and to be passed into cattle. Alternatively, bovine prions were present at low levels prior to modification of the rendering process and with the processing change survived in sufficient numbers to initiate the BSE epidemic when inoculated back into cattle orally through MBM. Against the latter hypothesis is the widespread geographical distribution throughout England of the initial 17 cases of BSE, which occurred almost simultaneously (282285286). Furthermore, there is no evidence of a preexisting prion disease of cattle, either in Great Britain or elsewhere.

Origin of BSE prions?

Brain extracts from BSE cattle cause disease in cattle, sheep, mice, pigs, and mink after intracerebral inoculation (291295), but prions in brain extracts from sheep with scrapie fed to cattle produced illness substantially different from BSE (296). However, no exhaustive effort has been made to test different strains of sheep prions or to examine the disease after bovine-to-bovine passage. In July 1988, the practice of feeding MBM to sheep and cattle was banned. Recent statistics argue that the epidemic is now disappearing as a result of this ruminant feed ban (Fig. 8A) (281), reminiscent of the disappearance of kuru in the Fore people of New Guinea (242253) (Fig. 8B).

Monitoring cattle for BSE prions.

Although many plans have been offered for the culling of older cattle to minimize the spread of BSE (281), it seems more important to monitor the frequency of prion disease in cattle as they are slaughtered for human consumption. No reliable, specific test for prion disease in live animals is available, but immunoassays for PrPSc in the brainstems of cattle might provide a reasonable approach to establishing the incidence of subclinical BSE in cattle entering the human food chain (176209,289297299345). Determining how early in the incubation period PrPSc can be detected by immunological methods is now possible, since a reliable bioassay has been created by expressing the BoPrP gene in Tg mice (180). Prior to development of Tg(BoPrP)Prnp0/0 mice, non-Tg mice inoculated intracerebrally with BSE brain extracts required more than 300 days to develop disease (67295300301). Depending on the titer of the inoculum, the structures of PrPC and PrPSc, and the structure of protein X, the number of inoculated animals developing disease can vary over a wide range (Table 3).

Have bovine prions been transmitted to humans?

In 1994, the first cases of CJD in teenagers and young adults that were eventually labeled new variant (v) CJD occurred in Britain (304). Besides these patients being young (305306), their brains showed numerous PrP amyloid plaques surrounded by a halo of intense spongiform degeneration (Fig. 10) (307 – 309).

Figure 10


The restricted geographical occurrence and chronology of vCJD have raised the possibility that BSE prions have been transmitted to humans. That only ≈25 vCJD cases have been recorded and the incidence has remained relatively constant make establishing the origin of vCJD difficult. It is noteworthy that epidemiological studies over the past three decades have failed to find evidence for transmission of sheep prions to humans (912). Attempts to predict the future number of cases of vCJD, assuming exposure to bovine prions prior to the offal ban, have been uninformative because so few cases of vCJD have occurred (312314). Are we at the beginning of a human prion disease epidemic in Britain like those seen for BSE and kuru (Fig. 8), or will the number of vCJD cases remain small as seen with iCJD caused by cadaveric HGH (255256)?

Strain of BSE prions.

Was a particular conformation of bovine PrPSc selected for heat resistance during the rendering process and then reselected multiple times as cattle infected by ingesting prion-contaminated MBM were slaughtered and their offal rendered into more MBM? Recent studies of PrPSc from brains of patients who died of vCJD show a pattern of PrP glycoforms different from those found for sCJD or iCJD (315316). But the utility of measuring PrP glycoforms is questionable in trying to relate BSE to vCJD (317,318) because PrPSc is formed after the protein is glycosylated (138140) and enzymatic deglycosylation of PrPSc requires denaturation (319320). Alternatively, it may be possible to establish a relationship between the conformations of PrPSc from cattle with BSE and those from humans with vCJD by using Tg mice as was done for strains generated in the brains of patients with FFI or fCJD (27180).

Yeast and Other Prions.

Although prions were originally defined in the context of an infectious pathogen (58), it is now becoming widely accepted that prions are elements that impart and propagate variability through multiple conformers of a normal cellular protein. Such a mechanism must surely not be restricted to a single class of transmissible pathogens. Indeed, it is likely that the original definition will need to be extended to encompass other situations in which a similar mechanism of information transfer occurs.

