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Lesson 7 of Cell Signaling 7 Motility: Tubulin and Tutorial Quizes for #TUBiol3373

Stephen J. Williams, Ph.D.

This lesson (lesson 7) will discuss the last type of cytoskeletal structure: microtubules and tubulin.  In addition I want to go over the last quiz answers and also introduce some new poll quizes.

I had given the lecture 7 over Canvas and each of you can download and go over the lecture but I will highlight a few slides in the lecture.

Let’s first review:

Remember that microtubules are the largest of the three cytoskeletal structures:

actin microfilaments < intermediate filaments < microtubules

This is very important to understand as the microtubules, as shown later, shuttle organelles and cellular structures like synaptic vesicles, as well as forming the centrisome and spindle fibers of mitosis.

 

 

 

 

 

 

 

 

 

 

 

 

 

Now remember the quiz question from last time

Remember that actin monomers (the G actin binds ATP)  while tubulin, the protein which makes up the microtubules binds GTP {although it is a little more complex than that as the following diagram shows}

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

See how the growth at the plus end is dependent on tubulin heterodimer GTP while when GDP is only bound to tubulin (both forms) you get a destabilization of the plus end and removal of tubulin dimers (catastrophe) if there is no source of tubulin GTP dimers (alpha tubulin GTP with beta tubulin GTP).

 

 

 

 

Also remember that like actin microfilaments you can have treadmilling (the plus end  continues growing while minus end undergoes catasrophe).  The VIDEO below describes these processes:

 

 

 

Certain SNPs and mutants of tubulin are found and can result in drastic phenotypic changes in microtubule stability and structure. Below is an article where a mutation in tubulin can result in microtubule catastrophe or destabilization of microtubule structures.

 

From: A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics;, E.A. Geyer et al..; elife 2015;4:e10113

Abstract

Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking.

https://doi.org/10.7554/eLife.10113.001

 

Remember the term allosterism: change in the affinity for binding of a ligand or substrate that is caused by the binding of another ligand away from the active site (for example like 2,3 DPG effect on oxygen binding to hemoglobin

 

Cellular transport of organelles and vesicles: a function of microtubules

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Now the above figure (figure 9 in your Powerpoint) shows the movement of organelles and vesicles in two different types of cells along microtubules.

Note the magenta arrow which goes from the nucleus toward the plus end of the microtubule (at cell membrane) is referred to as anterograde transport and is movement away from center of cell to the periphery.  Retrograde transport is movement of organelles and vesicles from periphery of cell to the center of the cell.

Note that kinesin is involved in anterograde transport while dyenin is involved in retrograde transport

Also refer to the Wiki page which shows a nice cartoon of this walking down a microtubule on the right hand side of the page

https://en.wikipedia.org/wiki/Axonal_transport

 

 

 

 

 

 

 

Cilia; a cellular structure of microtubules (we will talk about cilia later)

for more information on structure of Cillia please see https://www.ncbi.nlm.nih.gov/books/NBK21698/

This is from a posting by Dr. Larry Bernstein of Yale University at https://pharmaceuticalintelligence.com/2015/11/04/cilia-and-tubulin/

 

RESEARCHERS VIDEO AND MEASURE TUBULIN TRANSPORT IN CILIA FOR THE FIRST TIME.

http://health-innovations.org/2015/01/27/researchers-image-and-measure-tubulin-transport-in-cilia/

 

 

https://michellepetersen76.files.wordpress.com/2015/01/uga-researchers-image-and-measure-tubulin-transport-in-cilia-healthinnovations1.png

 

Defective cilia can lead to a host of diseases and conditions in the human body, from rare, inherited bone malformations to blindness, male infertility, kidney disease and obesity. It is known that these tiny cell organelles become deformed and cause these diseases because of a problem related to their assembly, which requires the translocation of vast quantities of the vital cell protein tubulin. What they didn’t know was how tubulin and another cell organelle known as flagella fit into the process.

Now, a new study from University of Georgia shows the mechanism behind tubulin transport and its assembly into cilia, including the first video imagery of the process. The study was published in the Journal of Cell Biology.

