Archive for the ‘Pharmaceutical Analytics’ Category

Projected Sales in 2020 of World’s Top Ten Oncology Drugs

Reporter: Aviva Lev-Ari, PhD, RN


UPDATED on 1/17/2017

The top 15 best-selling cancer drugs in 2022

All told, by 2022, the top 15 cancer drugs are expected to collectively make almost $90 billion in sales. To put that in perspective, that represents about one-fourth of the entire U.S. pharma market in 2014, according to QuintilesIMS data. It’s also bigger than pharma’s haul in Japan or China that year.

It will likely be no revelation that three drugs among the top six on our list—provided courtesy of EvaluatePharma and Chempetitive—come from the highly touted PD-1/PD-L1 or checkpoint inhibitor class.

The top 15 best-selling cancer drugs in 2022



Projected Sales in 2020 of World’s Top Ten Oncology Drugs


Top Oncology Medicines

Projected Sales in 2020

Revlimid (Celgene)

Generic Name: Lenalidomide

$10,110 Million


Imbruvica (AbbVie/J&J)

Generic Name: Ibrutinib

$8,213 Million


Avastin (Roche)

Generic Name: Bevacizumab

$ 6,733 Million


Opdivo (BMS)

Generic Name: Nivolumab

$ 6,201 Million


Xtandi (Medivation & Astellas)

Generic Name: Enzalutamide

$5,700 Million


Rituxan (Roche)

Generic Name: Rituximab

$5,407 Million


Ibrance (Pfizer)

Generic Name: Palbociclib

$4,722 Million


Perjeta (Roche)

Generic Name: Pertuzumab

$4,669 Million


Herceptin (Roche)

Generic Name: Trastuzumab

$4,573 Million


Keytruda (Merck)

Generic Name: Pembrolizumab

$3,560 Million



World’s Top Ten Cancer Drugs by 2020

Igea gives professionals, patients and investors interested in pharmaceuticals, biotechnology, healthcare technology, diagnostics and medical devices the most relevant, actionable news, information and analysis available anywhere. Our goal is to provide expert insights, analysis and information from industry leaders with a deep understanding of life sciences, medicine and healthcare. Created and curated by Luca Dezzani, MD, Global Medical Director at Novartis Oncology*, Igea offers an insider’s view on the most important developments in life sciences, healthcare technology, digital health and more.


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Milestones in Physiology & Discoveries in Medicine and Genomics: Request for Book Review Writing on Amazon.com


Milestones in Physiology

Discoveries in Medicine, Genomics and Therapeutics

Patient-centric Perspective 





Author, Curator and Editor

Larry H Bernstein, MD, FCAP

Chief Scientific Officer

Leaders in Pharmaceutical Business Intelligence




Chapter 1: Evolution of the Foundation for Diagnostics and Pharmaceuticals Industries

1.1  Outline of Medical Discoveries between 1880 and 1980

1.2 The History of Infectious Diseases and Epidemiology in the late 19th and 20th Century

1.3 The Classification of Microbiota

1.4 Selected Contributions to Chemistry from 1880 to 1980

1.5 The Evolution of Clinical Chemistry in the 20th Century

1.6 Milestones in the Evolution of Diagnostics in the US HealthCare System: 1920s to Pre-Genomics


Chapter 2. The search for the evolution of function of proteins, enzymes and metal catalysts in life processes

2.1 The life and work of Allan Wilson
2.2  The  evolution of myoglobin and hemoglobin
2.3  More complexity in proteins evolution
2.4  Life on earth is traced to oxygen binding
2.5  The colors of life function
2.6  The colors of respiration and electron transport
2.7  Highlights of a green evolution


Chapter 3. Evolution of New Relationships in Neuroendocrine States
3.1 Pituitary endocrine axis
3.2 Thyroid function
3.3 Sex hormones
3.4 Adrenal Cortex
3.5 Pancreatic Islets
3.6 Parathyroids
3.7 Gastointestinal hormones
3.8 Endocrine action on midbrain
3.9 Neural activity regulating endocrine response

3.10 Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious Depression


Chapter 4.  Problems of the Circulation, Altitude, and Immunity

4.1 Innervation of Heart and Heart Rate
4.2 Action of hormones on the circulation
4.3 Allogeneic Transfusion Reactions
4.4 Graft-versus Host reaction
4.5 Unique problems of perinatal period
4.6. High altitude sickness
4.7 Deep water adaptation
4.8 Heart-Lung-and Kidney
4.9 Acute Lung Injury

4.10 Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics


Chapter 5. Problems of Diets and Lifestyle Changes

5.1 Anorexia nervosa
5.2 Voluntary and Involuntary S-insufficiency
5.3 Diarrheas – bacterial and nonbacterial
5.4 Gluten-free diets
5.5 Diet and cholesterol
5.6 Diet and Type 2 diabetes mellitus
5.7 Diet and exercise
5.8 Anxiety and quality of Life
5.9 Nutritional Supplements


Chapter 6. Advances in Genomics, Therapeutics and Pharmacogenomics

6.1 Natural Products Chemistry

6.2 The Challenge of Antimicrobial Resistance

6.3 Viruses, Vaccines and immunotherapy

6.4 Genomics and Metabolomics Advances in Cancer

6.5 Proteomics – Protein Interaction

6.6 Pharmacogenomics

6.7 Biomarker Guided Therapy

6.8 The Emergence of a Pharmaceutical Industry in the 20th Century: Diagnostics Industry and Drug Development in the Genomics Era: Mid 80s to Present

6.09 The Union of Biomarkers and Drug Development

6.10 Proteomics and Biomarker Discovery

6.11 Epigenomics and Companion Diagnostics


Chapter  7

Integration of Physiology, Genomics and Pharmacotherapy

7.1 Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

7.2 Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

7.3 Diagnostics and Biomarkers: Novel Genomics Industry Trends vs Present Market Conditions and Historical Scientific Leaders Memoirs

7.4 Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

7.5 Diagnosing Diseases & Gene Therapy: Precision Genome Editing and Cost-effective microRNA Profiling

7.6 Imaging Biomarker for Arterial Stiffness: Pathways in Pharmacotherapy for Hypertension and Hypercholesterolemia Management

7.7 Neuroprotective Therapies: Pharmacogenomics vs Psychotropic drugs and Cholinesterase Inhibitors

7.8 Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes

7.9 Preserved vs Reduced Ejection Fraction: Available and Needed Therapies

7.10 Biosimilars: Intellectual Property Creation and Protection by Pioneer and by

7.11 Demonstrate Biosimilarity: New FDA Biosimilar Guidelines


Chapter 7.  Biopharma Today

8.1 A Great University engaged in Drug Discovery: University of Pittsburgh

8.2 Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

8.3 Predicting Tumor Response, Progression, and Time to Recurrence

8.4 Targeting Untargetable Proto-Oncogenes

8.5 Innovation: Drug Discovery, Medical Devices and Digital Health

8.6 Cardiotoxicity and Cardiomyopathy Related to Drugs Adverse Effects

8.7 Nanotechnology and Ocular Drug Delivery: Part I

8.8 Transdermal drug delivery (TDD) system and nanotechnology: Part II

8.9 The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

8.10 Natural Drug Target Discovery and Translational Medicine in Human Microbiome

8.11 From Genomics of Microorganisms to Translational Medicine

8.12 Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad


Chapter 9. BioPharma – Future Trends

9.1 Artificial Intelligence Versus the Scientist: Who Will Win?

9.2 The Vibrant Philly Biotech Scene: Focus on KannaLife Sciences and the Discipline and Potential of Pharmacognosy

9.3 The Vibrant Philly Biotech Scene: Focus on Computer-Aided Drug Design and Gfree Bio, LLC

9.4 Heroes in Medical Research: The Postdoctoral Fellow

9.5 NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

9.6 1st Pitch Life Science- Philadelphia- What VCs Really Think of your Pitch

9.7 Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

9.8 Heroes in Medical Research: Green Fluorescent Protein and the Rough Road in Science

9.9 Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

9.10 The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research


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Targeting amyloidopathy

Larry H. Bernstein, MD, FCAP



Targeting a rare amyloidotic disease through rationally designed polymer conjugates

Inmaculada Conejos–Sánchez, Isabel Cardoso, Maria J. Saraiva, María J.Vicent
Journal of Controlled Release 178 (2014), 95–100
Saraiva et al. discovered in 2006 a RAGE-based peptide sequence capable of preventing transthyretin (TTR) aggregate-induced cytotoxicity, hallmark of initial stages of an inherited rare amyloidosis known as Familial Amyloidotic Polyneuropathy (FAP). To allow clinical progression of this peptidic sequence as FAP treatment, a family of polymer conjugates has been designed, synthesised and fully characterised. This approach fulfills the strategies defined in the Polymer Therapeutics area as an exhaustive physico-chemical characterisation fitting activity output towards a novel molecular target that is described here. RAGE peptide acts extracellularly, therefore, nointracellular drug delivery was necessary. PEG was selected as carrier and polymer–drug linker optimisation was then carried out by means of biodegradable (disulphide) and non-biodegradable (amide) covalent bonds. Conjugate size in solution, stability under invitro and in vivo scenarios and TTR binding affinity through surface plasmon resonance (SPR) was also performed with all synthesised conjugates. In their in vitro evaluation by monitoring the activation of caspase-3 in Schwann cells, peptide derivatives demonstrated retention of peptide activity reducing TTR aggregates (TTRagg) cytotoxicity upon conjugation and a greater plasma stability than the parent free peptide. The results also confirmed that a more stable polymer–peptide linker (amide) is required to secure therapeutic efficiency.

Polymer therapeutics are well established as successful first generation nanomedicines for treatment of infectious diseases and cancer[1]. Polymer–protein, drug and aptamer conjugates are innovative chemical entities capable of improving bioactive compound properties and thus increasing efficacy and decreasing toxicity[2,3]. Design of second generation of conjugates is now focussing on improved polymer structures, polymer–based combination therapy and novel molecular targets with great potential to further progress the clinical importance of these unique technologies [4]. Novel conjugates for the treatment of neuropathological disorders are proposed in this study. Amyloidosis is well known in the form of Alzheimer’s and Parkinson’s disease, but the target disease here is a rarer pathological disorder named familial amyloid polyneuropathy (FAP). FAPs constitute an important group of inherited amyloidosis diseases, and one of the most commonFAPs is caused by a mutated protein called transthyretin (TTR), which forms amyloid deposits, mainly in the peripheral nervous system [5]. The aggregation cascade of this mutated protein, produces a TTR aggregate (TTRagg) able to trigger neurodegeneration through engagement with the receptor-for-advanced-glycation-end-products (RAGE) which is present on peripheral neurons. RAGE signalling has been defined to be involved in many human pathologies such as Alzhehimer’s disease, diabetes and ageing, among others. This receptor is also up-regulated in tissues fromFAP patients [6]. The secreted RAGE form, named soluble RAGE (sRAGE), acts as a decoy to trap ligands and prevent interaction with cell surface receptors. sRAGE was shown to have important inhibitory effects in several cell cultures and transgenic mouse models, in which it prevented or reversed full-length RAGE signalling.

Saraiva et al. [7] discovered a specific peptidic sequence (named RAGE peptide) that is able to suppress TTRagg-induced cytotoxicity in cell culture. A reduced version of that peptide was proved to maintain the activity and the affinity of the initial peptide. The final peptide (compound A) contains 6 amino acids and responds to the sequence (from N to C terminus): YVRVRY. Although this provides an opportunity to design novel therapeutics for FAP treatment, peptide therapeutics themselves display well known challenges for in vivo use, e.g. low stability, poor pharmacokinetics and potential immunogenicity. Moreover the RAGE peptide demonstrates low solubility in plasma limiting its potential for i.v.administration.


Herein, novel specific nanoconjugates for the treatment of amyloidosis, and in particular familial amyloidotic polyneuropathy are reported. Apart from the research reported by Prof Arima et al. [22] using a hepatocyte-targeted FAP siRNA complex with lactosylated dendrimer (G3)/α-cyclodextrin(Lac-α-CDE(G3)), no other type of polymer therapeutic has been reported up to now for the treatment of this chronic degenerative family of diseases. Our rational design started from an active biomolecule of peptidic nature (RAGE peptide) that recognises the TTR prefibrillar aggregates responsible to promote cell death in FAPpatients [7]. The clinical progress of this promising inhibitor was masked by the well-known limitations of peptides, such as low solubility, low stability and possible immunogenicity. PEGylation through various linking strategies was successfully accomplished here as a solution for the named drawbacks, using a systematic approach to maintain peptide activity and receptor binding specificity. The data relating toTTR binding affinity, conjugate linker stability and the conjugate size distribution in solution of PEG– RAGE peptide conjugates indicate that the conjugates containing amide linkers have the greatest potential for further development as FAP inhibitors. Moreover, this novel conjugate has promising possibilities as a FAP therapeutic to be used alone in the early stages of the disease or as part of rationally designed combination therapy [23,24]. Preliminary in vivo studies (biodistribution) are shown in the supporting information demonstrating the enhanced plasma stability of the peptide upon conjugation (Fig.5S) , showing nospecific accumulation in any organ and renal excretion. More exhaustive in vivo experiments are currently ongoing with selected conjugates.


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Familial transthyretin amyloid polyneuropathy

Curator: Larry H. Bernstein, MD, FCAP



First-Ever Evidence that Patisiran Reduces Pathogenic, Misfolded TTR Monomers and Oligomers in FAP Patients

We reported data from our ongoing Phase 2 open-label extension (OLE) study of patisiran, an investigational RNAi therapeutic targeting transthyretin (TTR) for the treatment of TTR-mediated amyloidosis (ATTR amyloidosis) patients with familial amyloidotic polyneuropathy (FAP). Alnylam scientists and collaborators from The Scripps Research Institute and Misfolding Diagnostics, Inc. were able to measure the effects of patisiran on pathogenic, misfolded TTR monomers and oligomers in FAP patients. Results showed a rapid and sustained reduction in serum non-native conformations of TTR (NNTTR) of approximately 90%. Since NNTTR is pathogenic in ATTR amyloidosis and the level of NNTTR reduction correlated with total TTR knockdown, these results provide direct mechanistic evidence supporting the therapeutic hypothesis that TTR knockdown has the potential to result in clinical benefit. Furthermore, complete 12-month data from all 27 patients that enrolled in the patisiran Phase 2 OLE study showed sustained mean maximum reductions in total serum TTR of 91% for over 18 months and a mean 3.1-point decrease in mNIS+7 at 12 months, which compares favorably to an estimated increase in mNIS+7 of 13 to 18 points at 12 months based upon analysis of historical data sets in untreated FAP patients with similar baseline characteristics. Importantly, patisiran administration continues to be generally well tolerated out to 21 months of treatment.

Read our press release

View the non-native TTR poster (480 KB PDF)

View the complete 12-month patisiran Phase 2 OLE data presentation (620 KB PDF)

We are encouraged by these new data that provide continued support for our hypothesis that patisiran has the potential to halt neuropathy progression in patients with FAP. If these results are replicated in a randomized, double-blind, placebo-controlled study, we believe that patisiran could emerge as an important treatment option for patients suffering from this debilitating, progressive and life-threatening disease.


Hereditary ATTR Amyloidosis with Polyneuropathy (hATTR-PN)

ATTR amyloidosis is a progressive, life-threatening disease caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in multiple organs, but primarily in the peripheral nerves and heart. ATTR amyloidosis can lead to significant morbidity, disability, and mortality. The TTR protein is produced primarily in the liver and is normally a carrier for retinol binding protein – one of the vehicles used to transport vitamin A around the body.  Mutations in the TTR gene cause misfolding of the protein and the formation of amyloid fibrils that typically contain both mutant and wild-type TTR that deposit in tissues such as the peripheral nerves and heart, resulting in intractable peripheral sensory neuropathy, autonomic neuropathy, and/or cardiomyopathy.

Click to Enlarge


ATTR represents a major unmet medical need with significant morbidity and mortality. There are over 100 reported TTR mutations; the particular TTR mutation and the site of amyloid deposition determine the clinical manifestations of the disease whether it is predominantly symptoms of neuropathy or cardiomyopathy.

Specifically, hereditary ATTR amyloidosis with polyneuropathy (hATTR-PN), also known as familial amyloidotic polyneuropathy (FAP), is an inherited, progressive disease leading to death within 5 to 15 years. It is due to a mutation in the transthyretin (TTR) gene, which causes misfolded TTR proteins to accumulate as amyloid fibrils predominantly in peripheral nerves and other organs. hATTR-PN can cause sensory, motor, and autonomic dysfunction, resulting in significant disability and death.

It is estimated that hATTR-PN, also known as FAP, affects approximately 10,000 people worldwide.  Patients have a life expectancy of 5 to 15 years from symptom onset, and the only treatment options for early stage disease are liver transplantation and TTR stabilizers such as tafamidis (approved in Europe) and diflunisal.  Unfortunately liver transplantation has limitations, including limited organ availability as well as substantial morbidity and mortality. Furthermore, transplantation eliminates the production of mutant TTR but does not affect wild-type TTR, which can further deposit after transplantation, leading to cardiomyopathy and worsening of neuropathy. There is a significant need for novel therapeutics to treat patients who have inherited mutations in the TTR gene.

Our ATTR program is the lead effort in our Genetic Medicine Strategic Therapeutic Area (STAr) product development and commercialization strategy, which is focused on advancing innovative RNAi therapeutics toward genetically defined targets for the treatment of rare diseases with high unmet medical need.  We are developing patisiran (ALN-TTR02), an intravenously administered RNAi therapeutic, to treat the hATTR-PN form of the disease.