Two notable prion-like determinants, [URE3] and [PSI], have already been described in yeast and one in another fungus denoted [Het-s*] (321326). Studies of candidate prion proteins in yeast may prove particularly helpful in the dissection of some of the events that feature in PrPSc formation. Interestingly, different strains of yeast prions have been identified (327). Conversion to the prion-like [PSI] state in yeast requires the molecular chaperone Hsp104; however, no homolog of Hsp104 has been found in mammals (322328). The NH2-terminal prion domains of Ure2p and Sup35 that are responsible for the [URE3] and [PSI] phenotypes in yeast have been identified. In contrast to PrP, which is a GPI-anchored membrane protein, both Ure2p and Sup35 are cytosolic proteins (329). When the prion domains of these yeast proteins were expressed in E. coli, the proteins were found to polymerize into fibrils with properties similar to those of PrP and other amyloids (323325).

Prevention of and Therapeutics for Prion Diseases.

As our understanding of prion propagation increases, it should be possible to design effective therapeutics. Because people at risk for inherited prion diseases can now be identified decades before neurologic dysfunction is evident, the development of an effective therapy for these fully penetrant disorders is imperative (334335). Although we have no way of predicting the number of individuals who may develop neurologic dysfunction from bovine prions in the future (313), it would be prudent to seek an effective therapy now (28336). Interfering with the conversion of PrPC into PrPSc seems to be the most attractive therapeutic target (60). Either stabilizing the structure of PrPC by binding a drug or modifying the action of protein X, which might function as a molecular chaperone (Fig. 5B), is a reasonable strategy. Whether it is more efficacious to design a drug that binds to PrPC at the protein X binding site or one that mimics the structure of PrPC with basic polymorphic residues that seem to prevent scrapie and CJD remains to be determined (178233).

The production of domestic animals that do not replicate prions may also be important with respect to preventing prion disease. Sheep encoding the R/R polymorphism at position 171 seem to be resistant to scrapie (199232338344); presumably, this was the genetic basis of Parry’s scrapie eradication program in Great Britain 30 years ago (4446). A more effective approach using dominant negatives for producing prion-resistant domestic animals, including sheep and cattle, is probably the expression of PrP transgenes encoding R171 as well as additional basic residues at the putative protein X binding site (Fig. 5B) (178).

Whether gene therapy for the human prion diseases by using the dominant-negative approach described above for prion-resistant animals will prove feasible depends on the availability of efficient vectors for delivery of the transgene to the CNS.

Concluding Remarks—Looking to the Future.

While learning the details of the structures of PrPs and deciphering the mechanism of PrPC transformation into PrPSc will be important, the fundamental principles of prion biology have become reasonably clear. Though some investigators prefer to view the composition of the infectious prion particle as unresolved (336346). But the discovery of prion-like phenomena mediated by proteins unrelated to PrP in yeast and fungi serves not only to strengthen the prion concept but also to widen it (329).

Hallmark of prion diseases.

The hallmark of all prion diseases—whether sporadic, dominantly inherited, or acquired by infection—is that they involve the aberrant metabolism and resulting accumulation of the prion protein (Table 1) (23). The conversion of PrPC into PrPSc involves a conformation change whereby the α-helical content diminishes and the amount of β-sheet increases (25).

Understanding how PrPC unfolds and refolds into PrPSc will be of paramount importance in transferring advances in the prion diseases to studies of other degenerative illnesses. The mechanism by which PrPSc is formed must involve a templating process whereby existing PrPSc directs the refolding of PrPC into a nascent PrPSc with the same conformation. Indeed, the expanding list of prion diseases and their novel modes of transmission and pathogenesis (Table1), as well as the unprecedented mechanisms of prion propagation and information transfer (Tables 5 and6), indicate that much more attention to these fatal disorders of protein conformation is urgently needed.

Multiple conformers.

The discovery that proteins may have multiple biologically active conformations may prove no less important than the implications of prions for diseases. How many different tertiary structures can PrPSc adopt? This query not only addresses the issue of the limits of prion diversity (345) but also applies to proteins as they normally function within the cell or act to affect homeostasis in multicellular organisms. The expanding list of chaperones that assist the folding and unfolding of proteins promises much new knowledge about this process. For example, it is now clear that proproteases can carry their own chaperone activity where the pro portion of the protein functions as a chaperone in cis to guide the folding of the proteolytically active portion before it is cleaved (347). Such a mechanism might well feature in the maturation of polypeptide hormones. Interestingly, mutation of the chaperone portion of prosubtilisin resulted in the folding of a subtilisin protease with different properties than the one folded by the wild-type chaperone. Such chaperones have also been shown to work in trans (347). Additionally, apoptosis during development and throughout adult life might also be regulated, at least in part, by alternative tertiary structures of proteins.


