It’s unknown why misfolded aggregates appear in cells cytosol during neurodegenerative diseases. If the forming mechanism was elucidated it would enable designing new and efficient therapies. One of those protein aggregates is composed of misfolded TDP-43. Aggregates hyper-phosphorylated, ubiquitinated and cleaved form of TDP-43 are found in frontotemporal dementia, in amyotrophic lateral sclerosis and in some cases of Alzheimer and Parkinson.

My feeling is that scientists, from Academy of Scientific and Innovative Research (AcSIR) in India, achieved one of the most important milestone since 2006, when Virginia Lee provided evidence of involvement of TDP-43 in ALS.

For the proper functioning of the cells, neutral pH is required. However during normal metabolism, all foods create waste products which are acidic. Accumulated waste and toxins ages the cell, sometimes causes it to change to a sick or abnormal cell. For example the cytosol of yeast cells acidifies during aging.

Cells experience a variety of stress-like conditions, in particular, nutrient starvation stress acidifies the cytosol and increases the cytosolic proton ion concentration due to the reduced efficiency of ATP proton pump.

In chemistry, protonation describes the addition of a proton to a molecule, forming an acid. Some proteins or protein domains inside the cells can function as biosensors. It has been proposed that cells sense starvation stress at the molecular level by protonating the side chains of biosensor protein molecules.

The scientists Divya Patni  and  Santosh Kumar Jha observed in a previous study that one domain of TDP-43 (tRRM) could function as a biosensor and sense pH stress. They shown that under low-pH conditions, mimicking starvation stress, TDP-43tRRM undergoes a conformational change named "L structure". enter image description here The L form structure is held by weak interactions and eventually fully misfolds and oligomerizes to form a β-sheet rich "β form". The unstructured regions of the protein gain structure during L ⇌ β conversion.

TDP-43 consists of 4 domains:

  • An N-terminal domain
  • Two RNA recognition motifs RRM1 and RRM2 working as a tandem (tRMM)
  • An unstructured C-terminal domain.

In this paper, Patni  and  Jha showed that the monomeric N form of TDP-43tRRM forms a misfolded amyloid-like protein assembly, β form, in a pH-dependent manner.

The side chains of the ionizable amino acid residues buried inside the protein structure can protonate or deprotonate only upon partial or complete unfolding.

They are promising candidates to function as gatekeeper residues for protein aggregation as it often begins with partial unfolding of the protein.

The scientists from the Academy of Scientific and Innovative Research in Ghaziabad India, hypothesized that ionization of a protein side-chain buried in the protein structure might be coupled to the formation of the misfolded β form.

An examination of the protein structure revealed that out of all of the amino acid residues whose side chain could titrate in the acidic pH range, only D105, H166, and H256 are almost completely buried in the protein structure. They systematically mutated these residues to neutral amino acids whose side chains cannot undergo protonation-deprotonation reaction ( D105A, H166Q and H256Q).

Patni  and  Jha observed that D105A and H256Q behaved like TDP-43tRRM in their pH-dependent misfolding behavior. However, H166Q retained the N-like secondary structure under low-pH conditions and did not show pH-dependent misfolding to the β form.

These results indicate that H166 is the critical side-chain residue whose protonation triggers the misfolding of TDP-43tRRM.

These results indicate that the protonation of H166 functions as a critical trigger switch that controls the amyloid-like misfolding of TDP-43tRRM upon pH stress sensing. It appears that the protonation of H166 results in proximal or distal conformational changes that initiate the misfolding of the protein.

It’s suspected since a long time that the RNA-Recognition motifs of TDP-43 may play a role in the aberrant self-assembly of the protein. Now a clear mechanism of action had been described, while it may not be the only one, hopefully it will enable the design of new therapies.

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This book retraces the main achievements of ALS research over the last 30 years, presents the drugs under clinical trial, as well as ongoing research on future treatments likely to be able stop the disease in a few years and to provide a complete cure in a decade or two.