Patisiran for the Treatment hATTR-PN

APOLLO Phase 3 Trial

In 2012, Alnylam entered into an exclusive alliance with Genzyme, a Sanofi company, to develop and commercialize RNAi therapeutics, including patisiran and revusiran, for the treatment of ATTR amyloidosis in Japan and the broader Asian-Pacific region. In early 2014, this relationship was extended as a significantly broader alliance to advance RNAi therapeutics as genetic medicines. Under this new agreement, Alnylam will lead development and commercialization of patisiran in North America and Europe while Genzyme will develop and commercialize the product in the rest of world.


Hereditary ATTR Amyloidosis with Cardiomyopathy (hATTR-CM)

ATTR amyloidosis is a progressive, life-threatening disease caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in multiple organs, but primarily in the peripheral nerves and heart. ATTR amyloidosis can lead to significant morbidity, disability, and mortality. The TTR protein is produced primarily in the liver and is normally a carrier for retinol binding protein – one of the vehicles used to transport vitamin A around the body.  Mutations in the TTR gene cause misfolding of the protein and the formation of amyloid fibrils that typically contain both mutant and wild-type TTR that deposit in tissues such as the peripheral nerves and heart, resulting in intractable peripheral sensory neuropathy, autonomic neuropathy, and/or cardiomyopathy.

Click to Enlarge                            http://www.alnylam.com/web/assets/tetramer.jpg

ATTR represents a major unmet medical need with significant morbidity and mortality. There are over 100 reported TTR mutations; the particular TTR mutation and the site of amyloid deposition determine the clinical manifestations of the disease, whether it is predominantly symptoms of neuropathy or cardiomyopathy.

Specifically, hereditary ATTR amyloidosis with cardiomyopathy (hATTR-CM), also known as familial amyloidotic cardiomyopathy (FAC), is an inherited, progressive disease leading to death within 2 to 5 years. It is due to a mutation in the transthyretin (TTR) gene, which causes misfolded TTR proteins to accumulate as amyloid fibrils primarily in the heart. Hereditary ATTR amyloidosis with cardiomyopathy can result in heart failure and death.

While the exact numbers are not known, it is estimated hATTR-CM, also known as FAC affects at least 40,000 people worldwide.  hATTR-CM is fatal within 2 to 5 years of diagnosis and treatment is currently limited to supportive care.  Wild-type ATTR amyloidosis (wtATTR amyloidosis), also known as senile systemic amyloidosis, is a nonhereditary, progressive disease leading to death within 2 to 5 years. It is caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in the heart. Wild-type ATTR amyloidosis can cause cardiomyopathy and result in heart failure and death. There are no approved therapies for the treatment of hATTR-CM or SSA; hence there is a significant unmet need for novel therapeutics to treat these patients.

Our ATTR program is the lead effort in our Genetic Medicine Strategic Therapeutic Area (STAr) product development and commercialization strategy, which is focused on advancing innovative RNAi therapeutics toward genetically defined targets for the treatment of rare diseases with high unmet medical need.  We are developing revusiran (ALN-TTRsc), a subcutaneously administered RNAi therapeutic for the treatment of hATTR-CM.

Revusiran for the Treatment of hATTR-CM

ENDEAVOUR Phase 3 Trial

In 2012, Alnylam entered into an exclusive alliance with Genzyme, a Sanofi company, to develop and commercialize RNAi therapeutics, including patisiran and revusiran, for the treatment of ATTR amyloidosis in Japan and the broader Asian-Pacific region. In early 2014, this relationship was extended as a broader alliance to advance RNAi therapeutics as genetic medicines. Under this new agreement, Alnylam and Genzyme have agreed to co-develop and co-commercialize revusiran in North America and Europe, with Genzyme developing and commercializing the product in the rest of world. This broadened relationship on revusiran is aimed at expanding and accelerating the product’s global value.

Pre-Clinical Data and Advancement of ALN-TTRsc02 for Transthyretin-Mediated Amyloidosis

We presented pre-clinical data with ALN-TTRsc02, an investigational RNAi therapeutic targeting transthyretin (TTR) for the treatment of TTR-mediated amyloidosis (ATTR amyloidosis).  In pre-clinical studies, including those in non-human primates (NHPs), ALN-TTRsc02 achieved potent and highly durable knockdown of serum TTR of up to 99% with multi-month durability achieved after just a single dose, supportive of a potentially once quarterly dose regimen. Results from studies comparing TTR knockdown activity of ALN-TTRsc02 to that of revusiran showed that ALN-TTRsc02 has a markedly superior TTR knockdown profile.  Further, in initial rat toxicology studies, ALN-TTRsc02 was found to be generally well tolerated with no significant adverse events at doses as high as 100 mg/kg.

Read our press release

View the presentation



Emerging Therapies for Transthyretin Cardiac Amyloidosis Could Herald a New Era for the Treatment of HFPEF

Oct 14, 2015   |  Adam Castano, MDDavid Narotsky, MDMathew S. Maurer, MD, FACC


Heart failure with a preserved ejection fraction (HFPEF) is a clinical syndrome that has no pharmacologic therapies approved for this use to date. In light of failed medicines, cardiologists have refocused treatment strategies based on the theory that HFPEF is a heterogeneous clinical syndrome with different etiologies. Classification of HFPEF according to etiologic subtype may, therefore, identify cohorts with treatable pathophysiologic mechanisms and may ultimately pave the way forward for developing meaningful HFPEF therapies.1

A wealth of data now indicates that amyloid infiltration is an important mechanism underlying HFPEF. Inherited mutations in transthyretin cardiac amyloidosis (ATTRm) or the aging process in wild-type disease (ATTRwt) cause destabilization of the transthyretin (TTR) protein into monomers or oligomers, which aggregate into amyloid fibrils. These insoluble fibrils accumulate in the myocardium and result in diastolic dysfunction, restrictive cardiomyopathy, and eventual congestive heart failure (Figure 1). In an autopsy study of HFPEF patients, almost 20% without antemortem suspicion of amyloid had left ventricular (LV) TTR amyloid deposition.2 Even more resounding evidence for the contribution of TTR amyloid to HFPEF was a study in which 120 hospitalized HFPEF patients with LV wall thickness ≥12 mm underwent technetium-99m 3,3-diphosphono-1,2-propranodicarboxylic acid (99mTc-DPD) cardiac imaging,3,4 a bone isotope known to have high sensitivity and specificity for diagnosing TTR cardiac amyloidosis.5,6 Moderate-to-severe myocardial uptake indicative of TTR cardiac amyloid deposition was detected in 13.3% of HFPEF patients who did not have TTR gene mutations. Therefore, TTR cardiac amyloid deposition, especially in older adults, is not rare, can be easily identified, and may contribute to the underlying pathophysiology of HFPEF.

Figure 1

As no U.S. Food and Drug Administration-approved drugs are currently available for the treatment of HFPEF or TTR cardiac amyloidosis, the development of medications that attenuate or prevent TTR-mediated organ toxicity has emerged as an important therapeutic goal. Over the past decade, a host of therapies and therapeutic drug classes have emerged in clinical trials (Table 1), and these may herald a new direction for treating HFPEF secondary to TTR amyloid.

Table 1

TTR Silencers (siRNA and Antisense Oligonucleotides)


Ribonucleic acid interference (RNAi) has surfaced as an endogenous cellular mechanism for controlling gene expression. Small interfering RNAs (siRNAs) delivered into cells can disrupt the production of target proteins.7,8 A formulation of lipid nanoparticle and triantennary N-acetylgalactosamine (GalNAc) conjugate that delivers siRNAs to hepatocytes is currently in clinical trials.9 Prior research demonstrated these GalNAc-siRNA conjugates result in robust and durable knockdown of a variety of hepatocyte targets across multiple species and appear to be well suited for suppression of TTR gene expression and subsequent TTR protein production.

The TTR siRNA conjugated to GalNAc, ALN-TTRSc, is now under active investigation as a subcutaneous injection in phase 3 clinical trials in patients with TTR cardiac amyloidosis.10 Prior phase 2 results demonstrated that ALN-TTRSc was generally well tolerated in patients with significant TTR disease burden and that it reduced both wild-type and mutant TTR gene expression by a mean of 87%. Harnessing RNAi technology appears to hold great promise for treating patients with TTR cardiac amyloidosis. The ability of ALN-TTRSc to lower both wild-type and mutant proteins may provide a major advantage over liver transplantation, which affects the production of only mutant protein and is further limited by donor shortage, cost, and need for immunosuppression.

Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) are under clinical investigation for their ability to inhibit hepatic expression of amyloidogenic TTR protein. Currently, the ASO compound, ISIS-TTRRx, is under investigation in a phase 3 multicenter, randomized, double-blind, placebo-controlled clinical trial in patients with familial amyloid polyneuropathy (FAP).11 The primary objective is to evaluate its efficacy as measured by change in neuropathy from baseline relative to placebo. Secondary measures will evaluate quality of life (QOL), modified body mass index (mBMI) by albumin, and pharmacodynamic effects on retinol binding protein. Exploratory objectives in a subset of patients with LV wall thickness ≥13 mm without a history of persistent hypertension will examine echocardiographic parameters, N-terminal pro–B-type natriuretic peptide (NT-proBNP), and polyneuropathy disability score relative to placebo. These data will facilitate analysis of the effect of antisense oligonucleotide-mediated TTR suppression on the TTR cardiac phenotype with a phase 3 trial anticipated to begin enrollment in 2016.

TTR Stabilizers (Diflunisal, Tafamidis)


Several TTR-stabilizing agents are in various stages of clinical trials. Diflunisal, a traditionally used and generically available nonsteroidal anti-inflammatory drug (NSAID), binds and stabilizes familial TTR variants against acid-mediated fibril formation in vitro and is now in human clinical trials.12,13 The use of diflunisal in patients with TTR cardiac amyloidosis is controversial given complication of chronic inhibition of cyclooxygenase (COX) enzymes, including gastrointestinal bleeding, renal dysfunction, fluid retention, and hypertension that may precipitate or exacerbate heart failure in vulnerable individuals.14-17 In TTR cardiac amyloidosis, an open-label cohort study suggested that low-dose diflunisal with careful monitoring along with a prophylactic proton pump inhibitor could be safely administered to compensated patients.18 An association was observed, however, between chronic diflunisal use and adverse changes in renal function suggesting that advanced kidney disease may be prohibitive in diflunisal therapy.In FAP patients with peripheral or autonomic neuropathy randomized to diflunisal or placebo, diflunisal slowed progression of neurologic impairment and preserved QOL over two years of follow-up.19 Echocardiography demonstrated cardiac involvement in approximately 50% of patients.20 Longer-term safety and efficacy data over an average 38 ± 31 months in 40 Japanese patients with hereditary ATTR amyloidosis who were not candidates for liver transplantation showed that diflunisal was mostly well tolerated.12 The authors cautioned the need for attentive monitoring of renal function and blood cell counts. Larger multicenter collaborations are needed to determine diflunisal’s true efficacy in HFPEF patients with TTR cardiac amyloidosis.


Tafamidis is under active investigation as a novel compound that binds to the thyroxine-binding sites of the TTR tetramer, inhibiting its dissociation into monomers and blocking the rate-limiting step in the TTR amyloidogenesis cascade.21 The TTR compound was shown in an 18-month double-blind, placebo-controlled trial to slow progression of neurologic symptoms in patients with early-stage ATTRm due to the V30M mutation.22 When focusing on cardiomyopathy in a phase 2, open-label trial, tafamidis also appeared to effectively stabilize TTR tetramers in non-V30M variants, wild-type and V122I, as well as biochemical and echocardiographic parameters.23,24 Preliminary data suggests that clinically stabilized patients had shorter disease duration, lower cardiac biomarkers, less myocardial thickening, and higher EF than those who were not stabilized, suggesting early institution of therapy may be beneficial. A phase 3 trial has completed enrollment and will evaluate the efficacy, safety, and tolerability of tafamidis 20 or 80 mg orally vs. placebo.25 This will contribute to long-term safety and efficacy data needed to determine the therapeutic effects of tafamidis among ATTRm variants.

Amyloid Degraders (Doxycycline/TUDCA and Anti-SAP Antibodies)


While silencer and stabilizer drugs are aimed at lowering amyloidogenic precursor protein production, they cannot remove already deposited fibrils in an infiltrated heart. Removal of already deposited fibrils by amyloid degraders would be an important therapeutic strategy, particularly in older adults with heavily infiltrated hearts reflected by thick walls, HFPEF, systolic heart failure, and restrictive cardiomyopathy. Combined doxycycline and tauroursodeoxycholic acid (TUDCA) disrupt TTR amyloid fibrils and appeared to have an acceptable safety profile in a small phase 2 open-label study among 20 TTR patients. No serious adverse reactions or clinical progression of cardiac or neuropathic involvement was observed over one year.26 An active phase 2, single-center, open-label, 12-month study will assess primary outcome measures including mBMI, neurologic impairment score, and NT-proBNP.27 Another phase 2 study is examining the tolerability and efficacy of doxycycline/TUDCA over an 18-month period in patients with TTR amyloid cardiomyopathy.28 Additionally, a study in patients with TTR amyloidosis is ongoing to determine the effect of doxycycline alone on neurologic function, cardiac biomarkers, echocardiographic parameters, modified body mass index, and autonomic neuropathy.29

Anti-SAP Antibodies

In order to safely clear established amyloid deposits, the role of the normal, nonfibrillar plasma glycoprotein present in all human amyloid deposits, serum amyloid P component (SAP), needs to be more clearly understood.30 In mice with amyloid AA type deposits, administration of antihuman SAP antibody triggered a potent giant cell reaction that removed massive visceral amyloid deposits without adverse effects.31 In humans with TTR cardiac amyloidosis, anti-SAP antibody treatments could be feasible because the bis-D proline compound, CPHPC, is capable of clearing circulating human SAP, which allow anti-SAP antibodies to reach residual deposited SAP. In a small, open-label, single-dose-escalation, phase 1 trial involving 15 patients with systemic amyloidosis, none of whom had clinical evidence of cardiac amyloidosis, were treated with CPHPC followed by human monoclonal IgG1 anti-SAP antibody.32 No serious adverse events were reported and amyloid deposits were cleared from the liver, kidney, and lymph node. Anti-SAP antibodies hold promise as a potential amyloid therapy because of their potential to target all forms of amyloid deposits across multiple tissue types.

Mutant or wild-type TTR cardiac amyloidoses are increasingly recognized as a cause of HFPEF. Clinicians need to be aware of this important HFPEF etiology because the diverse array of emerging disease-modifying agents for TTR cardiac amyloidosis in human clinical trials has the potential to herald a new era for the treatment of HFPEF.


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  10. U.S. National Institutes of Health. Phase 2 Study to Evaluate ALN-TTRSC in Patients With Transthyretin (TTR) Cardiac Amyloidosis (ClinicalTrials.gov website). 2014. Available at: https://www.clinicaltrials.gov/ct2/show/NCT01981837. Accessed 8/19/2015.
  11. U.S. National Institutes of Health. Efficacy and Safety of ISIS-TTRRx in Familial Amyloid Polyneuropathy (Clinical Trials.gov Website. 2013. Available at: http://www.clinicaltrials.gov/ct2/show/NCT01737398. Accessed 8/19/2015.
  12. Sekijima Y, Dendle MA, Kelly JW. Orally administered diflunisal stabilizes transthyretin against dissociation required for amyloidogenesis. Amyloid 2006;13:236-49.
  13. Tojo K, Sekijima Y, Kelly JW, Ikeda S. Diflunisal stabilizes familial amyloid polyneuropathy-associated transthyretin variant tetramers in serum against dissociation required for amyloidogenesis. Neurosci Res 2006;56:441-9.
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  15. Wallace JL. Pathogenesis of NSAID-induced gastroduodenal mucosal injury. Best Pract Res Clin Gastroenterol 2001;15:691-703.
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  20. Quarta CCF, Solomon RH Suhr SD, et al. The prevalence of cardiac amyloidosis in familial amyloidotic polyneuropathy with predominant neuropathy: The Diflunisal Trial. International Symposium on Amyloidosis 2014:88-9.
  21. Hammarstrom P, Jiang X, Hurshman AR, Powers ET, Kelly JW. Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity. Proc Natl Acad Sci U S A 2002;99 Suppl 4:16427-32.
  22. Coelho T, Maia LF, Martins da Silva A, et al. Tafamidis for transthyretin familial amyloid polyneuropathy: a randomized, controlled trial. Neurology 2012;79:785-92.
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  24. Maurer MS, Grogan DR, Judge DP, et al. Tafamidis in transthyretin amyloid cardiomyopathy: effects on transthyretin stabilization and clinical outcomes. Circ Heart Fail 2015;8:519-26.
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  26. Obici L, Cortese A, Lozza A, et al. Doxycycline plus tauroursodeoxycholic acid for transthyretin amyloidosis: a phase II study. Amyloid 2012;19 Suppl 1:34-6.
  27. U.S. National Institutes of Health. Safety, Efficacy and Pharmacokinetics of Doxycycline Plus Tauroursodeoxycholic Acid in Transthyretin Amyloidosis (ClinicalTrials.gov website). 2011. Available at: http://www.clinicaltrials.gov/ct2/show/NCT01171859. Accessed 8/19/2015.
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The Acid-Mediated Denaturation Pathway of Transthyretin Yields a Conformational Intermediate That Can Self-Assemble into Amyloid

Zhihong Lai , Wilfredo Colón , and Jeffery W. Kelly *
Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255
Biochemistry199635 (20), pp 6470–6482   http://dx.doi.org:/10.1021/bi952501g
Publication Date (Web): May 21, 1996  Copyright © 1996 American Chemical Society

Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0−3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand−loop−D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS−PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly β-sheet structure having a well-defined tertiary structure.