Cilia are found throughout the body, so defects in cilia formation affect cells that line airways, brain ventricles or the reproductive track.  One of the main causes of male infertility is the cilia won’t function properly.

The team used total internal reflection fluorescence microscopy to analyze moving protein particles inside the cilia of Chlamydomonas reinhardtii, a green alga widely used as a model for cilia analysis.

The team exploited the natural behaviour of the organism, which is to attach by its cilia to a smooth surface, such as a microscope glass cover. This positions the cilia within the 200-nanometer reach of the total internal reflection fluorescence microscope allowing for the imaging of individual proteins as they move inside the cilia.  A video explaining the process was published along with the study.

Tubulin is transported by this process called intraflagellar transport, or IFT.  Though it has long been suspected in the field and there was indirect evidence to support the theory, this is the first time it has been shown directly, through live imaging, that IFT does function as a tubulin pump.  The team observed that about 400,000 tubulin dimers need to be transported within 60 minutes to assemble a single cilium. Being able to see tubulin moving into cilia allowed for first insights into how this transport is regulated to make sure cilia will have the correct size.

The new findings are expected to have wide implications for a variety of diseases and conditions related to cilia defects in the body.  The team state that they are on the very basic side of this research.  But because more and more diseases are being connected to cilia-related conditions, including obesity and even diabetes, the number of people working on cilia has greatly expanded over the last few years.

 

So here are the answer to last weeks polls

  1. Actin filaments are the SMALLEST of the cytoskeletal structures.  As shown in this lecture it is tubulin that binds GTP.  Actin binds ATP.
  2.  ARP2/3 or actin related proteins 2 and 3 are nucleating proteins that assist in initiating growth of branched chain micofiliment networks.  Formins are associated with unbranched actin formations.
  3.  The answer is GAPs or GTPase activating proteins.  Remember RAS in active state when GTP is bound and when you hydrolyze the GTP to GDP Ras is inactive state

 

 

 

 

 

4.  Okay so I did a type here but the best answer was acetylcholinesterase (AchE) degrading acetylcholine.  Acetylcholinesterase degrades the neurotransmitter acetylcholine into choline and acetate not as I accidentally put into acetylCoA.  The freed choline can then be taken back up into the presynaptic neuron and then, with a new acetyl group (with Coenzyme A) will form acetylcholine.

 

Synthesis of the neurotransmitter acetylcholine

 

 

 

The neuromuscular junction

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Thanks to all who took the quiz.  Remember it is for your benefit.

 

 

 

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Parkinson’s Disease (PD), characterized by both motor and non-motor system pathology, is a common neurodegenerative disorder affecting about 1% of the population over age 60. Its prevalence presents an increasing social burden as the population ages. Since its introduction in the 1960’s, dopamine (DA)-replacement therapy (e.g., L-DOPA) has remained the gold standard treatment. While improving PD patients’ quality of life, the effects of treatment fade with disease progression and prolonged usage of these medications often (>80%) results in side effects including dyskinesias and motor fluctuations. Since the selective degeneration of A9 mDA neurons (mDANs) in the substantia nigra (SN) is a key pathological feature of the disease and is directly associated with the cardinal motor symptoms, dopaminergic cell transplantation has been proposed as a therapeutic strategy.

 

Researchers showed that mammalian fibroblasts can be converted into embryonic stem cell (ESC)-like induced pluripotent stem cells (iPSCs) by introducing four transcription factors i.e., Oct4, Sox2, Klf4, and c-Myc. This was then accomplished with human somatic cells, reprogramming them into human iPSCs (hiPSCs), offering the possibility of generating patient-specific stem cells. There are several major barriers to implementation of hiPSC-based cell therapy for PD. First, probably due to the limited understanding of the reprogramming process, wide variability exists between the differentiation potential of individual hiPSC lines. Second, the safety of hiPSC-based cell therapy has yet to be fully established. In particular, since any hiPSCs that remain undifferentiated or bear sub-clonal tumorigenic mutations have neoplastic potential, it is critical to eliminate completely such cells from a therapeutic product.