ALS does not have a single cause, on the contrary every case of ALS is probably induced by a combination of factors that will lead to cellular stress. The response to this cellular stress (UPR and others) involves the shutdown of functions that are essential for the proper functioning of the body. Indeed in a state of stress response, there is no more protein production, while the protein consumption resulting from metabolism is continuing, so muscle mass is gradually destroyed.

Scientists know how to chemically or biologically induce conditions similar to ALS in model animals. One of the possibilities is to use β-Methylamino-L-alanine, or BMAA. It is a toxin from a cyanobacterium. Cyanobacteria are well known by the nickname green algae, which can be found in all places where there is stagnant water including maritime bays like that of Morlaix in France, the tidal bore of the Petitcodiac river (where there is currently an epidemic of unknown neurological disease) or aquariums. enter image description here

But toxins are not only found in standing water, they are found everywhere in the natural environment. For example, between 1990 and 2018, 14 cases of amyotrophic lateral sclerosis (ALS) were diagnosed in residents and visitors with a second home in a mountain hamlet in the French Alps. enter image description here (Frederic Coune)

A systematic investigation was then carried out. The official report is quite disappointing. It asserts that it is not possible to hypothesize that there is a link between the cases of ALS observed and a particular risk associated with this place. The report goes on to say, and this is obviously comfortable for the French administration, that there is no specific management measure whose implementation can prevent the onset of this disease.

Recent on-site investigations by Lagrange, Spencer and other colleagues from France and the United States, however, showed that all the patients had ingested wild mushrooms, especially poisonous false morels. Half of these patients had consumed Gyromitra gigas mushrooms.

Consumption of the neurotoxic fungus containing gyromitrin Gyromitra sp. (false morel), has sometimes been implicated in the sporadic genesis of amyotrophic lateral sclerosis.

There are several fungal toxins that can cause organic damage in the human body. Tricholoma equestre may contain myotoxin and repeated ingestion may cause severe rhabdomyolysis. Ingestion of Amanita smithiana and A. proxima causes kidney damage. Gyromitrin, a toxic compound that is converted to hydrazines in the stomach, is present in some species of Gyromitra. It is primarily neurotoxic, but can also induce moderate liver damage and hemolysis.

Gyromitrin is a toxin and carcinogen found in several members of the fungal genus Gyromitra, such as G. esculenta. Poisoning causes nausea, stomach cramps, and diarrhea, while severe poisoning can lead to seizures, jaundice, and even coma or death. The gyromitrin content of false morels is said to be between 40 and 732 milligrams of gyromitrin per kilogram of mushroom (wet weight). The median lethal dose (LD50) of gyromitrin is 30 to 50 mg / kg in humans. That is, in some cases a hundred grams of the fungus would be enough to reach the lethal dose.

Scientists have even shown that high consumption of fresh or dried true morels Morchella spun causes temporary neurological syndrome (SN) with cerebellar signs. For safety, they recommend to avoid consuming real morels or button mushrooms raw or undercooked, fresh or dried.

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This book retraces the main achievements of ALS research over the last 30 years, presents the drugs under clinical trial, as well as ongoing research on future treatments likely to be able stop the disease in a few years and to provide a complete cure in a decade or two.

The insulin signaling pathway plays a crucial role in regulating the growth and metabolism of neurons. Deregulation of IGF-R signaling has been linked to a variety of neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's disease.

However, the role of insulin signaling in C9orf72 ALS / FTD is not yet clear. A positive correlation of the incidence of ALS with early-onset type 1 diabetes has been reported, and a decrease in insulin and IGF-1 in the blood and cerebrospinal fluid of patients with ALS, although the relevance of these findings to disease progression is unclear.

Intrathecal administration of IGF-1 improved motor performance, delayed disease onset, and prolonged survival in the SOD1G93A mouse model of ALS. However, three clinical trials of IGF-1 administered subcutaneously in ALS have reported conflicting results. The contradictory result of these trials may be due to insufficient administration of drugs to the brain and spinal cord and the fact that ALS has heterogeneous genetic risk factors.