Prevention of Transthyretin Amyloid Disease by Changing Protein Misfolding Energetics

Per Hammarström*, R. Luke Wiseman*, Evan T. Powers, Jeffery W. Kelly   + Author Affiliations

Science  31 Jan 2003; 299(5607):713-716   http://dx.doi.org:/10.1126/science.1079589

Genetic evidence suggests that inhibition of amyloid fibril formation by small molecules should be effective against amyloid diseases. Known amyloid inhibitors appear to function by shifting the aggregation equilibrium away from the amyloid state. Here, we describe a series of transthyretin amyloidosis inhibitors that functioned by increasing the kinetic barrier associated with misfolding, preventing amyloidogenesis by stabilizing the native state. The trans-suppressor mutation, threonine 119 → methionine 119, which is known to ameliorate familial amyloid disease, also functioned through kinetic stabilization, implying that this small-molecule strategy should be effective in treating amyloid diseases.


Rational design of potent human transthyretin amyloid disease inhibitors

Thomas Klabunde1,2, H. Michael Petrassi3, Vibha B. Oza3, Prakash Raman3, Jeffery W. Kelly3 & James C. Sacchettini1

Nature Structural & Molecular Biology 2000; 7: 312 – 321.                http://dx.doi.org:/10.1038/74082

The human amyloid disorders, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and senile systemic amyloidosis, are caused by insoluble transthyretin (TTR) fibrils, which deposit in the peripheral nerves and heart tissue. Several nonsteroidal anti-inflammatory drugs and structurally similar compounds have been found to strongly inhibit the formation of TTR amyloid fibrils in vitro. These include flufenamic acid, diclofenac, flurbiprofen, and resveratrol. Crystal structures of the protein–drug complexes have been determined to allow detailed analyses of the protein–drug interactions that stabilize the native tetrameric conformation of TTR and inhibit the formation of amyloidogenic TTR. Using a structure-based drug design approach ortho-trifluormethylphenyl anthranilic acid and N-(meta-trifluoromethylphenyl) phenoxazine 4,6-dicarboxylic acid have been discovered to be very potent and specific TTR fibril formation inhibitors. This research provides a rationale for a chemotherapeutic approach for the treatment of TTR-associated amyloid diseases.


First European consensus for diagnosis, management, and treatment of transthyretin familial amyloid polyneuropathy

Adams, Davida; Suhr, Ole B.b; Hund, Ernstc; Obici, Laurad; Tournev, Ivailoe,f; Campistol, Josep M.g; Slama, Michel S.h; Hazenberg, Bouke P.i; Coelho, Teresaj; from the European Network for TTR-FAP (ATTReuNET)

Current Opin Neurol: Feb 2016; 29 – Issue – p S14–S26      http://dx.doi.org:/10.1097/WCO.0000000000000289

Purpose of review: Early and accurate diagnosis of transthyretin familial amyloid polyneuropathy (TTR-FAP) represents one of the major challenges faced by physicians when caring for patients with idiopathic progressive neuropathy. There is little consensus in diagnostic and management approaches across Europe.

Recent findings: The low prevalence of TTR-FAP across Europe and the high variation in both genotype and phenotypic expression of the disease means that recognizing symptoms can be difficult outside of a specialized diagnostic environment. The resulting delay in diagnosis and the possibility of misdiagnosis can misguide clinical decision-making and negatively impact subsequent treatment approaches and outcomes.

Summary: This review summarizes the findings from two meetings of the European Network for TTR-FAP (ATTReuNET). This is an emerging group comprising representatives from 10 European countries with expertise in the diagnosis and management of TTR-FAP, including nine National Reference Centres. The current review presents management strategies and a consensus on the gold standard for diagnosis of TTR-FAP as well as a structured approach to ongoing multidisciplinary care for the patient. Greater communication, not just between members of an individual patient’s treatment team, but also between regional and national centres of expertise, is the key to the effective management of TTR-FAP.


Transthyretin familial amyloid polyneuropathy (TTR-FAP) is a highly debilitating and irreversible neurological disorder presenting symptoms of progressive sensorimotor and autonomic neuropathy [1▪,2▪,3]. TTR-FAP is caused by misfolding of the transthyretin (TTR) protein leading to protein aggregation and the formation of amyloid fibrils and, ultimately, to amyloidosis (commonly in the peripheral and autonomic nervous system and the heart) [4,5]. TTR-FAP usually proves fatal within 7–12 years from the onset of symptoms, most often due to cardiac dysfunction, infection, or cachexia [6,7▪▪].

The prevalence and disease presentation of TTR-FAP vary widely within Europe. In endemic regions (northern Portugal, Sweden, Cyprus, and Majorca), patients tend to present with a distinct genotype in large concentrations, predominantly a Val30Met substitution in the TTR gene [8–10]. In other areas of Europe, the genetic footprint of TTR-FAP is more varied, with less typical phenotypic expression [6,11]. For these sporadic or scattered cases, a lack of awareness among physicians of variable clinical features and limited access to diagnostic tools (i.e., pathological studies and genetic screening) can contribute to high rates of misdiagnosis and poorer patient outcomes [1▪,11]. In general, early and late-onset variants of TTR-FAP, found within endemic and nonendemic regions, present several additional diagnostic challenges [11,12,13▪,14].

Delay in the time to diagnosis is a major obstacle to the optimal management of TTR-FAP. With the exception of those with a clearly diagnosed familial history of FAP, patients still invariably wait several years between the emergence of first clinical signs and accurate diagnosis [6,11,14]. The timely initiation of appropriate treatment is particularly pertinent, given the rapidity and irreversibility with which TTR-FAP can progress if left unchecked, as well as the limited effectiveness of available treatments during the later stages of the disease [14]. This review aims to consolidate the existing literature and present an update of the best practices in the management of TTR-FAP in Europe. A summary of the methods used to achieve a TTR-FAP diagnosis is presented, as well as a review of available treatments and recommendations for treatment according to disease status.

Patients with TTR-FAP can present with a range of symptoms [11], and care should be taken to acquire a thorough clinical history of the patient as well as a family history of genetic disease. Delay in diagnosis is most pronounced in areas where TTR-FAP is not endemic or when there is no positive family history [1▪]. TTR-FAP and TTR-familial amyloid cardiomyopathy (TTR-FAC) are the two prototypic clinical disease manifestations of a broader disease spectrum caused by an underlying hereditary ATTR amyloidosis [19]. In TTR-FAP, the disease manifestation of neuropathy is most prominent and definitive for diagnosis, whereas cardiomyopathy often suggests TTR-FAC. However, this distinction is often superficial because cardiomyopathy, autonomic neuropathy, vitreous opacities, kidney disease, and meningeal involvement all may be present with varying severity for each patient with TTR-FAP.

Among early onset TTR-FAP with usually positive family history, symptoms of polyneuropathy present early in the disease process and usually predominate throughout the progression of the disease, making neurological testing an important diagnostic aid [14]. Careful clinical examination (e.g., electromyography with nerve conduction studies and sympathetic skin response, quantitative sensation test, quantitative autonomic test) can be used to detect, characterize, and scale the severity of neuropathic abnormalities involving small and large nerve fibres [10]. Although a patient cannot be diagnosed definitively with TTR-FAP on the basis of clinical presentation alone, symptoms suggesting the early signs of peripheral neuropathy, autonomic dysfunction, and cardiac conduction disorders or infiltrative cardiomyopathy are all indicators that further TTR-FAP diagnostic investigation is warranted. Late-onset TTR-FAP often presents as sporadic cases with distinct clinical features (e.g., milder autonomic dysfunction) and can be more difficult to diagnose than early-onset TTR-FAP (Table 2) [1▪,11,12,13▪,14,20].


Genetic testing is carried out to allow detection of specific amyloidogenic TTR mutations (Table 1), using varied techniques depending on the expertise and facilities available in each country (Table S2, http://links.lww.com/CONR/A39). A targeted approach to detect a specific mutation can be used for cases belonging to families with previous diagnosis. In index cases of either endemic and nonendemic regions that do not have a family history of disease, are difficult to confirm, and have atypical symptoms, TTR gene sequencing is required for the detection of both predicted and new amyloidogenic mutations [26,27].

Following diagnosis, the neuropathy stage and systemic extension of the disease should be determined in order to guide the next course of treatment (Table 4) [3,30,31]. The three stages of TTR-FAP severity are graded according to a patient’s walking disability and degree of assistance required [30]. Systemic assessment, especially of the heart, eyes, and kidney, is also essential to ensure all aspects of potential impact of the disease can be detected [10].

Table 4


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The goals of cardiac investigations are to detect serious conduction disorders with the risk of sudden death and infiltrative cardiomyopathy. Electrocardiograms (ECG), Holter-ECG, and intracardiac electrophysiology study are helpful to detect conduction disorders. Echocardiograms, cardiac magnetic resonance imaging, scintigraphy with bone tracers, and biomarkers (e.g., brain natriuretic peptide, troponin) can all help to diagnose infiltrative cardiomyopathy[10]. An early detection of cardiac abnormalities has obvious benefits to the patient, given that the prophylactic implantation of pacemakers was found to prevent 25% of major cardiac events in TTR-FAP patients followed up over an average of 4 years [32▪▪]. Assessment of cardiac denervation with 123-iodine meta-iodobenzylguanidine is a powerful prognostic marker in patients diagnosed with FAP [33].



Tafamidis is a first-in-class therapy that slows the progression of TTR amyloidogenesis by stabilizing the mutant TTR tetramer, thereby preventing its dissociation into monomers and amyloidogenic and toxic intermediates [55,56]. Tafamidis is currently indicated in Europe for the treatment of TTR amyloidosis in adult patients with stage I symptomatic polyneuropathy to delay peripheral neurological impairment [57].

In an 18-month, double-blind, placebo-controlled study of patients with early-onset Val30Met TTR-FAP, tafamidis was associated with a 52% lower reduction in neurological deterioration (P = 0.027), a preservation of nerve function, and TTR stabilization versus placebo [58▪▪]. However, only numerical differences were found for the coprimary endpoints of neuropathy impairment [neuropathy impairment score in the lower limb (NIS-LL) responder rates of 45.3% tafamidis vs 29.5% placebo; P = 0.068] and quality of life scores [58▪▪]. A 12-month, open-label extension study showed that the reduced rates of neurological deterioration associated with tafamidis were sustained over 30 months, with earlier initiation of tafamidis linking to better patient outcomes (P = 0.0435) [59▪]. The disease-slowing effects of tafamidis may be dependent on the early initiation of treatment. In an open-label study with Val30Met TTR-FAP patients with late-onset and advanced disease (NIS-LL score >10, mean age 56.4 years), NIS-LL and disability scores showed disease progression despite 12 months of treatment with tafamidis, marked by a worsening of neuropathy stage in 20% and the onset of orthostatic hypotension in 22% of patients at follow-up [60▪].

Tafamidis is not only effective in patients exhibiting the Val30Met mutation; it also has proven efficacy, in terms of TTR stabilization, in non-Val30Met patients over 12 months [61]. Although tafamidis has demonstrated safe use in patients with TTR-FAP, care should be exercised when prescribing to those with existing digestive problems (e.g., diarrhoea, faecal incontinence) [60▪].

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Diflunisal is a nonsteroidal anti-inflammatory drug (NSAID) that, similar to tafamidis, slows the rate of amyloidogenesis by preventing the dissociation, misfolding, and misassembly of the mutated TTR tetramer [62,63]. Off-label use has been reported for patients with stage I and II disease, although diflunisal is not currently licensed for the treatment of TTR-FAP.

Evidence for the clinical effectiveness of diflunisal in TTR-FAP derives from a placebo-controlled, double-blind, 24-month study in 130 patients with clinically detectable peripheral or autonomic neuropathy[64▪]. The deterioration in NIS scores was significantly more pronounced in patients receiving placebo compared with those taking diflunisal (P = 0.001), and physical quality of life measures showed significant improvement among diflunisal-treated patients (P = 0.001). Notable during this study was the high rate of attrition in the placebo group, with 50% more placebo-treated patients dropping out of this 2-year study as a result of disease progression, advanced stage of the disease, and varied mutations.

One retrospective analysis of off-label use of diflunisal in patients with TTR-FAP reported treatment discontinuation in 57% of patients because of adverse events that were largely gastrointestinal [65]. Conclusions on the safety of diflunisal in TTR-FAP will depend on further investigations on the impact of known cardiovascular and renal side-effects associated with the NSAID drug class [66,67].





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Recent progress in neurodegenerative diseases and gliomas

Curator: Larry H. Bernstein, MD, FCAP




Alzheimer’s Protein Not All Bad, Says MassGen Study

A controversial idea—that amyloid-beta (Aβ) protein fights bacterial infections in the brain—has gained additional support from a new study. Previously, the idea seemed worthy of investigation, if a bit of a stretch, on the basis of cell culture results. Now, thanks to the efforts of a scientific team lead by researchers based at Massachusetts General Hospital, it has been reinforced by observations of how the Aβ protein functions in animals’ brains.

Details of the new study appeared May 25 in the journal Science Translational Medicine, in an article entitled, “Amyloid-β Peptide Protects against Microbial Infection in Mouse and Worm Models of Alzheimer’s Disease.” The article suggests that the tendency of Aβ protein to form insoluble aggregates is not, as has been widely assumed, intrinsically abnormal, even though the aggregates are recognized as a hallmark of Alzheimer’s disease. Rather, Aβ protein appears to be a natural antibiotic that can trap and imprison bacterial pathogens that manage to pass the blood–brain barrier, which becomes increasingly “leaky” with age.

“We present in vivo data showing that Aβ expression protects against fungal and bacterial infections in mouse, nematode, and cell culture models of AD,” wrote the article’s authors. “We show that Aβ oligomerization, a behavior traditionally viewed as intrinsically pathological, may be necessary for the antimicrobial activities of the peptide.”

The MassGen scientists and their colleagues found that transgenic mice expressing human Aβ survived significantly longer after the induction of Salmonella infection in their brains than did mice with no genetic alteration. Mice lacking the amyloid precursor protein died even more rapidly. Transgenic Aβ expression also appeared to protect C. elegans roundworms from either Candida orSalmonella infection. Similarly, human Aβ expression protected cultured neuronal cells from Candida. In fact, human Aβ expressed by living cells appears to be 1000 times more potent against infection than does the synthetic Aβ used in previous studies.

That superiority appears to relate to properties of Aβ that have been considered part of Alzheimer’s disease pathology—the propensity of small molecules to form oligomers and then aggregate into Aβ plaques. This propensity, suggests the MassGen-led team, may indicate that Aβ acts like an antimicrobial peptide (AMP).

While AMPs fight infection through several mechanisms, a fundamental process involves forming oligomers that bind to microbial surfaces and then clump together into aggregates that both prevent the pathogens from attaching to host cells and allow the AMPs to kill microbes by disrupting their cellular membranes. The synthetic Aβ preparations used in earlier studies did not include oligomers. In the current study, however, oligomeric human Aβ not only showed an even stronger antimicrobial activity, its aggregation into the sorts of fibrils that form Aβ plaques was also seen to entrap microbes in both mouse and roundworm models.

“Our findings raise the intriguing possibility that β-amyloid may play a protective role in innate immunity and infectious or sterile inflammatory stimuli may drive amyloidosis,” the study’s authors concluded. “These data suggest a dual protective/damaging role for Aβ, as has been described for other antimicrobial peptides.”

One of the study’s co-corresponding authors, Rudolph Tanzi, Ph.D., director of the Genetics and Aging Research Unit in the MassGeneral Institute for Neurodegenerative Disease (MGH-MIND), pointed out that AMPs are known to play a role in the pathologies of a broad range of major and minor inflammatory disease. “For example, LL-37, which has been our model for Aβ’s antimicrobial activities, has been implicated in several late-life diseases, including rheumatoid arthritis, lupus, and atherosclerosis,” he elaborated. “The sort of dysregulation of AMP activity that can cause sustained inflammation in those conditions could contribute to the neurodegenerative actions of Aβ in Alzheimer’s disease.”

The study’s other co-corresponding author, Robert Moir, M.D., also of the MGH-MIND Genetics and Aging unit, noted that the study’s findings may lead to potential new therapeutic strategies. He also indicated that therapies designed to eliminate amyloid plaques from patient’s brains may have their limitations.

“It does appear likely that the inflammatory pathways of the innate immune system could be potential treatment targets, Dr. Moir explained. “If validated, our data also warrant the need for caution with therapies aimed at totally removing Aβ plaques. Amyloid-based therapies aimed at dialing down but not wiping out Aβ in the brain might be a better strategy.”

It remains to be determined, however, whether Aβ typically fights real infections or is apt to behave errantly, forming aggregates as though microbes are present, even if they are, in fact, not. “Our findings raise the intriguing possibility that Alzheimer’s pathology may arise when the brain perceives itself to be under attack from invading pathogens,” said Dr. Moir. “Further study will be required to determine whether or not a bona fide infection is involved.”Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer’s disease

Deepak Kumar, Vijaya Kumar, Se Hoon Choi, Kevin J. Washicosky, et al.
Science Translational Medicine  25 May 2016;  8 (340): 340ra72

Rehabilitation of a β-amyloid bad boy

A protein called Aβ is thought to cause neuronal death in Alzheimer’s disease (AD). Aβ forms insoluble aggregates in the brains of patients with AD, which are a hallmark of the disease. Aβ and its propensity for aggregation are widely viewed as intrinsically abnormal. However, in new work, Kumar et al. show that Aβ is a natural antibiotic that protects the brain from infection. Most surprisingly, Aβ aggregates trap and imprison bacterial pathogens. It remains unclear whether Aβ is fighting a real or falsely perceived infection in AD. However, in any case, these findings identify inflammatory pathways as potential new drug targets for treating AD.