 

In the present study the researchers established human induced pluripotent stem cell (hiPSC)-based autologous cell therapy. Researchers reported a platform of core techniques for the production of mDA progenitors as a safe and effective therapeutic product. First, by combining metabolism-regulating microRNAs with reprogramming factors, a method was developed to more efficiently generate clinical grade iPSCs, as evidenced by genomic integrity and unbiased pluripotent potential. Second, a “spotting”-based in vitro differentiation methodology was established to generate functional and healthy mDA cells in a scalable manner. Third, a chemical method was developed that safely eliminates undifferentiated cells from the final product. Dopaminergic cells thus produced can express high levels of characteristic mDA markers, produce and secrete dopamine, and exhibit electrophysiological features typical of mDA cells. Transplantation of these cells into rodent models of PD robustly restored motor dysfunction and reinnervated host brain, while showing no evidence of tumor formation or redistribution of the implanted cells.

 

Together these results supported the promise of these techniques to provide clinically applicable personalized autologous cell therapy for PD. It was recognized by researchers that this methodology is likely to be more costly in dollars and manpower than techniques using off-the-shelf methods and allogenic cell lines. Nevertheless, the cost for autologous cell therapy may be expected to decrease steadily with technological refinement and automation. Given the significant advantages inherent in a cell source free of ethical concerns and with the potential to obviate the need for immunosuppression, with its attendant costs and dangers, it was proposed that this platform is suitable for the successful implementation of human personalized autologous cell therapy for PD.

 

References:

 

https://www.jci.org/articles/view/130767/pdf?elqTrackId=2fd7d0edee744f9cb6d70a686d7b273b

 

https://www.ncbi.nlm.nih.gov/pubmed/31714896

 

https://www.ncbi.nlm.nih.gov/pubmed/23666606

 

https://www.ncbi.nlm.nih.gov/pubmed/27343168

 

https://www.ncbi.nlm.nih.gov/pubmed/21495962

 

https://www.ncbi.nlm.nih.gov/pubmed/28083784

 

https://www.ncbi.nlm.nih.gov/pubmed/20336395

 

https://www.ncbi.nlm.nih.gov/pubmed/28585381

 

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Contributions to Neuronal Systems by University Professors Eve Marder and Irv Epstein at Brandeis University

Reporter: Aviva Lev-Ari, PhD, RN

Oscillators: Chemicals, Neurons and People: A Celebration of Eve Marder, Irving Epstein and the Volen Center for Complex Systems
Sunday, Nov. 17, 2019, 1 – 5:30 p.m.
Location Schwartz
Room Auditorium
Event Sponsor(S) Office of the Provost
Website www.brandeis.edu…

A celebration for new university professors Eve Marder and Irving Epstein, and the 25th anniversary of the Volen Center!

Schedule of Events

  • 1p.m. – Opening remarks: Leslie Griffith, Nancy Lurie Marks Professor of Neuroscience and Director of the Volen National Center for Complex Systems
  • 1:30 p.m. – Panel Discussion: “Oscillators: Chemicals, Neurons and People”
    • Moderator: Gina Turrigiano, Joseph Levitan Professor of Vision Science
    • Panelist: Jorge Golowasch, Professor, Department of Biological Sciences, New Jersey Institute of Technology
    • Panelist: Nancy Kopell, Professor of Mathematics, Boston University
    • Panelist: Horacio Rotstein, Professor of Mathematical Biology & Computational Neuroscience, Department of Biological Sciences, New Jersey Institute of Technology
    • Panelist: Frances Skinner, Senior Scientist, Krembil Research Institute and Professor, Division of Neurology, Department of Medicine and Department of Physiology, University of Toront
  • 2:30 p.m. – Coffee
  • 3 p.m. – Irving Epstein: “How I Wandered into an Oscillatory State”
  • 4 p.m. – Eve Marder: “The Challenges Posed by Neuronal Oscillators that are both Stable and Plastic”
  • 5 p.m. – Closing remarks: Leslie Griffith

 

Eve Marder and Irv Epstein recall the collaborations that started it all

The University Professors will lead a public symposium on Nov. 17

headshots of University Professors Eve Marder, left, and Irv EpsteinPhoto/Mike LovettUniversity professors Eve Marder ’69, Left, and Irving R. Epstein

University Professors Eve Marder ’69 and Irv Epstein will help celebrate the 25th anniversary of the Volen National Center for Complex Systems in a public symposium November 17.