Atilano, Isaacs, Partridge, and colleagues have shown in a recent article that insulin/IGF signaling is reduced in C9orf72 fly models using adult brain RNA sequencing. They further demonstrated that activation of insulin/IGF signaling can attenuate several neurodegenerative phenotypes in flies expressing expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR.

Poly-GR levels are reduced when components of the insulin/IGF signaling pathway are genetically activated in diseased flies, suggesting a rescue mechanism. This effect on poly-GR levels was confirmed in a mammalian cell model. Modulation of insulin signaling in mammalian cells also lowers poly-GR levels. Remarkably, the systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Their data suggest that modulation of insulin/IGF signaling may be an effective therapeutic approach against C9orf72 ALS/FTD.

PTEN acts as a tumor suppressor gene through the action of its protein phosphatase product. This phosphatase is involved in the regulation of the cell cycle, preventing cells from growing and dividing too quickly. It is a target of many anticancer drugs. Pten reduction has also been reported to reduce the toxicity of C9orf72 repeats expressed in a mammalian cell line, again in agreement with the results described by scientists and the potential therapeutic benefit of modulating this pathway. It is also interesting to note that the process of brain aging has been linked to decreased insulin signaling as well as impaired insulin binding, which may partly explain why aging is a problem. risk factor for the disease.

The authors found that intrathoracic insulin administration prolonged the survival of flies expressing G4C2 repeats. Although robust, the lengthening of the lifespan was relatively modest, which could be explained by the transient nature of the insulin treatment. Insulin and IGF-1 ligands have already been tested in neurodegenerative diseases.

Overall, the researchers' study suggests that modulation of the insulin / IGF signaling pathway could be an effective therapeutic intervention against hexanucleotide repeat extension associated with C9orf72 neurodegenerative diseases, with InR being a genetic modifier. It will be interesting in the future to study the need for downstream effectors of insulin signaling in the rescue of toxicity. It is important to note that in the Drosophila, there is a single insulin-like system that has the dual function of insulin / IGF signaling; thus, the mechanism of toxicity described in the researchers' model could also be linked to IGFs. Therefore, it will be important to test whether treatment with insulin or IGF can save the survival of other C9orf72 ALS / FTD vertebrate model organisms.

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This book retraces the main achievements of ALS research over the last 30 years, presents the drugs under clinical trial, as well as ongoing research on future treatments likely to be able stop the disease in a few years and to provide a complete cure in a decade or two.

In March 2021 the European drug agency granted Orphan status to Ganglioside GM1 for treatment of ALS. It was presented by 3R Pharma Consulting GmbH, a consultancy organization acting as proxy for another organization. Why the European drug agency granted Orphan status is still unknown, but it seems that this agency is much more permissive than it's US counterpart, the FDA.

By coincidence, a new article on June 12, was about how ganglioside GM1 may be the cause of Guillain–Barré Syndrome after infection. enter image description here

The quality of life after an attack of Guillain–Barré syndrome can be significantly impaired. About a fifth of patients are unable to walk unaided after six months, and many experience chronic pain, fatigue and difficulty with work, education, hobbies and social activities. But usually quality of life improves significantly in the first year. Yet in 5% of cases Guillain–Barré syndrome (GBS) can lead to death as a result of many complications.

While GBS is a disease of the peripheral nervous system, ALS is a disease of the motor tracks in the central nervous system. Yet, many patients with ALS also recount that their disease started with an infection.

Some investigators have suggested that mechanisms resulting from molecular mimicry between viral proteins and human proteins participate in the pathogenesis of GBS.

Ganglioside GM1 has important physiological properties and impacts neuronal plasticity and repair mechanisms, and the release of neurotrophins in the brain. Because of GM1's close role in neuron repair mechanisms, it has been investigated as a possible drug to slow or even reverse the progression of a wide range of neurodegenerative conditions. Controlled phase II studies have indicated that GM1 can ease the symptoms of Parkinson's disease

Weirdly for the complement immune system GM1 is sometimes identified as a virus fragment.