The amyloid-β peptide (Aβ) is a key protein in Alzheimer’s disease (AD) pathology. We previously reported in vitro evidence suggesting that Aβ is an antimicrobial peptide. We present in vivo data showing that Aβ expression protects against fungal and bacterial infections in mouse, nematode, and cell culture models of AD. We show that Aβ oligomerization, a behavior traditionally viewed as intrinsically pathological, may be necessary for the antimicrobial activities of the peptide. Collectively, our data are consistent with a model in which soluble Aβ oligomers first bind to microbial cell wall carbohydrates via a heparin-binding domain. Developing protofibrils inhibited pathogen adhesion to host cells. Propagating β-amyloid fibrils mediate agglutination and eventual entrapment of unatttached microbes. Consistent with our model, Salmonella Typhimurium bacterial infection of the brains of transgenic 5XFAD mice resulted in rapid seeding and accelerated β-amyloid deposition, which closely colocalized with the invading bacteria. Our findings raise the intriguing possibility that β-amyloid may play a protective role in innate immunity and infectious or sterile inflammatory stimuli may drive amyloidosis. These data suggest a dual protective/damaging role for Aβ, as has been described for other antimicrobial peptides.


CRISPR Crossing New Barriers

Researchers Are Developing Ways to Edit Some of the Most Difficult-to-Edit DNA-Neuronal DNA



Confocal microscopic image of the hippocampus showing immunoreactivities for mEGFP (magenta) and the HA tag (green) fused to ß-Actin.

Ryohei Yasuda, Ph.D., scientific director, and his team at the Max Planck Florida Institute of Neuroscience (MPFI) are working to understand the way individual cells in our brains change as we learn and form memories. One of their main goals is to understand how different proteins behave and impact the structure and function of an individual cell, but, much like the field of genetics was once limited by the inability to visualize the structure of DNA, their research has been limited by their ability to locate and visualize the many different types of proteins within a single cell. Current imaging methods do not provide contrast and specificity high enough to see distinct proteins. Plus, the best methods are time-consuming and expensive; it can take a year or more to develop engineered models.

Over the past few years, the development of CRISPR technology has helped scientists overcome countless genetic engineering challenges, and allowed them to edit genes with unmatched precision and speed, massively increasing clarity and cutting the cost of research requiring genetic engineering. The technique has been used in myriad ways to increase understanding and treatment of diseases and disorders, but some cells are more difficult to edit than others. Brain cells have proven especially difficult to manipulate using CRISPR.

Recently, MPFI researchers Takayasu Mikuni, Ph.D., M.D., and Jun Nishiyama, Ph.D., M.D., and Dr. Yasuda were able to harness the power of the CRISPR/Cas9 system in order to create a quick, scalable, and high-resolution technique to edit neuronal DNA, which they called “SLENDR,” (single-cell labeling of endogenous proteins by CRISPR/Cas9-mediated homology-directed repair.) Using the technique, the researchers labeled several distinct proteins with fluorescence, and were able to observe protein localization in the brain that was previously invisible. That’s just the start of what researchers may be able to accomplish using this reliable, new technique for inserting genes into neurons.

CRISPR/Cas9 and Neurons

CRISPR is a tool built into bacterial DNA that the organisms use to fight infections. When a virus invades and attempts to insert its infectious DNA into that of a bacterial cell, a special section of the bacterial DNA, called CRISPR, cuts the viral DNA and renders it unable to wreak havoc on the bacteria. The organism then inserts a copy of the viral DNA into its own DNA to work as a type of adaptive immune system, to better recognize and defeat the invader in the future. As scientists have begun to understand how this system works, they have manipulated it to target and damage specific, functional genes in a variety of organisms, and in some cases, insert a new gene in its place.

Once the section of DNA is damaged, the technique relies on the cell to naturally repair its own DNA. There are two methods that the cell might use to accomplish this. One is homology-directed repair (HDR), the other is non-homologous end joining (NHEJ). HDR rebuilds or replaces the damaged locus of the genome, whereas NHEJ reattaches the damaged ends. When the reattachment occurs following the degradation of the ends, it often leads to the deletion of function of the gene (“knock-out” the gene). If a cell uses HDR to repair itself, scientists can include a desired gene in the CRISPR system that will be inserted into the DNA to replace the damaged gene.

Despite the impressive power of CRISPR system, its use in brain cells has been limited because by the time the brain has developed, its cells are no longer dividing. Most mature brain cells will repair themselves using NHEJ. The researcher can’t give the cell a gene to insert if it’s not going to insert one to begin with. While scientists can use CRISPR relatively easily to damage and knock out certain genes through NHEJ in the brain, the lack of cell division has made it very difficult for them to knock indesired sequences to genes, through HDR, with reliable precision. That’s where the SLENDR technique comes in.


SLENDR combines the power of the CRISPR/Cas9 system with the specificity and timing of in utero electroporation. Electroporation is a well-known technique used for introducing new material into cells and creating genetic knock-outs and knock-ins. Using in utero electroporation allows researchers to insert the CRISPR/CAS9 system into prenatal models, where brain cells are still developing and dividing. Thus, the broken DNA is still being repaired via HDR, giving researchers the opportunity to precisely modify a gene. This is a big deal. “I believe that SLENDR will be a standard tool for molecular and cellular neurobiology,” said Dr. Yasuda. “SLENDR provides a valuable means to determine subcellular localization of proteins, and will help researchers to determine the function of the proteins.”

In the recent study, the researchers at MPFI inserted a gene that made proteins of interest fluoresce under the microscope. They were even able to reliably label two different proteins with distinct colors at the same time in the same cell. The researchers were able to use the technique to visualize the proteins both in vivo and in vitro. And they were able to do it in a matter of days rather than years.

With existing knowledge of how brains develop, researchers can adjust the timing and position of the electroporation in utero to accurately target cells that will go on to populate particular cortical layers of the brain, even if they haven’t differentiated and moved to that layer yet.

The recent study used the technique primarily to tag certain proteins within brain cells and observe their behavior. But, with continued optimization, the method has the potential to elucidate immeasurable brain activities in both normal and diseased brains, and lead to a deeper understanding of brain function. “The most important part is that precise genome editing is possible in the brain. That’s what’s important,” said Dr.  Nishiyama, post-doctoral researcher who worked on the study. “That’s the biggest thing.” Neuroscientists would be remiss to ignore its worth and not explore its potential.

Emma Yasinski is a scientific writer at Max Planck Florida Institute for Neuroscience. Correspondence should be directed to Ryohei Yasuda, Ph.D. (ryohei.yasuda@mpfi.org), scientific director, Max Planck Florida Institute for Neuroscience.


Altered Metabolism of Four Compounds Drives Glioblastoma Growth

Findings suggest new ways to treat the malignancy, slow its progression and reveal its extent more precisely.


The altered metabolism of two essential amino acids helps drive the development of the most common and lethal form of brain cancer, according to a new study led by researchers at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James).

The study shows that in glioblastoma (GBM), the essential amino acids methionine and tryptophan are abnormally metabolized due to the loss of key enzymes in GBM cells.

The altered methionine metabolism leads to activation of oncogenes, while the changes in tryptophan metabolism shield GBM cells from detection by immune cells. Together, the changes promote tumor progress and cancer-cell survival.

“Our findings suggest that restricting dietary intake of methionine and tryptophan might help slow tumor progression and improve treatment outcomes,” says first author and OSUCCC – James researcher Kamalakannan Palanichamy, PhD, research assistant professor in Radiation Oncology.

“While we need to better understand how these abnormally regulated metabolites activate oncogenic proteins, our intriguing discovery suggests novel therapeutic targets for this disease,” says principal investigator and study leader Arnab Chakravarti, MD, chair and professor of Radiation Oncology and co-director of the Brain Tumor Program.

“For example, restoring the lost enzymes in the two metabolic pathways might slow tumor progression and reduce aggressiveness by inactivating oncogenic kinases and activating immune responses,” says Chakravarti, who holds the Max Morehouse Chair in Cancer Research.

Chakravarti further notes that because GBM cells take up methionine much faster than normal glioma cells, positron emission tomography that uses methionine as a tracer (MET-PET) might help map GBM tumors more accurately, allowing more precise surgical removal and radiation therapy planning. (MET-PET is currently an experimental imaging method.)

More than 11,880 new cases of GBM were estimated to occur in 2015, with overall survival averaging 12 to 15 months, so there is an urgent need for more effective therapies.

Amino acids are the building blocks of proteins. Tryptophan and methionine are essential amino acids – the diet must provide them because cells cannot make them. Normally, the lack of an essential amino acid in the diet can lead to serious diseases and even death. Foods rich in tryptophan and methionine include cheese, lamb, beef, pork, chicken, turkey, fish, eggs, nuts and soybeans.

Palanichamy, Chakravarti and their colleagues conducted this study using 13 primary GBM cell lines derived from patient tumors, four commercially available GBM cell lines and normal human astrocyte cells. Metabolite analyses were done using liquid chromatography coupled with mass spectrometry.



An investigation carried out at The Ohio State University Comprehensive Cancer Center (OH, USA) has uncovered abnormal metabolism of the essential amino acids methionine and tryptophan in glioblastoma.

The study suggests that this abnormal amino acid metabolism aids in the development of the disease. Furthermore, the findings, published recently in Clinical Cancer Research, hint at novel methods to potentially treat the malignancy, slow its progression and reveal its extent more precisely.

According to the study, it is the loss of key enzymes within glioblastoma cells that results in this abnormal metabolism. Modified methionine metabolism is described as promoting the activation of oncogenes, and the changes in tryptophan aid in masking the malignant cells from the immune system.

“While we need to better understand how these abnormally regulated metabolites activate oncogenic proteins, our intriguing discovery suggests novel therapeutic targets for this disease,” commented principal investigator and study leader Arnab Chakravarti (The Ohio State University Comprehensive Cancer Center).


Rapid eye movement sleep (dreaming) shown necessary for memory formation

Rapid eye movement sleep (dreaming) shown necessary for memory formation
A study published in the journal Science by researchers at the Douglas Mental Health University Institute at McGill University and the University of Bern provides the first evidence that rapid eye movement (REM) sleep — the phase where dreams appear — is directly involved in memory formation (at least in mice). “We already knew that … more…

May 16, 2016

Inhibition of  media septum GABA neurons during rapid eye movement (REM) sleep reduces theta rhythm (a characteristic of REM sleep). Schematic of the in vivo recording configuration: an optic fiber delivered orange laser light to the media septum part of the brain, allowing for optogenetic inhibition of media septum GABA neurons while recording the local field potential signal from electrodes implanted in hippocampus area CA1. (credit: Richard Boyce et al./Science)

A study published in the journal Science by researchers at the Douglas Mental Health University Institute at McGill University and the University of Bern provides the first evidence that rapid eye movement (REM) sleep — the phase where dreams appear — is directly involved in memory formation (at least in mice).

“We already knew that newly acquired information is stored into different types of memories, spatial or emotional, before being consolidated or integrated,” says Sylvain Williams, a researcher and professor of psychiatry at McGill*. “How the brain performs this process has remained unclear until now. We were able to prove for the first time that REM sleep (dreaming) is indeed critical for normal spatial memory formation in mice,” said Williams.

Dream quest

Hundreds of previous studies have tried unsuccessfully to isolate neural activity during REM sleep using traditional experimental methods. In this new study, the researchers instead used optogenetics, which enables scientists to precisely target a population of neurons and control its activity by light.

“We chose to target [GABA neurons in the media septum] that regulate the activity of the hippocampus, a structure that is critical for memory formation during wakefulness and is known as the ‘GPS system’ of the brain,” Williams says.

To test the long-term spatial memory of mice, the scientists trained the rodents to spot a new object placed in a controlled environment where two objects of similar shape and volume stand. Spontaneously, mice spend more time exploring a novel object than a familiar one, showing their use of learning and recall.

Shining orange laser light on media septum (MS) GABA neurons during REM sleep reduces frequency and power (purple section) of neuron signals in dorsal CA1 area of hippocampus (credit: Richard Boyce et al./Science)

When these mice were in REM sleep, however, the researchers used light pulses to turn off their memory-associated neurons to determine if it affects their memory consolidation. The next day, the same rodents did not succeed the spatial memory task learned on the previous day. Compared to the control group, their memory seemed erased, or at least impaired.

“Silencing the same neurons for similar durations outside of REM episodes had no effect on memory. This indicates that neuronal activity specifically during REM sleep is required for normal memory consolidation,” says the study’s lead author, Richard Boyce, a PhD student.

Implications for brain disease

REM sleep is understood to be a critical component of sleep in all mammals, including humans. Poor sleep quality is increasingly associated with the onset of various brain disorders such as Alzheimer’s and Parkinson’s disease.

In particular, REM sleep is often significantly perturbed in Alzheimer’s diseases (AD), and results from this study suggest that disruption of REM sleep may contribute directly to memory impairments observed in AD, the researchers say.

This work was partly funded by the Canadian Institutes of Health Research (CIHR), the Natural Science and Engineering Research Council of Canada (NSERC), a postdoctoral fellowship from Fonds de la recherche en Santé du Québec (FRSQ) and an Alexander Graham Bell Canada Graduate scholarship (NSERC).

* Williams’ team is also part of the CIUSSS de l’Ouest-de-l’Île-de-Montréal research network. Williams co-authored the study with Antoine Adamantidis, a researcher at the University of Bern’s Department of Clinical Research and at the Sleep Wake Epilepsy Center of the Bern University Hospital.

Abstract of Causal evidence for the role of REM sleep theta rhythm in contextual memory consolidation

Rapid eye movement sleep (REMS) has been linked with spatial and emotional memory consolidation. However, establishing direct causality between neural activity during REMS and memory consolidation has proven difficult because of the transient nature of REMS and significant caveats associated with REMS deprivation techniques. In mice, we optogenetically silenced medial septum γ-aminobutyric acid–releasing (MSGABA) neurons, allowing for temporally precise attenuation of the memory-associated theta rhythm during REMS without disturbing sleeping behavior. REMS-specific optogenetic silencing of MSGABA neurons selectively during a REMS critical window after learning erased subsequent novel object place recognition and impaired fear-conditioned contextual memory. Silencing MSGABA neurons for similar durations outside REMS episodes had no effect on memory. These results demonstrate that MSGABA neuronal activity specifically during REMS is required for normal memory consolidation.


Quantifying Consciousness

By Tanya Lewis

Overall brain metabolic rate can distinguish between pathological states of human consciousness, a study shows.


Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics

Abedini A, Plesner A, Cao P, Ridgway Z, et al.
eLife May 23, 2016; 10.7554/eLife.12977. http://dx.doi.org/10.7554/eLife.12977

Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.


NIH study visualizes proteins involved in cancer cell metabolism

Cryo-EM methods can determine structures of small proteins bound to potential drug candidates.


Scientists using a technology called cryo-EM (cryo-electron microscopy) have broken through a technological barrier in visualizing proteins with an approach that may have an impact on drug discovery and development. They were able to capture images of glutamate dehydrogenase, an enzyme found in cells, at a resolution of 1.8 angstroms, a level of detail at which the structure of the central parts of the enzyme could be visualized in atomic detail. The scientists from the National Cancer Institute (NCI), part of the National Institutes of Health, and their colleagues also reported achieving another major milestone, by showing that the shapes of cancer target proteins too small to be considered within the reach of current cryo-EM capabilities can now be determined at high resolution.

The research team was led by NCI’s Sriram Subramaniam, Ph.D., with contributions from scientists at the National Center for Advancing Translational Sciences (NCATS), also part of NIH. The findings appeared online May 26, 2016, in Cell.

“These advances demonstrate a real-life scenario in which drug developers now could potentially use cryo-EM to tweak drugs by actually observing the effects of varying drug structure — much like an explorer mapping the shoreline to find the best place to dock a boat — and alter its activity for a therapeutic effect,” said Doug Lowy, M.D., acting director, NCI.

Both discoveries have the potential to have an impact on drug discovery and development. Cryo-EM imaging enables analysis of structures of target proteins bound to drug candidates without first needing a step to coax the proteins to form ordered arrays. These arrays were needed for the traditional method of structure determination using X-ray crystallography, a powerful technique that has served researchers well for more than a half century. However, not all proteins can be crystallized easily, and those that do crystallize may not display the same shape that is present in their natural environment, either since the protein shape can be modified by crystallization additives or by the contacts that form between neighboring proteins within the crystal lattice.

“It is exciting to be able to use cryo-EM to visualize structures of complexes of potential drug candidates at such a high level of detail.”

Sriram Subramaniam, Ph.D.,National Caner Institute

“It is exciting to be able to use cryo-EM to visualize structures of complexes of potential drug candidates at such a high level of detail,” said Subramaniam. “The fact that we can obtain structures of small cancer target proteins bound to drug candidates without needing to form 3D crystals could revolutionize and accelerate the drug discovery process.”

Two of the small proteins the researchers imaged in this new study, isocitrate dehydrogenase (IDH1) and lactate dehydrogenase (LDH), are active targets for cancer drug development. Mutations in the genes that code for these proteins are common in several types of cancer. Thus, imaging the surfaces of these proteins in detail can help scientists identify molecules that will bind to them and aid in turning the protein activity off.

In publications in the journal Science last year and this year, Subramaniam and his team reported resolutions of 2.2 angstroms and 2.3 angstroms in cryo-EM with larger proteins, including a complex of a cancer target protein with a small molecule inhibitor. Of note, the journal Nature Methods deemed cryo-EM as the “Method of the Year” in January 2016. “Our earlier work showed what was technically possible,” Subramaniam said. “This latest advance is a delivery of that promise for small cancer target proteins.” For more information on cryo-EM, go to http://electron.nci.nih.gov.


Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Abedini A, Plesner A, Cao P, Ridgway Z, et al.
eLife May 23, 2016; 10.7554/eLife.12977. http://dx.doi.org/10.7554/eLife.12977

Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.