Marder, the Victor and Gwendolyn Beinfield Professor of Neuroscience, and Epstein, the Henry F. Fischbach Professor of Chemistry, were named University Professors last spring in recognition of their pioneering interdisciplinary achievements. At the symposium, they will each deliver a lecture drawing on their collaborative research on oscillators. In the case of Epstein, these are oscillating chemical reactions. In Marder’s case, it is rhythmically active neurons and/or circuits.   

But some 35 years ago, before they were leaders in their fields, Epstein and Marder saw the benefit of sharing ideas. A mutual colleague noticed that the chemical reactions recorded on the chart recorder Epstein was using looked intriguingly similar to the neuronal signals Marder was recording in her research.

“It’s relatively unusual behavior for chemistry, but it’s sort of the essence of what goes on in neuronal systems,” said Epstein, who soon learned some rudiments of neuroscience from Marder and began mathematically modeling groups of neurons. For her part, Marder “got an appreciation for what interacting with theorists could bring, to help her answer the kind of questions she wanted to answer,” said Epstein.

These days, Marder’s research on small neural circuits found in lobsters and crabs is credited with revolutionizing understanding of the fundamental nature of neuronal circuit operation, including how neuromodulators control behavioral outputs and how the stability of circuits is maintained over time. She has won many top prizes in neuroscience, including the Gruber Award in Neuroscience, the Kavli Prize in Neuroscience, and the National Academy of Sciences Neuroscience Prize. In March, she will receive the Carnegie Prize in Mind and Brain Science from Carnegie Mellon University.

Epstein, a Howard Hughes Medical Institute professor, pioneered the field of chemical oscillators. “When we got into the field of oscillating reactions, there were just three that were known, and they were all discovered accidentally,” said Epstein. “We decided that if we really understood these systems, we should be able to design them.”

Although it took him several years and three unsuccessful grant applications to secure funding for his ideas, Epstein and his lab ultimately won funding, and within a few months succeeded in developing their first novel chemical oscillating reaction.

Writ large, Marder and Epstein collaboratively demonstrate that the kinds of phenomena seen in neurons are also found in chemical and physical systems. “What you learn from modeling chemical reactions can help you understand how neurons work, and vice versa,” said Epstein.

“Volen is a place where you get all kinds of collaborations,” said Marder. “One of Brandeis’ strengths is its interactivity; those early days were quite catalytic.”

 

Categories: ResearchScience and Technology

SOURCE

https://www.brandeis.edu/now/2019/november/eve-irv-volen.html

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

The relationship between gut microbial metabolism and mental health is one of the most intriguing and controversial topics in microbiome research. Bidirectional microbiota–gut–brain communication has mostly been explored in animal models, with human research lagging behind. Large-scale metagenomics studies could facilitate the translational process, but their interpretation is hampered by a lack of dedicated reference databases and tools to study the microbial neuroactive potential.

 

Out of all the many ways, the teeming ecosystem of microbes in a person’s gut and other tissues might affect health. But, its potential influences on the brain may be the most provocative for research. Several studies in mice had indicated that gut microbes can affect behavior, and small scale studies on human beings suggested this microbial repertoire is altered in depression. Studies by two large European groups have found that several species of gut bacteria are missing in people with depression. The researchers can’t say whether the absence is a cause or an effect of the illness, but they showed that many gut bacteria could make substances that affect the nerve cell function—and maybe the mood.

 

Butyrate-producing Faecalibacterium and Coprococcus bacteria were consistently associated with higher quality of life indicators. Together with DialisterCoprococcus spp. was also depleted in depression, even after correcting for the confounding effects of antidepressants. Two kinds of microbes, Coprococcus and Dialister, were missing from the microbiomes of the depressed subjects, but not from those with a high quality of life. The researchers also found the depressed people had an increase in bacteria implicated in Crohn disease, suggesting inflammation may be at fault.