Alas Ganglioside GM1 is not the sole case of autoimmunity. Many autoimmune diseases, some of them ALS mimics, are caused by autoantibodies. An autoantibody is an antibody produced by the immune system that is directed against one or more of the individual's own proteins.

To complicate the matter, some autoantibodies are needed to maintain tissue and protein homeostasis through adaptive debris clearance. For example autoantibodies against TDP-43 are found lacking in Patients With Amyotrophic Lateral Sclerosis.

Autoantibody tests may be ordered as part of an investigation. ANA is a marker of the autoimmune process – it is positive with a variety of different autoimmune diseases but not specific. Consequently, if an ANA test is positive, it is followed up with other tests.

Plasmapheresis and intravenous immunoglobulins (IVIG) are the two main immunotherapy treatments for GBS. Plasmapheresis attempts to reduce the body's attack on the nervous system by filtering antibodies out of the bloodstream. Similarly, administration of IVIG neutralizes harmful antibodies and inflammation.

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This book retraces the main achievements of ALS research over the last 30 years, presents the drugs under clinical trial, as well as ongoing research on future treatments likely to be able stop the disease in a few years and to provide a complete cure in a decade or two.

Metabolic disorders are associated with the progression of amyotrophic lateral sclerosis. This new study by Tanya S McDonald and colleagues from the University of Queensland is very interesting because it focuses on physiology and not on molecular phenomenas.

Throughout the progression of ALS disease in laboratory mice, researchers have identified increased glucose uptake, possibly due to insulin-independent mechanisms. This glucose was then stored as glycogen in tissues such as the liver, rather than being used as an energy source. This might explain ALS' hypermetabolism.

Normally, in a healthy human, the postprandial state (after-meal) elevates glucose levels and triggers the release of insulin from the pancreas. As insulin levels rise, there is an increase in glucose uptake and then storage of excess glucose in peripheral tissues.

Glycogen is one of two forms of energy storage, with glycogen being short-term storage and the other being triglyceride stores in adipose tissue (i.e. body fat) for long term storage.

Patients with ALS cannot maintain their weight, and experience rapid muscle loss. Curiously, this muscle loss is not the subject of much attention from scientists who are interested only in motor neurons. They often deplore a lack of biomarkers, while the loss of muscle mass is an obvious biomarker. This article suggests that ALS is a form of diabetes, although this is not formally expressed in the article.

Rapid weight loss in patients with ALS is associated with rapid disease progression, while conversely, a higher body mass index (~ 27) tends to increase the survival rate. Studies also suggest that insulin resistance plays a role in disease progression in patients and animal models of ALS.

Glucose homeostasis is fundamental for the human body and is mainly regulated by the levels of 4 major hormones: 1. Insulin 2. Glucagon 3. Cortisol 4. Epinephrine The ratios of these circulating hormones will dictate the activity of specific metabolic pathways that control glucose homeostasis. There are many other hormones (thyroid hormone, growth hormone, etc.) and adipokines (adiponectin, leptin, etc.) that can influence glucose homeostasis, as well as neural mechanisms that control higher level functions such as hunger and satiety.

Insulin secretion depends on oxidative metabolism. In humans, glycogen is made and stored primarily in liver cells and skeletal muscle cells.

SOD1G93A mice exhibit loss of body weight and lean body mass with reduced activity and increased oxygen uptake in the mid-symptomatic stage of disease.

McDonald and his colleagues first investigated whether the weight loss frequently observed in SOD1G93A mice was due to reduced food intake or increased energy expenditure.

At the onset of the disease, the mice showed no difference in body weight, but still had a 10% loss of their lean body mass (body mass other than fat, including bones, muscles, blood, skin, etc.). That is, fat was substituted for muscle mass.

At the mid-symptomatic stage, the SOD1G93A mice weighed significantly less than their normal counterparts, with a loss of 8 and 10% of total body weight and lean body mass, respectively.