Single domain antibodies (sdAbs) aid in x-ray crystallography of mammalian serotonin 5-HT3 receptor

Serotonin 5-HT3 is part of the cys-loop receptor family, the mechanism of this family is not well understood due to difficulties in obtaining high resolution crystal structures. Serotonin 5-HT3 receptor is an important druggable target in alleviating nausea and vomiting induced by chemotherapy or anesthesia, as well as psychiatric disorders. It’s structure is critical in discovering new drugs to modulate its activity.

Previously, electron microscopy imaging of non-mammalian homologs of Cys-loop receptors provided basic understanding of extracellular ligand binding sites and pore forming domains. Little was known about intracellular domains and the way they interact with cellular scaffolding proteins, as they are absent in non-mammalian homologs. A recent publication in Nature extends our understanding behind the mechanism of serotonin 5-HT3 receptors, by resolving a 3.5A crystal structure.

Mouse 5-HT3 exists as a homopentamer and is difficult to express, purify and crystallize. To overcome this challenge, researchers split the receptor by proteolyzing each subunit into two fragments. In addition, an sdAb chaperone, which acts as an inhibitor locking the channel into a non-conducting conformation, was used to stabilized the pentameric structure, enabling resolution of a 3.5A crystal structure. Most importantly the split receptor displays an intracellular domain that is tightly coupled to the membrane domain, which provides important structural information that will lead to further understanding of the physiological conformation of 5-HT3 and Cys-loop receptors.

Hassaine G. et al. X-ray structure of the mouse serotonin 5-HT3 receptor Nature. Aug 2014. 512(7514):276-281


UCLA animal study shows how brain connects memories across time

Wednesday, May 25, 2016

Using a miniature microscope that opens a window into the brain, UCLA neuroscientists have identified in mice how the brain links different memories over time–and this may help develop new drugs in the future for memory-robbing diseases such as Alzheimer’s.


FDA approves new antibody drug for treating pediatric neuroblastoma

Pediatric neuroblastoma is a rare and difficult to treat cancer that forms from immature nerve cells. This form of cancer occurs in 1 in 100,000 children, with 650 new cases each year in the United States. Current therapies, which are non-specific, only provide 40-50% long term survival rate to patients suffering from high-risk neuroblastoma, making this form of cancer an area of high medical unmet need.

A new drug, called dinutuxumab was granted priority review and orphan drug designation by the FDA. It is the first drug of its kind to be approved that specifically treats pediatric neuroblastoma. In addition to the approval, the FDA also issued a rare pediatric review priority voucher to the makers of the drug, for future groundbreaking therapies in pediatric neuroblastoma.

Dinutuxumab (formerly called ch14.18) is a disialoganglioside (GD2) binding chimeric monoclonal antibody that works in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and 13-cis-retinoic acid (RA) for treating high-risk pediatric neuroblastoma.

Antibody therapeutics are highly efficacious and specific towards rare and difficult-to-treat cancers and discovery of new antibody therapeutics will help address critical needs. Antibody drug discovery may be challenging, but working with an experienced partner can help.

FDA approves first therapy for high-risk neuroblastoma


Electronic Biosensor Detects Molecules Linked to Cancer, Alzheimer’s, and Parkinson’s

5/20/2016  by Fundação de Amparo À Pesquisa Do Estado de São Paulo

A biosensor developed by researchers at the National Nanotechnology Laboratory (LNNano) in Campinas, São Paulo State, Brazil, has been proven capable of detecting molecules associated with neurodegenerative diseases and some types of cancer.

The device is basically a single-layer organic nanometer-scale transistor on a glass slide. It contains the reduced form of the peptide glutathione (GSH), which reacts in a specific way when it comes into contact with the enzyme glutathione S-transferase (GST), linked to Parkinson’s, Alzheimer’s and breast cancer, among other diseases. The GSH-GST reaction is detected by the transistor, which can be used for diagnostic purposes.

An inexpensive portable biosensor has been developed by researchers at Brazil’s National Nanotechnology Laboratory with FAPESP’s support. (Credit: LNNano)

The project focuses on the development of point-of-care devices by researchers in a range of knowledge areas, using functional materials to produce simple sensors and microfluidic systems for rapid diagnosis.

“Platforms like this one can be deployed to diagnose complex diseases quickly, safely and relatively cheaply, using nanometer-scale systems to identify molecules of interest in the material analyzed,” explained Carlos Cesar Bof Bufon, Head of LNNano’s Functional Devices & Systems Lab (DSF) and a member of the research team for the project, whose principal investigator is Lauro Kubota, a professor at the University of Campinas’s Chemistry Institute (IQ-UNICAMP).

In addition to portability and low cost, the advantages of the nanometric biosensor include its sensitivity in detecting molecules, according to Bufon.

“This is the first time organic transistor technology has been used in detecting the pair GSH-GST, which is important in diagnosing degenerative diseases, for example,” he explained. “The device can detect such molecules even when they’re present at very low levels in the examined material, thanks to its nanometric sensitivity.” A nanometer (nm) is one billionth of a meter (10-9 meter), or one millionth of a millimeter.

The system can be adapted to detect other substances, such as molecules linked to different diseases and elements present in contaminated material, among other applications. This requires replacing the molecules in the sensor with others that react with the chemicals targeted by the test, which are known as analytes.

The team is working on paper-based biosensors to lower the cost even further and to improve portability and facilitate fabrication as well as disposal.

The challenge is that paper is an insulator in its usual form. Bufon has developed a technique to make paper conductive and capable of transporting sensing data by impregnating cellulose fibers with polymers that have conductive properties.

The technique is based on in situ synthesis of conductive polymers. For the polymers not to remain trapped on the surface of the paper, they have to be synthesized inside and between the pores of the cellulose fibers. This is done by gas-phase chemical polymerization: a liquid oxidant is infiltrated into the paper, which is then exposed to monomers in the gas phase. A monomer is a molecule of low molecular weight capable of reacting with identical or different molecules of low molecular weight to form a polymer.

The monomers evaporate under the paper and penetrate the pores of the fibers at the submicrometer scale. Inside the pores, they blend with the oxidant and begin the polymerization process right there, impregnating the entire material.

The polymerized paper acquires the conductive properties of the polymers. This conductivity can be adjusted by manipulating the element embedded in the cellulose fibers, depending on the application for which the paper is designed. Thus, the device can be electrically conductive, allowing current to flow without significant losses, or semiconductive, interacting with specific molecules and functioning as a physical, chemical or electrochemical sensor.


Protein Oxidation in Aging: Not All Proteins Are Created Equal

Cancer, Alzheimer’s disease and other age-related diseases develop over the course of aging, and certain proteins are shown to play critical roles this process. Those proteins are subject to destabilization as a result of oxidation, which further leads to features of aging cells. It is estimated that almost 50% of proteins are damaged due to oxidation for people at their 80s. The oxidative damage mediated by free radicals occurs when converting food to energy in the presence of oxygen. Cellular structures, such as proteins, DNA, and lipids, are prone to these oxidation damages, which further contribute to the development of age-related diseases.

Using computational models with physics principles incorporated, de Graff el al. from Stony Brook University unfolded the molecular mechanism that how natural chemical process affects the aging of proteins. First, the authors revealed the major factor to explain stability loss in aging cells and organisms is likely to be random modification of the protein sidechains. Furthermore, through the evaluation and analysis on the protein electrostatics, the authors suggested that highly charged proteins are in particular subject to the oxidation induced destabilization. Even one single oxidation could lead to unfold the whole structure for these highly charged proteins. Old cells are enriched in those highly charged proteins, thus the destabilization effects are elevated in the aging cells. In addition, 20 proteins associated with aging are further identified to be at high risk of oxidation. The list includes telomerase proteins and histones, both of which play critical roles in the aging of cells and cancer development. The team is currently working on analyzing more proteins, with the hope to provide key information to aid targeted treatments against age-related diseases.

Further Reading: Emerging Opportunity for Treating Alzheimer Disease by Immunotherapy

Adam M.R. de Graff, Michael J. Hazoglou, Ken A. Dill. Highly Charged Proteins: The Achilles’ Heel of Aging Proteomes.Structure, 24, 285-292 (2016)

Baruch, K. et al. PD-1 Immune Checkpoint Blockade Reduces Pathology and Improves Memory in Mouse Models of Alzheimer’s Disease. Nat. Med. 22, 135-137 (2016)


Single domain antibodies shown to cross blood brain barrier and offers enhanced delivery of therapeutics to CNS targets

A major challenge in developing both small molecule and antibody therapeutics for CNS disorders including brain cancer and neurodegenerative diseases, is penetrating the blood brain barrier (BBB). A study published in FASEB demonstrated that monomeric variable heavy-chain domain of camel homodimeric antibodies (mVHH), can cross the BBB in-vivo, and recognize its intracellular target: glial fibrillary acidic protein (GFAP). The ability of mVHH to cross the BBB of normal animals and those undergoing pathological stress makes it a promising modality for treating CNS diseases as well as for brain imaging.

The investigators of this study expressed a recombinant fusion protein, VHH-GFP, which was able to cross the BBB in-vivo and specifically label astrocytes. GenScript is fully engaged in single-domain antibody lead generation and optimization. With our one-stop services, we are determined to be your best partner in antibody drug discovery from gene synthesis to in-vivo characterization of candidate antibodies. All you need to provide is the Genbank accession number of the antigen protein!

Li T. et al. Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging. FASEB. Oct 2012. 26:3969-79


New insight behind the success of fighting cancer by targeting immune checkpoint proteins

Immune checkpoint blockade has proven to be highly successful in the clinic at treating aggressive and difficult-to-treat forms of cancer. The mechanism of the blockade, targeting CTLA-4 and PD-1 receptors which act as on/off switches in T cell-mediated tumor rejection, is well understood. However, little is known about the tumor antigen recognition profile of these affected T-cells, once the checkpoint blockade is initiated.

In a recent published study, the authors used genomics and bioinformatics approaches to identify critical epitopes on 3-methylcholanthrene induced sarcoma cell lines, d42m1-T3 and F244. CD8+ T cells in anti-PD-1 treated tumor bearing mice were isolated and fluorescently labeled with tetramers loaded with predicted mutant epitopes. Out of 66 predicted mutants, mLama4 and mAlg8 were among the highest in tetramer-positive infiltrating T-cells. To determine whether targeting these epitopes alone would yield similar results as anti-PD-1 treatment, vaccines against these two epitopes were developed and tested in mice. Prophylactic administration of the combined vaccine against mLama4 and mAlg8 yielded an 88% survival in tumor bearing mice, thus demonstrating that these two epitopes are the major antigenic targets from checkpoint-blockade and therapies against these two targets are similarly efficacious.

In addition to understanding the mechanism, identification of these tumor-specific mutant antigens is the first step in discovering the next wave of cancer immunotherapies via vaccines or antibody therapeutics. Choosing the right antibody platform can speed the discovery of a new therapeutics against these new targets. Single domain antibodies have the advantage of expedited optimization, flexibility of incorporating multiple specificity and functions, superior stability, and low COG over standard antibody approaches.

Gubin MM. et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature. Nov 2014. 515:577-584


Anti-PD-1 is poised to be a blockbuster, which other immune-checkpoint targeting drugs are on the horizon?

Clinical studies of anti-immune-checkpoint protein therapeutics have shown not only an improved overall survival, but also a long-term durable response, compared to chemotherapy and genomically-targeted therapy. To expand the success of immune-checkpoint therapeutics into more tumor types and improving efficacy in difficult-to-treat tumors, additional targets involved in checkpoint-blockade need to be explored, as well as testing the synergy between combining approaches.

Currently, CTLA-4 and PD-1/PD-L1 are furthest along in development, and have shown very promising results in metastatic melanoma patients. This is just a fraction of targets involved in the checkpoint-blockade pathway. Several notable targets include:

  • LAG-3 – Furthest along in clinical development with both a fusion protein and antibody approach, antibody apporach being tested in combination with anti-PD-1
  • TIM-3 – Also in clinical development. Pre-clinical studies indicate that it co-expresses with PD-1 on tumor-infiltrating lymphocytes. Combination with anti-PD-improves anti-tumor response
  • VISTA – Antibody targeting VISTA was shown to improve anti-tumor immune response in mice

In addition, there are also co-stimulatory factors that are also being explored as viable therapeutic targets

  • OX40 – Both OX40 and 4-1BB are part of the TNF-receptor superfamily. Phase I data shows acceptable safety profile, and evidence of anti-tumor response in some patients
  • 4-1BB – Phase I/II data on an antibody therapeutic targeting OX40 shows promising clinical response for melanoma, renal cell carcinoma and ovarian cancer.
  • Inducible co-stimulator (ICOS) – Member of the CD28/B7 family. Its expression was found to increase upon T-cell activation. Anti-CTLA-4 therapy increases ICOS-positive effector T-cells, indicating that it may work in synergy with anti-CTLA-4. Clinical trials of anti-ICOS antibody are planned for 2015.

Sharma P and Allison JP. Immune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative Potential. Cell. April 2015;161:205-214


CTLA-4 found in dendritic cells suggests New cancer treatment possibilities

Both dendritic cells and T cells are important in triggering the immune response, whereas antigen presenting dendritic cells act as the “general” leading T cells “soldiers” to chase and eliminate enemies in the battle against cancer. The well-known immune checkpoint break, CTLA-4, is believed to be present only in T cells (and cells of the same lineage). However, a new study published in Stem Cells and Development suggests that CTLA-4 also presents in dendritic cells. It further explores the mechanism on how turning off the dendritic cells in the immune response against tumors.

Matthew Halpert, et al. Dendritic Cell Secreted CTLA-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7. Stem Cells and Development, 2016; DOI: 10.1089/scd.2016.0009


With a wide range of animal models to choose from, what are the crucial factors to consider?

A recent perspective published in Nature Medicine addresses these gaps by comparing the strengths and limitations of different tumor models, as well as best models to use for answering different biological questions and best practices for preclinical modeling.

Below is a summary of the authors’ key considerations:

  • It is important to choose a model based on the biology of the target. Several diverse tumor models may be required to address complex biology
  • If the biology of the target includes signaling between the tumor and the stroma, then it is crucial to understand drug efficacy in the presence of an appropriate tumor microenvironment with orthotopic models
  • Avoid overuse of models that are highly sensitive to the drug, unless there is clinically relevant biomarker data to support the findings
  • For studying agents that reduce pre-existing tumors, make sure that the tumors are established in the model prior to treatment
  • Understanding the pharmacokinetics of a drug in the model prior to studies is important to ensure that the dosing is within range, and that off-target and toxic side effects are not skewing anti-tumor activity.

Gould SE, Junttila MR and de Sauvage FJ. Translational value of mouse models in oncology drug development. Nat Med. May 2015. 21(5):431-439

Revolutionary Impact of Nanodrug Delivery on Neuroscience

Reza Khanbabaie1,2,3 and Mohsen Jahanshahi
Curr Neuropharmacol. 2012 Dec; 10(4): 370–392.   doi:  10.2174/157015912804143513

Brain research is the most expanding interdisciplinary research that is using the state of the art techniques to overcome limitations in order to conduct more accurate and effective experiments. Drug delivery to the target site in the central nervous system (CNS) is one of the most difficult steps in neuroscience researches and therapies. Taking advantage of the nanoscale structure of neural cells (both neurons and glia); nanodrug delivery (second generation of biotechnological products) has a potential revolutionary impact into the basic understanding, visualization and therapeutic applications of neuroscience. Current review article firstly provides an overview of preparation and characterization, purification and separation, loading and delivering of nanodrugs. Different types of nanoparticle bioproducts and a number of methods for their fabrication and delivery systems including (carbon) nanotubes are explained. In the second part, neuroscience and nervous system drugs are deeply investigated. Different mechanisms in which nanoparticles enhance the uptake and clearance of molecules form cerebrospinal fluid (CSF) are discussed. The focus is on nanodrugs that are being used or have potential to improve neural researches, diagnosis and therapy of neurodegenerative disorders.

Keywords: Nanodrug, Nanofabrication and purification, Neuroscience, Nervous system, Nano-nervous drugs.


The delivery of drugs to the nervous system is mainly limited by the presence of two anatomical and biochemical dynamic barriers: the blood–brain barrier (BBB) and blood–cerebrospinal fluid barrier (BCSFB) separating the blood from the cerebral parenchyma [1]. These barriers tightly seal the central nervous system (CNS) from the changeable milieu of blood. With the advancement of electron microscopy it is found that the ultrastructural localization of the blood–brain barrier is correlated with the capillary endothelial cells within the brain [2]. The BBB inhibits the free paracellular diffusion of water-soluble molecules by an elaborate network of complex tight junctions (TJs) that interconnects the endothelial cells. Similar to the endothelial barrier, the morphological correlate of the BCSFB is found at the level of unique apical tight junctions between the choroid plexus epithelial cells inhibiting paracellular diffusion of water-soluble molecules across this barrier [1, 3]. Beside its barrier function, it allows the directed transport of ions and nutrients into the cerebrospinal fluid (CSF) and removal of toxic agents out of the CSF using numerous transport systems.