Copper and its role on “progressive neurodegeneration” and death

Reported by: Dr. Venkat S. Karra, Ph.D.

Many of us are familiar with prion disease from its most startling and unusual incarnations—the outbreaks of “mad cow” disease (bovine spongiform encephalopathy) that created a crisis in the global beef industry. Or the strange story of Kuru, a fatal illness affecting a tribe in Papua New Guinea known for its cannibalism. Both are forms of prion disease, caused by the abnormal folding of a protein and resulting in progressive neurodegeneration and death.

While exactly how the protein malfunctions has been shrouded in mystery, scientists at The Scripps Research Institute now report in the journal Proceedings of the National Academy of Sciences (PNAS) that reducing copper in the body delays the onset of disease. Mice lacking a copper-transport gene lived significantly longer when infected with a prion disease than did normal mice.

“This conclusively shows that copper plays a role in the misfolding of the protein, but is not essential to that misfolding,” said Scripps Research Professor Michael Oldstone, who led the new study.

“We’ve known for many years that prion proteins bind copper,” said Scripps Research graduate student Owen Siggs, first author of the paper with former Oldstone lab member Justin Cruite. “But what scientists couldn’t agree on was whether this was a good thing or a bad thing during prion disease. By creating a mutation in mice that lowers the amount of circulating copper by 60%, we’ve shown that reducing copper can delay the onset of prion disease.”

Zombie Proteins
Unlike most infections, which are caused by bacteria, viruses, or parasites, prion disease stems from the dysfunction of a naturally occurring protein.

“We all contain a normal prion protein, and when that’s converted to an abnormal prion protein, you get a chronic nervous system disease,” said Oldstone. “That occurs genetically (spontaneously in some people) or is acquired by passage of infectious prions. Passage can occur by eating infected meat; in the past, by cannibalism in the Fore population in New Guinea through the ingestion or smearing of infectious brains; or by introduction of infectious prions on surgical instruments or with medical products made from infected individuals.”

When introduced into the body, the abnormal prion protein causes the misfolding of other, normal prion proteins, which then aggregate into plaques in the brain and nervous system, causing tremors, agitation, and failure of motor function, and leads invariably to death.

Like a prion, Alzheimer’s protein seeds itself in the brain

Misshapen amyloid-beta self-propagates in mice


The Alzheimer’s-related protein amyloid-beta is an infectious instigator in the brain, gradually contorting its harmless brethren into dangerous versions, new evidence suggests. The study adds to the argument that A-beta is a prion, a misfolded protein that behaves like the contagious culprits behind Creutzfeldt-Jakob disease in people, scrapie in sheep and mad cow disease.


Memories Cemented by Flux of Prion-Like Proteins

Persistent memories are doubly paradoxical. They are stable because they are built on physical connections that are dynamically maintained. Also, long-term memories are maintained through the work of prion-like proteins, even though prions are notorious for their contribution to neurodegenerative diseases—Alzheimer’s, Parkinson’s, and Huntington’s—and the destruction of memory.

Prion-like proteins, assert researchers at Columbia University, can have a functional role within neurons instead of contributing to disease. These researchers first identified a functional prion within Aplysia, a giant sea slug. Then they found a similar prion within mice. And now, in a new study, the researchers have proposed a mechanism for how this prion maintains long-term memories.

The new study—“The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3”—appeared June 17 in the journal Neuron. It explains how CPEB3, which stands for cytoplasmic polyadenylation element-binding protein, can account for the persistence of memory even though memories are built on molecular substrates that undergo rapid turnover.

A similar protein exists in humans, suggesting that human memories, too, rely on functional prions. This protein, said Eric Kandel, M.D., the leader of the Columbia team, may have the same role in memory. “Until this has been examined,” he prudently added, “we won’t know.”

When disease-causing prions form within a neuron, they cause damage by grouping together in sticky aggregates that disrupt cellular processes. Prion aggregates are highly stable and accumulate in infected tissue, causing tissue damage and cell death. The dying cell releases the prion proteins, which are then taken up by other cells—and are thus considered infectious.