 

Looking for something that could link microbes to mood, researchers compiled a list of 56 substances important for proper functioning of nervous system that gut microbes either produce or break down. They found, for example, that Coprococcus seems to have a pathway related to dopamine, a key brain signal involved in depression, although they have no evidence how this might protect against depression. The same microbe also makes an anti-inflammatory substance called butyrate, and increased inflammation is implicated in depression.

 

Still, it is very much unclear that how microbial compounds made in the gut might influence the brain. One possible channel is the vagus nerve, which links the gut and brain. Resolving the microbiome-brain connection might lead to novel therapies. Some physicians and companies are already exploring typical probiotics, oral bacterial supplements, for depression, although they don’t normally include the missing gut microbes identified in the new study.

 

References:

 

https://www.sciencemag.org/news/2019/02/evidence-mounts-gut-bacteria-can-influence-mood-prevent-depression?utm_source=Nature+Briefing

 

https://www.nature.com/articles/s41564-018-0337-x

 

https://www.ncbi.nlm.nih.gov/pubmed/22968153

 

https://www.ncbi.nlm.nih.gov/pubmed/24888394

 

https://www.ncbi.nlm.nih.gov/pubmed/27067014

 

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  1. Lungs can supply blood stem cells and also produce platelets: Lungs, known primarily for breathing, play a previously unrecognized role in blood production, with more than half of the platelets in a mouse’s circulation produced there. Furthermore, a previously unknown pool of blood stem cells has been identified that is capable of restoring blood production when bone marrow stem cells are depleted.

 

  1. A new drug for multiple sclerosis: A new multiple sclerosis (MS) drug, which grew out of the work of UCSF (University of California, San Francisco) neurologist was approved by the FDA. Ocrelizumab, the first drug to reflect current scientific understanding of MS, was approved to treat both relapsing-remitting MS and primary progressive MS.

 

  1. Marijuana legalized – research needed on therapeutic possibilities and negative effects: Recreational marijuana will be legal in California starting in January, and that has brought a renewed urgency to seek out more information on the drug’s health effects, both positive and negative. UCSF scientists recognize marijuana’s contradictory status: the drug has proven therapeutic uses, but it can also lead to tremendous public health problems.

 

  1. Source of autism discovered: In a finding that could help unlock the fundamental mysteries about how events early in brain development lead to autism, researchers traced how distinct sets of genetic defects in a single neuronal protein can lead to either epilepsy in infancy or to autism spectrum disorders in predictable ways.

 

  1. Protein found in diet responsible for inflammation in brain: Ketogenic diets, characterized by extreme low-carbohydrate, high-fat regimens are known to benefit people with epilepsy and other neurological illnesses by lowering inflammation in the brain. UCSF researchers discovered the previously undiscovered mechanism by which a low-carbohydrate diet reduces inflammation in the brain. Importantly, the team identified a pivotal protein that links the diet to inflammatory genes, which, if blocked, could mirror the anti-inflammatory effects of ketogenic diets.

 

  1. Learning and memory failure due to brain injury is now restorable by drug: In a finding that holds promise for treating people with traumatic brain injury, an experimental drug, ISRIB (integrated stress response inhibitor), completely reversed severe learning and memory impairments caused by traumatic brain injury in mice. The groundbreaking finding revealed that the drug fully restored the ability to learn and remember in the brain-injured mice even when the animals were initially treated as long as a month after injury.

 

  1. Regulatory T cells induce stem cells for promoting hair growth: In a finding that could impact baldness, researchers found that regulatory T cells, a type of immune cell generally associated with controlling inflammation, directly trigger stem cells in the skin to promote healthy hair growth. An experiment with mice revealed that without these immune cells as partners, stem cells cannot regenerate hair follicles, leading to baldness.

 

  1. More intake of good fat is also bad: Liberal consumption of good fat (monounsaturated fat) – found in olive oil and avocados – may lead to fatty liver disease, a risk factor for metabolic disorders like type 2 diabetes and hypertension. Eating the fat in combination with high starch content was found to cause the most severe fatty liver disease in mice.

 

  1. Chemical toxicity in almost every daily use products: Unregulated chemicals are increasingly prevalent in products people use every day, and that rise matches a concurrent rise in health conditions like cancers and childhood diseases, Thus, researcher in UCSF is working to understand the environment’s role – including exposure to chemicals – in health conditions.