One of the most challenging steps in neuroscience researches and therapy is the availability of techniques to penetrate these permeability barriers and delivering drugs to the CNS. Several strategies have been used to circumvent the barriers inhibiting CNS penetration. These strategies generally fall into one or more of the following three categories: manipulating drugs, disrupting the BBB (BBBD) and finding alternative routes for drug delivery. Drug manipulation methods include: Lipophilic Analogs, prodrugs, chemical drug delivery systems (CDDS), Carrier-mediated transport (CMT) and Receptor-mediated drug delivery. The drug manipulating strategy has been frequently employed, but the results have often been disappointing [46]. All of these methods have major limitations: they are invasive procedures, have toxic side effects and low efficiency, and are not sufficiently safe [7]. Two methods for disrupting the BBB have been reported: osmotic blood-brain barrier disruption and biochemical blood-brain barrier disruption. However, these procedures also break down the self-defense mechanism of the brain and make it vulnerable to damage or infection from all circulating chemicals or toxins. Since the above techniques aim to enhance the penetration of drugs to the CNS via circulatory system, they will increase the penetration of drugs throughout the entire body. This will frequently result in unwanted systemic side effects. In the other hand, systemically administered agents must penetrate the BBB to enter the CNS, which is a difficult task. Despite advances in rational CNS drug design and BBBD, many potentially efficacious drug molecules still cannot penetrate into the brain parenchyma at therapeutic concentrations. The alternative strategy to enhance CNS penetration of drug molecules is based on methodology that does not rely on the cardiovascular system. These strategies circumvent the BBB altogether and do not need drug manipulation to enhance BBB permeability and/or BBBD. Using alternative routes to deliver drugs to the CNS, e.g. intraventricular/intrathecal route and olfactory pathway, is one of these strategies.

One strategy for bypassing the BBB that has been studied extensively both in laboratory and in clinical trials is the intralumbar injection or intreventricular infusion of drugs directly into the CSF. Compared to vascular drug delivery, intra-CSF drug administration theoretically has several advantages. Intra-CSF administration bypasses the BCB and results in immediate high CSF drug concentrations. Due to the fact that the drug is somewhat contained within the CNS, a smaller dose can be used, potentially minimizing systemic toxicity. Furthermore, drugs in the CSF encounter minimize protein binding and decrease enzymatic activity relative to drugs in plasma, leading to longer drug half-life in the CSF. Finally, since the CSF freely exchanges molecules with the extracellular fluid of the brain parenchyma, delivering drugs into the CSF could theoretically result in therapeutic CNS drug concentrations [7, 8]. However, for several reasons this delivery was not as successful as predicted. These include a slow rate of drug distribution within the CSF and increase in intracranial pressure associated with fluid injection or infusion into small ventricular volumes.

Another CNS drug delivery route is the intranasal route. In this method drugs are transported intranasally along olfactory sensory neurons to yield significant concentrations in the CSF and olfactory bulb. An obvious advantage of this method is that it is noninvasive relative to other strategies. This method has received relatively little attention, since there are difficulties that have to be overcome. Among these obstacles is an enzymatically active, low pH nasal epithelium, the possibility of mucosal irritation or the possibility of large variability caused by nasal pathology, such as common cold.

Based on the advantages and disadvantages of aforementioned strategies, researchers are still looking for novel and better methods of CNS drug deliveries. The most direct way of circumventing the BBB is to deliver drugs directly to the brain interstitium which mainly includes the use of small colloidal particles like liposomes and nanoparticles [8]. By directing agents uniquely to an intracranial target, interstitial drug delivery can theoretically yield high CNS drug concentrations with minimal systemic exposure and toxicity. Furthermore, with this strategy, intracranial drug concentrations can be sustained, which is crucial in treatment with many chemotherapeutic agents. The basic reason of common acceptance of these carriers is due to their controlled profile or drug release nature as well as due to their selected targeting mechanism. Targeting action maybe due to the steric hindrance created by nano-vectors for achieving targeting ability. These carriers are usually administered through parenteral route and due to their steric phenomenon they conceal themselves from opsonisation event induced by tissue macrophages. By this way they achieve targeting ability to brain and other reticuloendothelial system (RES) organs like liver, spleen, etc.

Several approaches have been developed for delivering drugs directly to the brain interstitium like injections, catheters, and pumps. One such methodology is the Ommaya reservoir or implantable pump which achieves truly continuous drug delivery. Though interstitial drug delivery to the CNS has had only modest clinical impact, its therapeutic potential may soon be realized using new advances in polymer technologies to modify the aforementioned techniques. Polymeric or lipidbased devices that can deliver drug molecules at defined rates for specific periods of time are now making a tremendous impact in clinical medicine.

Among the strategies of direct drug delivery to the CNS, nanoparticles have attracted considerable interest from the last few decades. It has been shown that nano delivery systems have great potential to facilitate the movement of drugs across barriers (e.g., BBB). Nanosystems employed for the development of nano drug delivery systems in the treatment of CNS disorders include polymeric nanoparticles, nanospheres, nanosuspensions, nanoemulsions, nanogels, nano-micelles and nano-liposomes, carbon nanotubes, nanofibers and nanorobots, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and lipid drug conjugates (LDC). Although the exact mechanism of barrier opening by nanoparticles is not known, the novel properties such as tiny size, tailored surface, better solubility, and multi-functionality of nanoparticles present the capability to interact with composite cellular functions in new ways. In fact, nanotechnology has now emerged as an area of research for invention of newer approaches for the CNS drug delivery and a revolutionary method to improve diagnosis and therapy of neurodegenerative disorders.

In this line, an overview of preparation and characterization, purification and separation, loading and delivering of nanodrugs is the first subject of this review. Different types of nanoparticle bioproducts including carbon nanotubes as a drug delivery system and also as a novel tool in neuroscience research are explored. For instance, nanodrug delivery systems like human serum albumin (HSA) nanoparticles, bovine serum albumin (BSA)-Gum Arabic (Acacia) nanoparticles and α-lactalbumin nanoparticles are explained.

The impact of nanotechnology on neuroscience and drug delivery to the central nervous system (CNS) is the subject of the second part of this review. Different mechanisms in which nanoparticles enhance the uptake of molecules both hydrophilic and hydrophobic across the BBB and the impact of various physiochemical parameters of nanoparticles on its uptake and clearance form CSF are discussed. Also nanodrugs that are being used or have potential to improve neural researches, diagnosis and therapy of neurodegenerative disorders are investigated.


Nanotechnology started by the suggestion of a famous physicist, Richard Feynman, that it should be possible, in principle, to make nanoscale machines that “arrange the atoms the way we want”, and do chemical synthesis by mechanical manipulation [9, 10]. Nanotechnologies exploit materials and devices with a functional organization that has been engineered at the nanometer scale. In a broad sense, they can be defined as the science and engineering involved in the design, syntheses, characterization, and application of materials and devices whose smallest functional organization in at least one dimension is on the nanometer scale, ranging from a few to several hundred nanometers. A nanometer is roughly the size of a molecule itself (e.g., a DNA molecule is about 2.5 nm long while a sodium atom is about 0.2 nm) [10]. Nanotechnology is not in itself a single emerging scientific discipline but rather a meeting of traditional sciences such as chemistry, physics, materials science, and biology to bring together the required collective expertise needed to develop these novel technologies.

The application of nanotechnology in cell biology and physiology enables targeted interactions at a fundamental molecular level. Nanotechnology, in the context of nanomedicine, can be defined as the technologies for making nanocarriers of therapeutics and imaging agents, nanoelectronic biosensors, nanodevices, and microdevices with nanostructures. It also covers possible future applications of molecular nanotechnology (MNT) and nanovaccinology. Unlike the definition in core nanotechnology field, which restricts the “nano” to at least 1–100 nm in one dimension, nanocarriers in the biomedical field are often referred to as particles with a dimension a few nanometers to 1000 nm [8, 11]. Although, the initial properties of nanomaterials studied were for its physical, mechanical, electrical, magnetic, chemical and biological applications, recently, attention has been geared towards its pharmaceutical application, especially in the area of drug delivery [8]. There are a few challenges in use of large size materials in drug deliveries. Some of these challenges are poor bioavailability, in vivo stability, solubility, intestinal absorption, sustained and targeted delivery to site of action, therapeutic effectiveness, generalized side effects, and plasma fluctuations of drugs (see Table 11).

The most important innovations are taking place in nanopharmocology and drug delivery which involves developing nanoscale particles or molecules to improve bioavailability. These pharmacological applications of nanotechnology include: the formation of novel nanoscopic entities [11, 27], exploring and matching specific compounds to particular patients for maximum effectiveness; and advanced pharmaceutical delivery systems and discovery of new pharmacological molecular entities; selection of pharmaceuticals for specific individuals to maximize effectiveness and minimize side effects, and delivery of pharmaceuticals to targeted locations or tissues within the body. Examples of nanomaterials include nanotubes and nanofibers, liposomes, nanoparticles, polymeric micelles, block ionomer complexes, nanogels, and dendrimers.

Nanotubes [28, 29] and nanofibers mimic tubular structures that appear in nature, such as rod shaped bacteria or viruses, microtubules, ion channels, as well as axons and dendrites. They are low-dimensional nanostructures, having a very large axial ratio. Properties of a molecule in a nanotube or nanofiber structure can be different from those in the bulk or in other nanomaterials, such as spherical nanoparticles. These materials have a large surface–volume ratio, which results in a high exposure of the material components to the surrounding environment [30]. This makes nanotubes and nanofibers promising structures for biosensing and molecular recognition [31]. However, it provides a way to control drug release through the nanotubes wall, while the large hollow area inside nanotubes provides an excellent storage for drugs and other agents [32]. Furthermore, nanotubes can be synthesized to be open-ended, which can be exploited for certain biological applications.

Carbon nanotubes (CNTs) was discovered by Iijima [33] which are composed of carbon atoms arranged in hexagonal ring structures similar to graphite, with some five-membered or seven-membered rings providing the structure curvature [29, 34,35]. CNTs are compatible with biological tissues for scaffolding purposes and the charge carried by the nanotubes can be manipulated to control neurite outgrowth [36, 37]. It has also been suggested that CNTs functionalized with growth factors, such as nerve growth factor or brain-derived neurotrophic factor, can stimulate growth of neurons on the nanotube scaffold [3840]. In such application the toxicity of CNTs remains an issue that must be overcome [41, 42]. It has been reported that conductive polymer coatings for living neural cells has been generated using poly (3,4-ethylenedioxythiophene) PEDOT nanotubes [43]. The electric conductivity of PEDOT was used to enhance the electrical activity of the tissue with a long range aim of treating CNS disorders, which show sensory and motor impairments. These observations suggested that nanotube and nanofiber scaffolds have potential for neuroregeneration as well as treatment of CNS trauma [27, 44]. Nanomaterials suggest a promising strategy for neuroprotection [45]. Neuroprotection is an effect that may result in salvage, recovery, or regeneration of the nervous system.

The role of nanotechnology in targeted drug delivery and imaging was discussed in many reviews and papers [46, 47]. As a step towards a realistic system, a brief overview of preparation, characterization, delivery, loading, purification and separation of nanoparticles and nanodrugs are presented herein. In next two sections the fabrication methods of nanoparticle bioproducts and also the delivery systems of nanodrugs are explained. Subsequently we go back to the CNS nanodrugs for research and therapy and the delivery systems of nanodrugs for nervous system.



The major goals in designing nanoparticles as a delivery system are to control particle size, surface properties [85] and release of pharmacologically active agents in order to achieve the site-specific action of the drug at the therapeutically optimal rate and dose regimen [86]. If nanoparticles are considered to be used as drug delivery vehicles, it depends on many factors including: (a) size of nanoparticles required; (b) inherent properties of the drug, e.g., aqueous solubility; (c) surface characteristics such as charge and permeability; (d) degree of biodegradability, biocompatibility and toxicity; (e) drug release profile desired; and (f) antigenicity of the final product. The advantages of using nanoparticles as a drug delivery system might be summarized as follow [87]:

  1. Particle size and surface characteristics of nanoparticles can be easily manipulated to achieve both passive and active drug targeting after parenteral administration.
  2. They control and sustain release of the drug during the transportation and at the site of localization, altering organ distribution of the drug and subsequent clearance of the drug so as to achieve increase in drug therapeutic efficacy and reduction in side effects.
  3. Controlled release and particle degradation characteristics can be readily modulated by the choice of matrix constituents. Drug loading is relatively high and drugs can be incorporated into the systems without any chemical reaction; this is an important factor for preserving the drug activity.
  4. Site-specific targeting can be achieved by attaching targeting ligands to surface of particles or use of magnetic guidance.
  5. The system can be used for various routes of administration including oral, nasal, parenteral, intraocular etc.


The major goals in designing nanoparticles as a delivery system are to control particle size, surface properties [85] and release of pharmacologically active agents in order to achieve the site-specific action of the drug at the therapeutically optimal rate and dose regimen [86]. If nanoparticles are considered to be used as drug delivery vehicles, it depends on many factors including: (a) size of nanoparticles required; (b) inherent properties of the drug, e.g., aqueous solubility; (c) surface characteristics such as charge and permeability; (d) degree of biodegradability, biocompatibility and toxicity; (e) drug release profile desired; and (f) antigenicity of the final product. The advantages of using nanoparticles as a drug delivery system might be summarized as follow [87]:

  1. Particle size and surface characteristics of nanoparticles can be easily manipulated to achieve both passive and active drug targeting after parenteral administration.
  2. They control and sustain release of the drug during the transportation and at the site of localization, altering organ distribution of the drug and subsequent clearance of the drug so as to achieve increase in drug therapeutic efficacy and reduction in side effects.
  3. Controlled release and particle degradation characteristics can be readily modulated by the choice of matrix constituents. Drug loading is relatively high and drugs can be incorporated into the systems without any chemical reaction; this is an important factor for preserving the drug activity.
  4. Site-specific targeting can be achieved by attaching targeting ligands to surface of particles or use of magnetic guidance.
  5. The system can be used for various routes of administration including oral, nasal, parenteral, intraocular etc.


Nanomaterials and nanoparticles can interact with biological systems at fundamental and molecular levels [100, 101]. In neuroscience, the application of nanotechnologies entails specific interactions with neurons and glial cells. Nanodevices can target the cells and glia with a high degree of specificity. This unique molecular specificity enables the nanodrugs to stimulate and interact with tissues and neurons in controlled ways, while minimizing undesirable effects. There are two main types of nervous system drugs (neurodrugs): behavioural and molecular. Behavioural neurodrugs are for the study of how different drugs affect human behaviour and human brain. These drugs are usually used for diagnosis and therapy of neurodegeneration disorders [47, 102]. Molecular neurodrugs are used for the study of neurons and their neurochemical interactions. Since for the most part, neurons in the human brain communicate with one another by releasing chemical messengers called neurotransmitters, these drugs have to target specific transmitters and receptors to have beneficial effect on neurological functions. The preparation of these two types of drugs is closely connected. Researchers are studying the interactions of different neurotransmitters [103], neurohormones [104], neuromodulators [105], enzymes [106], second messengers [107], co-transporters [108, 109], ion channels [110], and receptor proteins [111] in the central and peripheral nervous systems to develop drugs to treat many different neurological disorders, including pain [112], neurodegenerative diseases such as Parkinson’s disease [113] and Alzheimer’s disease [114], psychological disorders [115], addiction [116], and many others.

The blood–brain barrier significantly hinders the passage of systemically delivered therapeutics and the brain extracellular matrix limits the distribution and longevity of locally delivered agents. Nanoparticles represent a promising solution to these problems. They can cross blood-brain barrier and enter the CNS. Although the applications of nanotechnology in basic and clinical neuroscience are only in the early stages of development, partly because of the complexities associated with interacting with neural cells and the mammalian nervous system, however the early results show an impressive potential of nanotechnologies to contribute to neuroscience research [117]. One area in which nanotechnology may have a significant clinical impact in neuroscience is the selective transport and delivery of drugs and other small molecules across the blood brain barrier that cannot cross otherwise.

Examples of current research include technologies that are designed to better interact with neural cells, advanced molecular imaging technologies [118, 119], materials and hybrid molecules used in neural regeneration [120], neuroprotection [121], and targeted delivery of drugs and small molecules across the blood–brain barrier [122, 123]. Among all these modern methods of drug delivery to the central nervous system (CNS), the design and application of bionanotechnologies aimed at the CNS provide revolutionary new approaches for studying cell and molecular biology and physiology. The successful and meaningful development of bionanotechnologies designed to interact with the CNS as research or clinical tools require an understanding of the relevant neurophysiology and neuropathology, an appreciation of the inherent ‘nanoscale’ structure of the CNS, and an understanding of the relevant chemistry and materials science and engineering. At nanoscale, consideration of individual molecules and interacting groups of molecules in relation to the bulk macroscopic properties of the material or device becomes important, since it is control over the fundamental molecular structure that allows control over the macroscopic chemical and physical properties [124]. Applications to neuroscience and physiology imply materials and devices designed to interact with the body at subcellular (i.e., molecular) scales with a high degree of specificity. This can potentially translate into targeted cellular and tissue-specific clinical applications designed to achieve maximal therapeutic affects with minimal side effects.

It started with controlled release strategy and the development of miniaturized delivery systems [125] and continued by the application of albumin nanoparticles for the first time in the Johns Hopkins Medical Institution in Baltimore [126]. Other nanoconstructs such as drug-polymer conjugates were first proposed in the 1970s [127] and developed pre-clinically in the 1980s [128]. Prof. Kreuter [129] proposed a definition of polymeric nanoparticles for pharmaceutical purposes for the first time that later was adopted by the Encyclopaedia of Pharmaceutical Technology [130] and the Encyclopedia of nanotechnology [131]. Today, more than 25 nanomedicines have already been approved for human use [102]. Usually the application of nanodrugs to neuroscience is divided into two parts: application in basic neuroscience [124], and in clinical neuroscience [27].

The development of nanotechnology products may play an important role in adding a new group of therapeutics to the products of pharmaceutical companies [132]. Nanotechnology enhances (1) delivery of poorly water-soluble drugs; (2) delivery of large macromolecule drugs to intracellular sites of action; (3) targeted delivery of drugs in a cell- or tissue-specific manner; (4) transcytosis of drugs across tight epithelial and endothelial barriers; (5) co-delivery of two or more drugs or therapeutic modality for combination therapy; (6) visualization of sites of drug delivery by combining therapeutic agents with imaging modalities; and (7) real-time read on the in vivo efficacy of a therapeutic agent [133]. Additionally, the manufacturing complexity of nanotechnology therapeutics may also create a significant hurdle for generic drug companies to develop equivalent therapeutics readily [132].