However, the total food intake was similar between normal mice and SOD1G93A mice at these two stages of the disease.

While at the initial stage there was no difference in oxygen uptake between mutated and normal mice, at the mid symptomatic stage the mean oxygen uptake in SOD1G93A mice was significantly higher than in normal mice.

This increase in oxygen uptake in the mid-symptomatic stage was not, however, due to an increase in average locomotor activity, as the reduction in locomotor activity was only measured during the dark cycle in the dying stages. onset and semi-symptomatic. Indeed, during the light phase, the mid-symptom SOD1G93A mice were 126% more active than their normal counterparts.

No correlation was found between the decrease in lean body mass and the average oxygen uptake over a 24-hour period.This increase in oxygen uptake at the mid-symptomatic stage is therefore unexplained.

Exogenous glucose uptake is increased in SOD1G93A mice at the mid-symptomatic stage of the disease

The scientists then set out to determine whether glucose management was impaired in SOD1G93A mice. At the onset of symptoms, SOD1G93A mice and their normal counterparts responded similarly to glucose. However, in the mid-symptomatic stage of the disease, SOD1G93A mice showed a faster rate of blood glucose clearance.

The authors then confirmed that the loss of body weight in SOD1G93A mice was not responsible for the decrease in blood glucose concentration. Although the baseline insulin concentration remained unchanged, the response of plasma insulin to exogenous glucose was significantly lower in SOD1G93A mice, with a 44% reduction in insulin concentrations.

At the onset of the disease and at its mid-symptomatic stage there was no difference in the immunoreactive zone of the glucagon-positive cells. But at the mid-symptomatic stage McDonald and his colleagues found in the pancreas of SOD1G93A mice, a 22% reduction in insulin-positive β cells compared to the pancreas of normal mice.

Despite this difference in baseline blood glucose concentrations, normal and SOD1G93A mice responded similarly to insulin. This is a major difference between diabetes and ALS.

Although the SOD1G93A mice weighed less at onset and during the middle of symptoms, the amount of insulin did not correlate with the inverse of blood sugar levels. This indicates that the detection of oxidative metabolism was inoperative, which is one of the characteristics of diabetes.

In addition to insulin and glucagon levels, the authors also demonstrated that glycogen concentrations were 210 and 480% higher in the liver of SOD1G93A mice at onset and mid-symptom stages, respectively.

Insulin tolerance is not affected in SOD1G93A mice, despite decreased fasting blood sugar

After an overnight fast, the accumulation of glycogen in the liver was still 400-500% higher in SOD1G93A mice amid symptoms compared to their normal counterparts. These changes are insulin independent because there was no difference in the elimination of glucose in response to exogenous insulin. In addition, SOD1G93A mice exhibit reduced insulin-expressing cell surface area and impaired insulin release in response to exogenous glucose. SOD1G93A mice also showed an accumulation of glycogen in the liver, despite increased circulating glucagon concentrations and gene expression data, suggesting a decrease in both glycogen synthesis and degradation.

This indicates that glucagon signaling may be altered in the liver of SOD1G93A mice. Finally, the gene expression profile of several metabolic enzymes suggested that the liver switches from using glucose to fatty acids as an energy source, which has already been found in skeletal muscle and CNS tissues in the body. SLA.

This confirms the results in other affected tissues which show a shift from the use of glucose to lipids as the primary fuel source for the TCA cycle. Although the exact trigger that leads to this change is unknown, it has been proposed that an increase in fatty acid metabolism occurs to compensate for the inability of tissues to use glucose and glycogen as energy substrates. Although this is a beneficial short-term compensatory mechanism, chronic dependence on fatty acid metabolism via β-oxidation can lead to the accumulation of toxic byproducts, especially reactive oxygen species. (ROS).