Sourced through Scoop.it from: www.genengnews.com

PLoS One. 2014; 9(2): e89286. doi:  10.1371/journal.pone.0089286

Increasing Prion Propensity by Hydrophobic Insertion

Aaron C. Gonzalez Nelson,# Kacy R. Paul,# Michelina PetriNoe FloresRyan A. RoggeSean M. Cascarina, and Eric D. Ross*

Prion formation involves the conversion of proteins from a soluble form into an infectious amyloid form. Most yeast prion proteins contain glutamine/asparagine-rich regions that are responsible for prion aggregation. Prion formation by these domains is driven primarily by amino acid composition, not primary sequence, yet there is a surprising disconnect between the amino acids thought to have the highest aggregation propensity and those that are actually found in yeast prion domains. Specifically, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobic residues are strongly under-represented. Here, we directly test the effects of hydrophobic and aromatic residues on prion formation. Remarkably, we found that insertion of as few as two hydrophobic residues resulted in a multiple orders-of-magnitude increase in prion formation, and significant acceleration of in vitro amyloid formation. Thus, insertion or deletion of hydrophobic residues provides a simple tool to control the prion activity of a protein.

Protein disorder, prion propensities, and self-organizing macromolecular collectives.

Malinovska L1, Kroschwald S, Alberti S.

Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition.

PAPA – a method for predicting prion forming propensity

Biologists Find Unexpected Role for Amyloid-Forming Protein
Yeast protein could offer clues to how Alzheimer’s plaques form in the brain.
Fibrous protein clumps known as amyloids are most often associated with diseases such as Alzheimer’s disease, where they form characteristic plaques in the brain.

Scientists first described amyloids about 150 years ago; they have since been tagged as key players in Parkinson’s disease, Huntington’s disease, and rheumatoid arthritis, as well as Alzheimer’s. However, recent findings suggest that this class of proteins may also have critical biological functions in healthy cells.

MIT biologists have discovered that yeast cells need to build amyloid-like structures during the production of reproductive cells called spores. Learning more about how yeast build and then break down these protein structures could help scientists develop drugs that destroy disease-causing amyloids, the researchers say.


“Amyloids in the brain persist for decades. We just can’t get rid of them, yet yeast cells seem to have a mechanism for getting rid of them in 15 minutes,” says Luke Berchowitz, a postdoc at MIT’s Koch Institute for Integrative Cancer Research and the paper’s lead author. “If we can harness that mechanism, and really understand it, that could lead to anti-amyloid therapeutic opportunities.”

Reproductive role

Berchowitz and colleagues came across the yeast amyloid-forming protein known as Rim4 while investigating how sexual reproduction works in yeast. Rim4 is a protein containing long regions of disorder and stretches rich in the amino acid asparagine, which is a hallmark of a type of amyloid-forming proteins known as prions.

Berchowitz and Amon had previously discovered that Rim4 latches onto messenger RNA (mRNA) molecules, which carry genetic information to the cell’s protein-building machinery. In the new Cell paper, the researchers found that Rim4 uses amyloid-like clusters to prevent these mRNA molecules from being transcribed into proteins.

This process regulates the formation of spores — reproductive cells that are analogous to eggs and sperm, the researchers found.

Yeast usually reproduce asexually, through a process called budding, but under certain high-stress conditions, they can also undergo sexual reproduction through creation of spores that fuse to form new cells. The MIT team found that as yeast cells near completion of sexual reproduction, Rim4 amyloid-like clusters are broken down, releasing mRNA required for the cells to complete meiosis — the specialized type of cell division that produces spores.

“None of us anticipated that the way Rim4 actually works is by formation of these aggregates,” says Scott Keeney, a member of the Memorial Sloan Kettering Cancer Center, who was not involved in the research. “We’re used to thinking of these as toxic aggregates, so to demonstrate that they actually have a useful function in cells is intriguing.”

The researchers also found preliminary data suggesting that an amyloid protein known as DAZL plays the same role in sperm formation in mice; they believe that similar proteins are probably found in every organism that reproduces sexually, including humans.

Berchowitz says it is still unclear why cells rely on amyloid-forming proteins for this type of regulation, but one advantage amyloids offer is their ability to withstand the harsh environments in which sexual reproduction cells are formed. “Amyloids are stable and they’re able to sequester things,” he says. “They’re very tough guardians.”

“A great opportunity”

Previously, scientists have found a few other examples of amyloid-forming proteins that have critical roles in normal cell functions: In fruit flies, persistence of memory can rely on formation of amyloid-like structures in the brain, and amyloids are also involved in the formation of the skin pigment melanin in humans.

Learning more about how cells break down those amyloids could help scientists develop new drugs for disease such as Alzheimer’s, Parkinson’s, Huntington’s, and rheumatoid arthritis. Berchowitz is now working on figuring out how yeast cells regulate the breakdown of Rim4 aggregates.

“It’s a great opportunity to study assembly, regulation, and function of amyloids in living cells,” he says. “It’s pretty exciting that we can form them rapidly, synchronously, and abundantly.”

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