Safe, site-specific, and efficient delivery of compounds to CNS disease sites remains a singular goal in achieving optimal therapeutic outcomes to combat neurodegenerative diseases. Treatment of CNS disorders by systemic administration or local delivery of drugs is currently inefficient in many cases. Furthermore, clinical neuroscience faces great challenges due to the extremely heterogeneous cellular and molecular environment and the complexities of the brain’s anatomical and functional “wiring” and associated information processing [224]. However, the emergence of nanotechnology provides hope that it will revolutionize diagnosis and treatment of CNS disorders. Neurodegenerative diseases are usually linked to a loss of brain and spinal cord cells. For example, the neuronal damage in AD and PD is associated with abnormal protein processing and accumulation and results in gradual cognitive and motor deterioration [225].





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Announcement from LPBI Group: key code LPBI16 for Exclusive Discount to attend Boston’s Discovery on Target (September 19-22, 2016, CRISPR: Mechanisms to Applications on 9/19/2016)


Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

As a proud partner of this event, Leaders in Pharmaceutical Business Intelligence Group has secured a special discounted price for you to attend, resulting in a $200 discount on a commercial registration and $100 discount on an academic registration!

*This offer is valid for new registrants only, does not apply to previously registered attendees or short courses, and cannot be combined with any other offer. You must mention key code LPBI16 to receive this discount.

Don’t miss your opportunity to network with 1,100+ of your peers at this year’s event. Special early registration savings are currently available through Friday, August 12.

Preliminary AGENDA and Registration Link


For sponsorship & exhibit information, please contact: Jon Stroup, Sr Business Development Manager,
(+1) 781-972-5483, jstroup@healthtech.com


See us in CHI’s Media Partners section online:


Contact: 617-244-4024, avivalev-ari@alum.berkeley.edu




Leaders in Pharmaceutical Business Intelligence (LPBI) Group, Boston


will cover in REAL TIME

Cambridge Healthtech Institute’s

Discovery on Target

September 19-22, 2016,

CRISPR: Mechanisms to Applications 

September 19, 2016

Westin Boston Waterfront, Boston, MA

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN




Stephen J Williams, PhD

Senior Editor




Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.


As a proud partner of this event, Leaders in Pharmaceutical Business Intelligence Group has secured a special discounted price for you to attend, resulting in a $200 discount on a commercial registration and $100 discount on an academic registration!

*This offer is valid for new registrants only, does not apply to previously registered attendees or short courses, and cannot be combined with any other offer. You must mention key code LPBI16 to receive this discount.

Don’t miss your opportunity to network with 1,100+ of your peers at this year’s event. Special early registration savings are currently available through Friday, June 3.


Preliminary AGENDA and Registration Link


For sponsorship & exhibit information, please contact: Jon Stroup, Sr Business Development Manager,
(+1) 781-972-5483, jstroup@healthtech.com


See us in CHI’s Media Partners section online:


Contact: 617-244-4024, avivalev-ari@alum.berkeley.edu



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Molecular basis for schizophrenia

Larry H. Bernstein, mD, FCAP, Curator




The proteome of schizophrenia

Juliana M Nascimento  & Daniel Martins-de-Souza
npj Schizophrenia 1, Article number: 14003 (2015)     http://dx.doi.org:/10.1038/npjschz.2014.3

On observing schizophrenia from a clinical point of view up to its molecular basis, one may conclude that this is likely to be one of the most complex human disorders to be characterized in all aspects. Such complexity is the reflex of an intricate combination of genetic and environmental components that influence brain functions since pre-natal neurodevelopment, passing by brain maturation, up to the onset of disease and disease establishment. The perfect function of tissues, organs, systems, and finally the organism depends heavily on the proper functioning of cells. Several lines of evidence, including genetics, genomics, transcriptomics, neuropathology, and pharmacology, have supported the idea that dysfunctional cells are causative to schizophrenia. Together with the above-mentioned techniques, proteomics have been contributing to understanding the biochemical basis of schizophrenia at the cellular and tissue level through the identification of differentially expressed proteins and consequently their biochemical pathways, mostly in the brain tissue but also in other cells. In addition, mass spectrometry-based proteomics have identified and precisely quantified proteins that may serve as biomarker candidates to prognosis, diagnosis, and medication monitoring in peripheral tissue. Here, we review all data produced by proteomic investigation in the last 5 years using tissue and/or cells from schizophrenic patients, focusing on postmortem brain tissue and peripheral blood serum and plasma. This information has provided integrated pictures of the biochemical systems involved in the pathobiology, and has suggested potential biomarkers, and warrant potential targets to alternative treatment therapies to schizophrenia.

Schizophrenia is a complex neuropsychiatric disorder that produces severe symptoms and significant lifelong disability, causing massive personal and societal burden.1,2 About 1% of the world’s population is affected by schizophrenia.3 Despite the strong genetic component, showing increasing risks for those related to schizophrenic patients,4 and the known role of environment as a trigger, schizophrenia signs and symptoms have unknown etiology. Currently, the disease diagnosis is essentially clinically defined by observed signs of psychosis, which often include paranoid delusions and auditory hallucinations,5 with onset during late adolescence and/or early adulthood.

Pharmacological treatments are available for schizophrenia; yet, most of the currently used antipsychotic medications were discovered in the 1950s, or are a variation of those medications, and since then no new major drug class has been introduced to the clinic. In addition, efficacy of medication is poor, and only about 40% of schizophrenic patients respond effectively to initial treatment with antipsychotics.6,7 Unfortunately, comprehensive studies on molecular mechanisms of schizophrenia have been scant; hence, current treatments are only partly beneficial to a subset of symptoms. The response to drugs is heterogenous, mainly because of individual variations of the disease, in addition to scarce knowledge on its pathophysiology, impairing both diagnosis and adequate treatment selection.8,9

Heterogenic and multifactorial aspects of schizophrenia have always hindered biochemical characterization studies and delayed the establishment of preclinical models of the disease.10 Several studies, including postmortem, imaging, pharmacological, and genetic studies, reported common traces of the disease, such as synaptic deficits, abnormal neural network, and changes in neurotransmission, involving dopamine, glutamate, and gamma-aminobutyric acid.2,11,12,13 Additional abnormalities, such as aberrant inflammatory responses, oligodendrocyte alterations, epigenetic changes, mitochondrial dysfunction, and reactive oxygen species (ROS) imbalance, are often described in schizophrenia.14,15,16

A complex cross talk between genetic and environmental factors during neurogenesis is responsible for promoting differences of gene and protein expression in schizophrenia, causing abnormal processes during neurodevelopment.2 Recent studies found reinforcement of genes associated with the major hypotheses of glutamatergic neurotransmission, such as DRD2 (dopamine receptor D2)—the main target of antipsychotic drugs17—among other potential targets, involving perturbation of specific neurotransmitter systems or pathways, which are yet to be studied. The complexity of schizophrenia reinforces the need to unravel molecular mechanisms, as those insights have been shown to be essential in identifying and validating drug targets and biomarkers.9 Therefore, unraveling models with relevance to the cause and onset of schizophrenia is essential toward improving treatments and outcomes for those with the disorder.

Here we review the advances of proteomics on schizophrenia research, toward a better understanding of disease mechanisms and response to treatment, and the efforts toward the discovery of biomarkers for diagnosis and disease evolution.

The role of proteomics in schizophrenia research

In the past century, psychiatric research was dedicated to understanding the nature of several disorders, including action of psychotherapeutics. It was also shown that schizophrenia is a highly heritable disease, indicating a strong genetic influence and an estimated heritability of 80–85%,18,19 more likely with a polygenic basis.20 Since the beginning of the twenty-first century, revolution of genomic technologies has allowed a deeper understanding of the genetic basis of diseases, and several genetic findings on psychiatric disorders have been reported,21 unraveling candidate genes linked to risk factors of psychiatric disorders, such as DISC1 (disrupted in schizophrenia 1),22 involved in neuronal development and synapse formation.23,24 In fact, the International Schizophrenia Consortium (ISC) found indication for a polygenic contribution to schizophrenia.25 While candidate gene studies are beneficial, in cases with a not yet well-understood biology, such as schizophrenia, a single gene only adds a small phenotype effect to the multifactorial etiology of the disease.20,26,27

Since 2008, genomic technology innovations have led to a better understanding of psychiatric disorders, providing information about numerous genes that have a role in brain development.21 Recent advances of next-generation sequencing have facilitated a higher coverage and sample throughput of schizophrenia studies.28,29,30Furthermore, international collaborations, which increased the number of participant subjects and samples, have combined efforts to provide deeper insight from comprehensive biological data sets, such as the Psychiatric GWAS (genome-wide association studies) Consortium (PGC; http://pgc.unc.edu).31 Most recently, two main studies, reporting comprehensive GWAS analysis, were able to identify 13 (ref. 27) and 108 schizophrenia-associated risk loci,17 the latter being the largest GWAS study on schizophrenia to date, with up to 36,989 cases and 113,075 controls. Unbiased GWAS,17,27,32,33 indicating genetic regions (loci) that contribute to disease susceptibility, and structural variation studies, such as copy number variants,30,34 are the main identification sources of gene variants with small effects on disease phenotype.35 For instance, copy number variants, including deletions and duplications of several DNA segments, confer significant risk increase in alleles of schizophrenia genome up to 10–25-fold.9,34,36Several of those findings support the leading etiological hypothesis of the disorder, and point to functionally related targets, such as DRD2, miRNA-137, N-methyl-D-aspartate receptor (NMDAR) complex, or calcium channel subunits.17,30,36,37 Information on genetic variations as a base will increase knowledge on mechanisms of schizophrenia and other psychiatric disorders.

Deciphering the human genome was a revolution in genetics, and the anticipated next step was to decode RNA complexity to understand how information was delivered, and its variety between individuals. Development of large-scale transcriptome analyses, such as cDNA microarrays, Serial Analysis of Gene Expression, and the analyses of Expressed Sequence Tag, and more recently the advance of whole transcriptome shotgun sequencing (or RNA-Seq), providing the presence and quantification of RNA at a given time in a genome, allowed a deeper insight into the dynamics of an organism. Transcriptome analyses revealed RNA implication in psychiatric diseases,38,39 including abnormalities resulting from alternative splicing, in addition to messenger RNA transcripts, such as total RNA and small RNA, including micro-RNA.40 Those abnormalities were observed in several biological processes, such as synaptic and mitochondrial/energetic function,41,42,43 cytoskeleton,44 immune and inflammation response,45,46,47 and the myelination pathway.48 Although not yet fully understood, the more the pieces of the puzzle discovered, the more comprehensive the pathology network becomes.

Genomic and transcriptomic studies generated significant data, although these changes cannot yet be translated into biomarkers. The main limitation of genetic approaches in schizophrenia is extrapolation to functional protein expression, as proteins undergo several modifications from transcription to posttranslation, and transcript abundance cannot really predict protein levels either in normal conditions or in response to stress, such as diseases.49,50 Therefore, proteomic techniques are being increasingly used in screening for identification of biomarkers in schizophrenia,51,52 providing several insights into the pathophysiology of the disease. Proteomics can show global expression of proteins or protein groups, and is more complex than genomics as it can change from each cell type at any given time or state.49 Also a high-throughput method, proteomic studies detect fewer expressed proteins than a transcriptomic detects expressed genes, but protein expression provides a precise functional profile and presents an unbiased current physiological state as a reflex of the complex interaction of gene versus environment. The importance of those interactions has been increasing in the research of schizophrenia and other neurological diseases.10,41,53

Regarding research into schizophrenia, numerous studies have investigated the proteome of postmortem brain tissue, including several brain regions such as the dorsolateral prefrontal cortex,41,54,55 frontal cortex,56thalamus,57 anterior cingulate cortex,58,59,60hippocampus,61,62 corpus callosum,63 and insular cortex.64 Postmortem brain tissue has yielded many valuable insights into the pathophysiology of schizophrenia, but less information on disease onset and development. Thus, other tissues and cells have been tested, providing data from naive patients as well, such as from cerebrospinal fluid (CSF),65,66,67,68blood serum and plasma,69,70,71,72,73 liver,68,74 and fibroblasts,75 which can be biopsied from living patients, among others,76,77aiming to reveal more about potential biomarkers of discovery and monitoring of the disease.

Proteomic methodologies used in schizophrenia research

A proteome comprises the entire set of proteins in a biological system (cell, tissue, or organism) in a particular state, at a given time.78 The need to understand all proteins derived from almost 20,000 genes identified by the Human Genome Project turns molecular biology studies toward proteomics. Because of the progress of mass spectrometry techniques, more fine and high-throughput methods are available, supporting the identification of hundreds (or thousands) of proteins in a single biological sample. In 2014, two major consortiums have delivered a draft of the human proteome,79,80 with a large-scale data set covering 84–92% of the protein-coded genes annotated for the human genome. The more information annotated on protein knowledge databases, the more unknown causes of diseases and biomarker identification can be performed.

In the first decade of proteomics, the main quantitative methods used were gel-based, such as two-dimensional gel electrophoresis (2DE), including the fluorescent two-dimensional differential gel electrophoresis (2D-DIGE). Despite its recognized usefulness,81 gel-based techniques have been consistently replaced by gel-free techniques with the introduction of the concept of shotgun proteomics, which employs basically liquid chromatography followed by mass spectrometry (LC/MS).82 The large scale was possible only because of the development of proteomics based on mass spectrometry, which offers insights into protein abundance, expression profiles according to cell type, posttranslational modifications, and protein–protein interactions, and the possibility to study modifications at the protein level.83

2DE was first described in 1975,84 and after intense enhancements in the 1980s85,86 it became widely used in the separation of complex protein mixtures according to their isoelectric point, in the first dimension by isoelectric focusing, and according to their molecular weight (MW), in the second dimension, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This separation leads to a protein profile comprising several spots, each of which, in theory, represents a single protein, providing information about intact proteins and isoforms. Protein visualization techniques include common post-run methods, such as Coomassie blue or silver staining, and also pre-labeling of samples with fluorescent dies, such as in the 2D-DIGE.87 Image analysis of the latter provides a more sensitive quantification method, as up to 10-fold lower amount of samples can be applied. In addition, 2D-DIGE allows co-running of different samples in the same gel, labeled with distinct fluorescent dies (i.e., Cy3, Cy2, Cy5), and might also include an internal control for cross-gel comparison purposes. Those techniques have significantly improved in the previous years with respect to reproducibility and robustness, allowing better comparison between samples and across different laboratories.

Furthermore, mass spectrometry (MS) revolutionized proteomic studies when combined with the 2DE/2D-DIGE workflow, improving sensitivity for identification of differentially expressed proteins, by measuring molecular mass-to-charge ratio of ions (m/z).88 Protein spots, excised from the gel, are digested (i.e., trypsin) and masses of these peptides measured on MS instruments, providing a peptide mass fingerprint of each protein, which is then compared with an in silico-digested database. Further fragmentation of each peptide, performed on an MS/MS instrument, provides the sequence of that peptide, assisting in protein identification. Disadvantages of 2DE/MS combination include the incompatibility to very low or very high molecular weight or isoelectric point, in addition to those proteins with low abundance, which will not be spotted.89 Nevertheless, several proteome studies in schizophrenia were performed using proteomic screening approaches such as 2DE/2D-DIGE, providing large-scale data on the pathophysiology of the disease.41,55,56,58,61,66,90

Hence, schizophrenia and other psychiatric disorder studies have intensively used shotgun proteomics for the analysis of peptides and proteins for profiling, and for quantification of protein modification analysis.54,59,72,75,77,91 For shotgun proteomics, proteins are first digested into peptides (i.e., using trypsin, as previously), which are next separated by high-performance liquid chromatography online-connected to a hybrid MS, providing a gel-free proteomic system (LC-MS/MS). Shotgun proteomics lead to the possibility of identifying more proteins, increasing sampling of low abundant and extreme-sized proteins.82 Most proteomic studies on schizophrenia have used label-free methods for quantification,59,75,92 which assume chromatographic peak areas correlated to the concentration of peptides,93and is one of the simplest ways to compare proteomics, allowing comparison among several samples at once.94Nevertheless, both in vitro and in vivo stable isotopic labeling methods are available in shotgun proteomics, for quantification accuracy of protein concentrations simultaneously in several biological samples. In vitroapproaches include isobaric tags for relative and absolute quantification95 and isotope-coded protein labeling, whereas in vivo metabolic methods, such as stable isotope labeling by amino acids in cell culture96 and stable isotope labeling in mammals, have been used in proteomic quantification.97 Some have been applied to neuropsychiatric disorders, in postmortem brains and CSF,54,57,98 and in animal and cell studies99,100 on proteomic research.

The power to identify and quantify proteins and protein sets at high resolution, among multiple samples, is essential to understand large case studies in biomedical research. Recent advances have been made in MS-based techniques, such as selected reaction monitoring/multiple reaction monitoring, which has just emerged as a promising technology for a more precise MS-based quantification of targeted protein,101,102 and was awarded Nature’s Method of the Year in 2012 on biological research methods.103 Selected reaction monitoring is specific, accurate, and sensitive, as it selects proteotypic peptides—those that uniquely identify the targeted protein—for its analysis, which might overcome several current validation issues, such as semiquantitative western blotting techniques, availability, and specificity.104 Furthermore, this ability to quantify specific proteins across several samples is particularly interesting with regard to biomarkers, as clinical validation of biomarker signatures for a given disease must be tested over a large sample set to achieve satisfactory statistical power. Indeed, proteomic studies in psychiatric disorders slowly start to validate pathways and biological functions that were found differentially expressed by selected reaction monitoring.105,106,107,108

Likewise, other proteomic methodologies have been extensively applied to schizophrenia research in order to discover and validate biomarkers, such as multiplex immunoassays,69 which use multiplexed dye-coded microspheres of selected protein sets, thus providing profile studies of cytokines, growth factors, or metabolic pathways, from blood serum or CSF samples.70,109,110,111Aiming to reach the broader spectrum of protein visualization, concerns regarding the possibility to obtain sub-proteomes (using fractionation methods)112 by depleting high-abundant proteins or enriching a group of proteins in a sample should be part of the design and technique choice. Protein separation and quantification using SELDI-TOF-MS ProteinChip analysis or metal ion affinity chromatography to select proteins from a mixture have been used in schizophrenia research lately.65,68,72 Regardless of the protein analysis method, study design and sample preparation choice are crucial steps in proteomic studies. Platforms using reduced number of analytes, but a broader number of clinical samples, provide a precise statistical interpretation.