 

  1. Cytomegalovirus found as common factor for diabetes and heart disease in young women: Cytomegalovirus is associated with risk factors for type 2 diabetes and heart disease in women younger than 50. Women of normal weight who were infected with the typically asymptomatic cytomegalovirus, or CMV, were more likely to have metabolic syndrome. Surprisingly, the reverse was found in those with extreme obesity.

 

References:

 

https://www.ucsf.edu/news/2017/12/409241/most-popular-science-stories-2017

 

https://www.ucsf.edu/news/2017/03/406111/surprising-new-role-lungs-making-blood

 

https://www.ucsf.edu/news/2017/03/406296/new-multiple-sclerosis-drug-ocrelizumab-could-halt-disease

 

https://www.ucsf.edu/news/2017/06/407351/dazed-and-confused-marijuana-legalization-raises-need-more-research

 

https://www.ucsf.edu/news/2017/01/405631/autism-researchers-discover-genetic-rosetta-stone

 

https://www.ucsf.edu/news/2017/09/408366/how-ketogenic-diets-curb-inflammation-brain

 

https://www.ucsf.edu/news/2017/07/407656/drug-reverses-memory-failure-caused-traumatic-brain-injury

 

https://www.ucsf.edu/news/2017/05/407121/new-hair-growth-mechanism-discovered

 

https://www.ucsf.edu/news/2017/06/407536/go-easy-avocado-toast-good-fat-can-still-be-bad-you-research-shows

 

https://www.ucsf.edu/news/2017/06/407416/toxic-exposure-chemicals-are-our-water-food-air-and-furniture

 

https://www.ucsf.edu/news/2017/02/405871/common-virus-tied-diabetes-heart-disease-women-under-50

 

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Memory Gene Goes Viral

Reporter: Irina Robu, PhD

A gene crucial for learning, called Arc can send genetic material from one neuron to another by using viruses was discovered by two independent team of scientist from University of Massachusetts Medical School and University of Utah which was published in Cell.  According to Dr. Edmund Talley, a program director at National Institute of Neurological Disorders and Stroke “this work is a great example of the importance of basic neuroscience research”.

Arc plays an important role in the brain’s ability to store new information, however little is known of how it works. According to the University of Utah scientists, research into the examination of the Arc gene began by introducing it into bacterial cells. When the cells made the Arc protein, it clumped together into a form that resembled a viral capsid, the shell that contains a virus’ genetic information. The Arc “capsids” appeared to mirror viral capsids in their physical structure in addition as their behavior and other properties.

At the same time, University of Massachusetts scientist led by Vivian Budnik, Ph. D and Travis Thomson, Ph.D. set out to scrutinize the contents of tiny sacks released by cells called extracellular vesicles. Their experiments in fruit flies revealed that motor neurons that control the flies’ muscles release vesicles containing a high concentration of the Arcgene’s messenger RNA (mRNA), the DNA-like intermediary molecule cells use to create the protein encoded by a DNA sequence.

Both groups similarly found evidence that Arc capsids contain Arc mRNA and that the capsids are released from neurons inside those vesicles. Also, both groups suggest that Arc capsids act like viruses by delivering mRNA to nearby cells. Furthermore, Dr. Shepherd’s team presented that the more active neurons are, the more of those vesicles they release. Dr. Shepherd’s group grew mouse neurons lacking the Arc gene in petri dishes filled with Arc-containing vesicles or Arc capsids alone. They revealed that the formerly Arc-less neurons took in the vesicles and capsids and used the Arc mRNA contained within to produce the Arc protein themselves. Finally, just like neurons that naturally manufacture the Arc protein, those cells made more of it when their electrical activity increased.

Both groups of scientists plan to examine why cells use this virus-like strategy to shuttle Arc mRNA between cells and which might allow the toxic proteins responsible for Alzheimer’s disease to spread through the brain.

SOURCE

http://www.sciencenewsline.com/news/2018011717480045.html

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Targeting amyloidopathy

Larry H. Bernstein, MD, FCAP

LPBI

 

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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