Conclusion What is described in this article is a reminder of the evolution of diabetes. When diabetes begins, the pancreas normally produces insulin. Muscle cells preferably use fatty acids as an energy source. Gradually the cells of the body responsible for collecting and using glucose become insensitive to insulin. Since glucose cannot enter the cells, the beta cells of the islets of Langerhans in the pancreas will produce more insulin to force the cells to take up glucose. In this article the mechanism is a little different, instead of making more insulin, the body stores glucose in the form of glycogen. The more diabetes progresses, the more beta cells are depleted, until they disappear. This disappearance was also noted in the article.

However, this does not explain the local appearance of the onset of ALS and the geographical progression of the disease, and it is a work on model mice for ALS. It is known that work on mice is rarely transferable to humans, particularly for neurodegenerative diseases.

Yet, this article is unique. This is an article that talks about physiology, it does not appeal to obscure molecules that are arbitrarily assigned biological roles, it suggests a mechanism for ALS that is down to earth.

Of course there are still many unknowns as to why ALS often starts with a specific muscle and then progresses. We don't know how to treat diabetes any more than we do with ALS, but I believe that an important step has been accomplished.

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This book retraces the main achievements of ALS research over the last 30 years, presents the drugs under clinical trial, as well as ongoing research on future treatments likely to be able stop the disease in a few years and to provide a complete cure in a decade or two.

As various pathogens have been reported in the blood, cerebrospinal fluid, and central nervous system of patients with ALS, a few scientific publications have suggested that infectious agents may play a role in neurodegenerative diseases, but these agents have never been identified.

In a new scientific publication, the authors report they have found an unidentified virus like signature in 120 whole blood RNA samples, in ALS patient as well in controls samples. However when they used other public databases they where unable to find this viral signature, so little have been learned.

The search for pathogens using sequencing data from blood samples in patients with ALS has already been done, but as sequencing techniques can only read tiny fragments of DNA at the same time (the "reads"), they must recourse to reference genomes to succeed in a reconstitution, and this reconstitution does not allow the discovery of genomes are not included in the reference bases.

To complicate matters further, the readings belong to the patient and not to an hypothetical microbe or virus.

So when Melnick, Prudencio and his colleagues at Boulder and Jacksonville set out to design a new sequencing pipeline they made sure it does not ignore "reads" that cannot be aligned with a known genome.

The authors developed a bioinformatics pipeline that identifies microbial sequences in mammalian RNA-seq data, including sequences without significant nucleotide similarity results in GenBank.

They opted for a de novo assembly of unmapped reads into contigs, followed by aligning unmapped reads to these contigs for quantification. The code used in this manuscript is available at https://github.com/Senorelegans/MysteryMiner

A total of 120 whole blood RNA samples were initially used. It included 30 healthy controls (from the general population who do not have blood relatives with ALS), 30 pre-symptomatic C9ORF72 mutant carriers, 30 symptomatic cases of C9ORF72 ALS, and 30 cases symptomatic C9ORF72 negative ALS.

The efficiency of this pipeline has been tested by the authors on public RNA-seq data. The scientists then applied this pipeline to a new RNA-seq dataset generated from a cohort of 120 samples from patients and controls with amyotrophic lateral sclerosis (ALS), and identified sequences corresponding to bacteria and known viruses, as well as new virus-like sequences.

The complete dataset contains 8.64 X 109,406 combined reads. About 2.7% (2.34 X 10 ^ 8) of the reads did not match the human genome. From these non-host reads, 2,976,988 contigs were assembled and 17,047 BLASTN (regular biome) contigs were identified. A total of 25,815 contigs did not match by BLASTN and after filtering they identified 2,980 dark biome contigs (identified by BLASTX) and 859 double dark biome contigs (no BLASTX or BLASTN hit).

In the dark biome contigs, Melnick and his colleagues noted many contigs with a region of protein sequence similar to the RNA-dependent RNA polymerase (RdRP) of several RNA viruses, showing the greatest similarity to the virus. velvet tobacco marbling. This was present in the control as well.

RdRP is an essential protein encoded in the genomes of all viruses containing RNA without a DNA stage, that is to say RNA viruses including SARS-CoV-2.