Indeed, statistics and bioinformatics are of extreme importance for proteomic studies, as different types of assays (2DE, shotgun-MS, or multiplex immunoassays) are required to precisely quantify changes in expression of hundreds (or thousands) of proteins. Therefore, those fields are improving, together with the development of new tools and methods for proteomic analysis, offering better algorithms and image analysis tools, in order to provide a more robust analysis from the growing number of data generated.


What do proteomics tell us about schizophrenia?

Proteomic technologies, mostly focusing on mass spectrometric analysis, are a valuable tool in psychiatric research. A simple search on PubMed using the terms ‘proteomics or proteome and schizophrenia’ provides a total of 218 articles since the first article on proteomics of schizophrenia in the beginning of the 2000s.56 Out of them, 124 articles (and growing) were published within the last 5 years (2010–2014) on human and animal studies, including some reviews, showing considerable increase in awareness of the importance of proteomics in the study of schizophrenia. We have focused, for the purpose of the review, on proteomic studies on human samples of schizophrenia patients compared with controls, from the last 5 years.

These studies, which are summarized in Table 1, have been using proteomic screening approaches such as shotgun-MS (10/23), 2DE/DIGE (7/23), and multiplex immunoassays (10/23), alone or combined. Although postmortem brains are the main studied tissue in schizophrenia research,57,58,59,61,113 influences of chronic medication or sample heterogeneity and age have impaired some interpretation of the molecular differences found in postmortem brain tissue of schizophrenia patients compared with control subjects.114Thus, current studies have been mostly focusing on more accessible peripheral tissues, with a preference for blood serum and plasma,72,73,109,115 and CSF,65,66 although there are studies on skin fibroblasts75 and saliva as well.76Those have become the main tissues used in proteomic studies of schizophrenia because of the possibility of multiple sampling, thus providing better characterization of disease onset, development, and response to treatment. This broader characterization could lead to a more complete understanding of the disease and to development of diagnostic/prognostic biomarkers. Indeed, an analysis of proteins that are common to brain, CSF, and blood samples from at least two studies presented in Table 1, using Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Qiagen, Redwood, CA, USA; www.ingenuity.comFigure 1), shows biomarker candidates of psychiatric disorders and their interactions, and is further discussed.

Table 1: Human proteomic studies from the last 5 years of different tissues and cells in schizophrenic patients

Figure 1 Protein network of regulated proteins in schizophrenia brain, CSF, and blood samples, analyzed by ingenuity pathways knowledge database. ALDOC, aldolase C; CSF, cerebrospinal fluid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


Figure 1


Neuronal transmission and synaptic function

Differentially expressed proteins in schizophrenia proteomic studies have been found to be involved in neuronal transmission, synaptic plasticity, and neurites outgrowth, including several cytoskeletal constituents. Most significant proteomic changes included downregulation of neuroreceptors such as NMDA receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), in addition to glutamatergic signaling molecules, such as neurofilaments (NEFL and NEFM), glutamate-ammonia ligase (GLUL), and guanine nucleotide-binding proteins (G proteins) (GNB1), or dihydropyrimidinase-related protein 2 (DPYSL2), which are involved in synaptic function, axon guidance, and signal transduction impairment in schizophrenia.59,113NEFL, in addition to its role in neuronal morphogenesis, is directly associated with NMDA receptors. NMDAR hypofunction was associated with neurotransmitter dysfunction in NR1 transgenic mice,105 with variations in bioactive peptides and proteins. As GLUL is responsible for removing glutamate from neuronal synapses, it is most likely involved in glutamate imbalance in schizophrenia.2

Other proteins related to NMDA functionality and synaptic plasticity, such as MAPK3, SYNPO, CYFIP2, VDAC, CAMK2B, PRDX1, and ESYT, were also observed differentially expressed in postsynaptic density-enriched samples of postmortem brain tissues.59 Data corresponding to a genomic study of schizophrenia34found an excess of copy number variants in schizophrenia, confirming several of the proteins differentially regulated with functions in the postsynaptic membrane.

Calcium homeostasis and signaling

Calcium signaling has also been found to be differentially regulated in schizophrenia proteomic studies.54,76,113,116Calcium is a pivotal metabolite for the dopaminergic hypothesis in schizophrenia, mainly because it has a central role in the function of dopamine receptors D1 and D2.117 Proteins such as calmodulin (CALM1, CALM2), calcium/calmodulin-dependent protein kinase II (CAMK2B, CAMK2D, CAMK2G), voltage-dependent anion channels (VDAC1, VDAC2), and the plasma membrane calcium-transporting ATPase 4 (PMCA-4) are some of the calcium-related proteins found downregulated in the brains of schizophrenia patients.59,116 Some proteins were found differentially expressed in secretion fluids of schizophrenic patients—for example, calmodulin-like proteins and the S100 family of calcium-binding proteins (S100A6, S100A12)—such as in eccrine sweat118 and saliva.76 Complementing these findings, S100B was found downregulated in the nuclear proteome of schizophrenia corpus callosum.119 In addition, calcium activated differential expression of calmodulin-dependent protein kinase II (CAMK2), and calcineurin A in phencyclidine-treated rats.113  

Energy metabolism

The brain has a high glucose uptake to supply its major metabolic activity rate. Thus, one of the most consistent dysfunctions underlying the pathophysiology of schizophrenia is in energy metabolism pathways, along with mitochondrial dysfunction and oxidative stress.120,121Glucose metabolism is confirmed by hyperglycemia, impaired glucose tolerance, and/or insulin resistance in first-onset, antipsychotic, naive schizophrenic patients.110,122 Numerous proteomic studies have identified the glycolysis–gluconeogenesis pathway as being consistently disrupted both in brain and CSF,41,57,58,60,123 and is followed by peripheral tissues.77,90,113,120 The expression of proteins associated with the energy metabolism pathway, such as aldolase C (ALDOC), enolase 1 (ENO1), neuronal enolase 2 (ENO2), lactate dehydrogenase B (LDHB), phosphoglycerate mutase 1 (brain) (PGAM1), phosphoglycerate kinase 1 (PGK1), pyruvate kinase isozyme R/L [PKLR], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are often significantly deregulated in schizophrenic patients compared with controls.55,77,120 The most consistent differentially expressed enzyme is aldolase C (ALDOC), which was found altered in several brain samples58,61,66 and also as a marker on blood serum samples.77 Likewise, pyruvate, the final product of glycolysis, and NADPH have been quantified in lower amounts in schizophrenic samples compared with controls, in the thalamus,57 and is replicated in phencyclidine-treated rats, a model of schizophrenia research.113 Whereas schizophrenia seems to be more associated with glycolysis, major depressive disorders are likely to be more associated with oxidative phosphorylation.124

DISC1, a major risk factor of the schizophrenia-susceptibility gene candidate,22 can affect mitochondrial morphology and axonal trafficking.125,126 Alterations of mitochondria morphology were reinforced by the imbalance of the oxidative phosphorilation system, including proteins such as NADH dehydrogenases (i.e., NDUFA1, NDUFV2, NDUFS3, NDUFB5), and ATPases (ATP5B, ATP6V1B2, ATP6V1A1), which have been previously shown altered in animal models of schizoprenia,113,127,128,129 but also had significant regulation in human brains.57,59 Other molecules such as dopamine have been shown to inhibit electron transport chain complex I (NADH dehydrogenase).130     

Oxidative stress

This overall imbalance of mitochondrial energy metabolism, associated with elevated calcium, leads to hazardous ROS concentrations and oxidative stress events in brain cells.131 The resultant ROS may cause oxidative damage in cellular DNA, RNA, proteins, and membrane lipids. Proteomics of the brain have shown several enzymes involved in redox activities (responsible for removing ROS and protecting cells against oxidative injury) to be differentially expressed in schizophrenia brain tissues. Proteins such as superoxide dismutase (which catalyze the dismutation of superoxide (O2) into oxygen and hydrogen peroxide), peroxiredoxins (PRDX1, PRDX2, and PRDX3) (which are responsible for reducing hydrogen peroxide), glutathione S-transferases (i.e., GSTM3, GSTTLp28, and GSTP1) (which are a family of multifunctional enzymes involved in cellular detoxification, glutathione reduction, and neutralization of ROS), and NADPH-dependent oxidoreductases such as carbonyl reductases (CBR1 and CBR2) and quinoid dihydropteridine reductase (QDPR) (which might be involved in the NADP/NADPH imbalance observed in the thalamus) were often found regulated in brain tissue,41,57,58,59,61,113 but could also be detected to be differentially regulated in peripheral tissues such as blood and fibroblast samples .70,73,75,132 Proteomics and combined metabolomics support evidence that slight imbalance in energy glucose metabolism, disrupting mitochondria and the oxidative phosphorylation system, results in compromised ATP production and oxidative stress, which is central in the pathophysiology of schizophrenia.41,113,133  


Cytoskeleton constituents are proteins that have shown broad differential expressions in schizophrenia—namely, microtubules such as tubulins (TBA1B, TUBB2A), microfilaments such as actins (ACTG1, ACTB) and actin-binding proteins such as tropomyosins (TPM1, TPM2, TPM3, TPM4), and intermediate filaments (i.e., GFAP, vimetin) and endocytosis proteins, such as dynamin (DNM1), a protein involved in clathrin-mediated endocytosis and other vesicular trafficking processes.55,58,59,113,134,135 Such modifications impact the cellular structure, axonal function, and neurite outgrowth, influencing synaptic plasticity and metabolism, all significantly influencing disturbed cytoskeleton arrangement in schizophrenia.44 Protein components of the cytoskeleton, such as the above-mentioned neurofilaments M and L (NEFL, NEFM) and DPYSL2, a regulator of cytoskeleton remodeling, have a role in axon guidance, neuronal growth, and cell migration. Glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes, was found to be strongly regulated in brain tissues, both up- and downexpressed, indicating a precise protein expression across the brain.55,59,61,135,136 In addition, actin was often found downregulated in brain tissues,41,61,62,75 but was upregulated in fibroblasts75 or liver74 of schizophrenic patients.

Immune system and inflammation

Several abnormalities were found in schizophrenia proteomics, including changes in immune- and inflammation-related pathways in first-onset schizophrenic patients compared with controls. Molecules such as α-defensins (DEFA1, DEFA2, DEFA3, DEFA4), migration inhibitory factor, and several interleukins (IL-1ra, IL-8, IL-10, IL-15, IL-16, IL-17, and IL-18), including growth factors such as brain-derived neurotrophic factor, have been differentially regulated in blood samples from schizophrenic patients compared with controls.70,73,76,109,115,132 In addition, extracellular calcium-binding S100A12 exhibits cytokine-like characteristics, recruiting inflammatory cells to the sites of tissue damage. Indeed, anti-inflammatory treatment with cyclooxygenase-2 (COX-2) inhibitors has shown diminished schizophrenic behavior by blocking the synthesis of proinflammatory prostaglandins.137 Multiplex immunoassay profiling studies of blood serum have found numerous components of inflammation signaling pathways.109,111,115 Levels of anti-inflammatory cytokines IL-1ra and IL-10 were decreased after treatment with atypical antipsychotics, which correlated with symptom improvement.109 In addition, profiling studies using a subset of cytokines found increased levels of interleukins (i.e., IL-1β) in the cerebrospinal fluid of first-episode schizophrenic patients, indicative of immune system activation in the brain of some patients.138 Therefore, a proper subset of those altered molecular inflammatory molecules could be included in a sensitive and specific biomarker panel, both for diagnosis and treatment follow-up response.

An overview

Diverse proteomic techniques provided non-biased screening analyses of postmortem brain tissue from schizophrenic patients, and insights into pathways affected in the disease.10,57 In addition, more accessible tissues, such as cerebrospinal fluid, blood serum and plasma, and others such as fibroblasts, liver, and urine,57,66,70,74,75,139 have complemented those findings, suggesting several proteins that could be used as biomarkers to improve diagnosis. We have not gone through the details of the role of oligodendrocytes in schizophrenia, as these were recently tackled somewhere else,140,141,142 although these are as important as all that are listed here.

Proteomic insights from naive first-onset patients’ impaired protein pathways confirm patterns of disease onset, which along with genetic predisposition could be used as biomarkers for stratification of patients, improving the diagnosis and treatment classification. Also, this valuable information can lead to a more individualized medication, selected according to specific molecular dysfunctions and phenotype observed in schizophrenic individuals. Understanding the pathways affected by medication might also lead to reliable analytical platforms to evaluate individual response to treatment in a personalized-medicine mode. Moreover, the ability to monitor levels of molecules in noninvasive body fluids, such as saliva, urine, or blood serum or plasma, is a great advance. In addition, knowledge of gene–protein pathway networks affected and impaired by the disease can give clues for the development of new and more efficient targeted drugs to those relevant pathways.51,121     


Psychiatric disorders are one of the biggest burdens to society,3 and consequently one of the most challenging fields of medical research, with complex and multifactorial characteristics, along with genetic, neurodevelopmental, environmental, and molecular components. Hence, proteomics can add valuable insights into revealing psychiatric disorder connections, as it is closely linked to phenotype, and, by definition, proteomics constitute one of the most suitable approaches for this purpose.143

In 2010, the Human Proteome Organization (HUPO) started a project aiming to map the entire human proteome, the Human Proteome Project (HPP) initiative, with joint initiatives such as the Chromosome-centric Human Proteome Project.144,145,146 Thus, at the beginning of 2014, two extensive drafts of this map were released,79,80 showing progress in the identification of proteins from high-quality proteomic data to complement genomic annotation. The Human Brain Proteome Project (HBPP) initiative, specifically addressing the proteomic landscape of the human brain, aims to study individuals affected by neurodegenerative diseases, understanding its many different cell types and their particular structure at the cellular and tissue level.147,148 Another main focus is to untangle the human plasma proteome149 on health and disease, to support biomarker validation and development of new tools for diagnosis, disease progression, and medication efficiency, considering the confounding factors present in those body fluids.

From the schizophrenia research point of view, this are exciting news, because of the potential of information that can be extracted, as, regardless of efforts in the search for biomarkers, by investigating the transcriptome and proteome in the post-genomic era, schizophrenia is one more psychiatric disorder without a reliable marker. Those recent advances in ‘omics’ technologies, such as genomics, transcriptomics, proteomics, and metabolomics, which are not only expanding coverage and resolution but also becoming cheaper and more accessible, present new prospects for a global comprehension of biological characteristics of disease mechanisms.150

While genomic and transcriptomic technologies have achieved single-nucleotide resolution, the protein coverage of the amino-acid sequence is still restricted. State-of-the-art shotgun mass spectrometry has improved immensely, such as targeted proteomic measurements, and is useful for biomarker identification. Although the detection of some protein variants, such as differential splice products and posttranslational modifications, remains a challenge for proteomics to get a more comprehensive picture of the whole proteome using a systematic approach. This high-throughput investigation of nucleic acids, proteins, and metabolites from particular tissues and cells provides essential data, which is basic to system biology studies, in order to create integral models of cellular processes.151 Therefore, integrating biological data from omics studies to the expertise of complementary disciplines such as mathematics, physics, and computational sciences, toward better conceptual analysis and predictive models, provides new tools for understanding biological systems at different levels. Hence, we can analyze the cellular space-time and hierarchical organization,152 aiming for complete understanding of psychiatric diseases and identifying candidate biomarkers, especially before and after the onset of clinical manifestations, as well as target metabolic pathways impaired and/or affected by antipsychotics, in order to distinguish subgroups of patients who respond to medication on the basis of their molecular profiles.51

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Heterochronic microRNAs in temporal specification of neural stem cells: application toward rejuvenation

Takuya Shimazaki  & Hideyuki Okano

npj Aging and Mechanisms of Disease 2, Article number: 15014 (2016)   http://dx.doi.org:/10.1038/npjamd.2015.14

Plasticity is a critical factor enabling stem cells to contribute to the development and regeneration of tissues. In the mammalian central nervous system (CNS), neural stem cells (NSCs) that are defined by their capability for self-renewal and differentiation into neurons and glia, are present in the ventricular neuroaxis throughout life. However, the differentiation potential of NSCs changes in a spatiotemporally regulated manner and these cells progressively lose plasticity during development. One of the major alterations in this process is the switch from neurogenesis to gliogenesis. NSCs initiate neurogenesis immediately after neural tube closure and then turn to gliogenesis from midgestation, which requires an irreversible competence transition that enforces a progressive reduction of neuropotency. A growing body of evidence indicates that the neurogenesis-to-gliogenesis transition is governed by multiple layers of regulatory networks consisting of multiple factors, including epigenetic regulators, transcription factors, and non-coding RNA (ncRNA). In this review, we focus on critical roles of microRNAs (miRNAs), a class of small ncRNA that regulate gene expression at the post-transcriptional level, in the regulation of the switch from neurogenesis to gliogenesis in NSCs in the developing CNS. Unraveling the regulatory interactions of miRNAs and target genes will provide insights into the regulation of plasticity of NSCs, and the development of new strategies for the regeneration of damaged CNS.

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