To validate that this virus-like sequence was not a contig assembly artifact or a contaminant introduced during library construction or sequencing, the authors used RT-PCR of the original patient samples to demonstrate that this sequence was present in positive samples identified by RNA-seq analysis and not detectable in negative samples.

The scientists then investigated whether similar results would be obtained from other ALS data sets. To this end, they examined five other publicly available ALS datasets.

However, they found no statistically significant difference between samples from patients with ALS and control samples for virus / bacteria genus / species in normal / dark biome for any of the remaining ALS datasets.

Here we review recent publications about ALS and try to connect the dots between autophagy, insulin resistance, C9orf72, FUS, proteopathies, mitophagy and defective neuromuscular junction. It seems autophagy dysregulation is central to all those aspects of ALS.

TDP-43

Over the past decade, it has become increasingly clear that the most notable neurodegenerative diseases, such as ALS, FTLD, and AD, share a common prominent pathological feature known as TAR DNA-binding protein 43 (TDP-43) proteinopathy, which is usually characterized by the presence of aberrant phosphorylation, ubiquitination, cleavage and/or nuclear depletion of TDP-43 in neurons and glial cells. The role of TDP-43 as a neurotoxicity trigger has been well documented in different in vitro and in vivo experimental models.

There is increasing evidence that autophagy is defective in neurodegenerative disorders, including motor neurons affected in amyotrophic lateral sclerosis (ALS). Restoring impaired autophagy in motor neurons may therefore represent a rational approach for ALS. In this publication the clinically approved anti-hypertensive drug rilmenidine was used to stimulate mTOR-independent autophagy in double transgenic TDP-43WTxQ331K mice to alleviate impaired autophagy.

Although rilmenidine treatment induced robust autophagy in spinal cords, this exacerbated the phenotype of TDP-43WTxQ331K mice, truncated lifespan, accelerated motor neuron loss, and pronounced nuclear TDP-43 clearance.

Importantly, rilmenidine significantly promoted mitophagy in spinal cords TDP-43WTxQ331K mice, evidenced by reduced mitochondrial markers and load in spinal motor neurons. These results suggest that autophagy induction accelerates the phenotype of this TDP-43 mouse model of ALS, most likely through excessive mitochondrial clearance in motor neurons.

C9orf72

The coordinated activities of autophagy and the ubiquitin proteasome system (UPS) are key to preventing the aggregation and toxicity of misfold-prone proteins which manifest in a number of neurodegenerative disorders.

Both C9ORF72 and androgen receptors regulate autophagy, while their aberrantly-expanded isoforms may lead to a failure in both autophagy and the UPS, further promoting protein aggregation and toxicity within motor neurons and skeletal muscles.

In fact, autophagy and the UPS intermingle with endocytic/secretory pathways to regulate axonal homeostasis and neurotransmission by interacting with key proteins which operate at the NMJ.

FUS

The mechanism by which FUS affects the translation of polyribosomes has not been established. In a recent publication, the authors show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, they show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1.

Moreover, they report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin. The scientists also show that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.

Stress granules

It was recently reported that the stress granule (SG) protein Staufen1 (STAU1) was overabundant in neurodegenerative disorder spinocerebellar ataxia type 2 (SCA2) patient cells, animal models, and ALS-TDP-43 fibroblasts, and provided a link between SG formation and autophagy.

The authors demonstrate STAU1 overabundance and increased total and phosphorylated mammalian target of rapamycin (mTOR) in fibroblast cells from patients with ALS with mutations in TDP-43, patients with dementia with PSEN1 mutations, a patient with parkinsonism with MAPT mutation, Huntington's disease (HD) mutations, and SCA2 mutations.

Increased STAU1 levels and mTOR activity were seen in human ALS spinal cord tissues as well as in animal models. Changes in STAU1 and mTOR protein levels were post-transcriptional. Exogenous expression of STAU1 in wildtype cells was sufficient to activate mTOR and downstream targets and form SGs.

mTOR

The mTOR pathway is a central regulator of mammalian metabolism and physiology, with important roles in the function of tissues including liver, muscle, white and brown adipose tissue, and the brain, and is dysregulated in human diseases, such as diabetes, obesity, depression, and certain cancers.

As usual it must be underlined that mTOR is important for living beings, and simply inhibiting it is out of question. It would simply further starve motor neurons and exacerbate the disease as shown above.

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This book retraces the main achievements of ALS research over the last 30 years, presents the drugs under clinical trial, as well as ongoing research on future treatments likely to be able stop the disease in a few years and to provide a complete cure in a decade or two.

1 doi: 10.3390/ijms21114021. Cell-Clearing Systems Bridging Repeat Expansion Proteotoxicity and Neuromuscular Junction Alterations in ALS and SBMA Fiona Limanaqi 1, Carla Letizia Busceti 2, Francesca Biagioni 2, Federica Cantini 1, Paola Lenzi 1, Francesco Fornai 1 2 2 doi: 10.1016/j.nbd.2021.105359. Online ahead of print. Stimulation of mTOR-independent autophagy and mitophagy by rilmenidine exacerbates the phenotype of transgenic TDP-43 mice Nirma D Perera 1, Doris Tomas 1, Nayomi Wanniarachchillage 1, Brittany Cuic 1, Sophia J Luikinga 1, Valeria Rytova 1, Bradley J Turner 2 3 doi: 10.1074/jbc.RA120.013801. Epub 2020 Oct 20. FUS contributes to mTOR-dependent inhibition of translation Myriam Sévigny 1, Isabelle Bourdeau Julien 1, Janani Priya Venkatasubramani 1, Jeremy B Hui 1, Paul A Dutchak 1, Chantelle F Sephton 2 4 doi: 10.1002/ana.26069. Online ahead of print. Staufen1 in Human Neurodegeneration Sharan Paul 1, Warunee Dansithong 1, Karla P Figueroa 1, Mandi Gandelman 1, Daniel R Scoles 1, Stefan M Pulst 1

Amyotrophic lateral sclerosis is a non-cell autonomous disease, and motor neuron degeneration is modulated by intracellular and intercellular damages. Or at least this is what tells some scientists, indeed there is an abundance of proposal for ALS etiology and no consensus.

Another dissension point between ALS scientists is if the disease starts in the brain, or in a muscle. The former is the mainstream hypothesis. Both camps have proven again and again that their proposal was the right one.

A third mystery is that scientists almost never bothered to explore the most obvious manifestation of ALS: The muscle wasting.

With the amelioration of tools' performance, scientist's attention is turning to extra cellular vesicles.

Extra cellular vesicles

In the Central Nervous System (CNS), intercellular crosstalk happens among neurons, between neurons and glia or cells of the innate immune system, through different modalities, involving the release into the extracellular space of molecules such as neurotransmitters, neurotrophic factors, metabolites, and mutant proteins encapsulated or not in vesicles.

C9orf72, which presents aberrant hexanucleotide (GGGGCC) expansion in the non-coding region in ALS patients, regulates vesicle trafficking. Other proteins such as SOD1, TDP-43 or FUS are found in vesicles in ALS.

Where we discuss of muscles

Although much less studied than for motor neurons, abnormalities have been also described in skeletal muscle from ALS patients.

Accumulation of misfolded mutant proteins is observed in skeletal muscle.

In line with the pivotal role of defective mitochondrial respiratory chain and oxidative stress in ALS skeletal muscle, increasing levels of PGC‐1α, a transcription coactivator that promotes mitochondrial biogenesis, can improve muscle function even at late stages of the disease.

Skeletal muscle is a major site of glucose storage in the form of glycogen, which is transformed into ATP through glycolysis. The dysfunction of fast‐twitch type IIb myofibres in ALS is consistent with glucose intolerance and insulin resistance reported in ALS patients.

Myofibres from transgenic mice over expressing wild‐type TDP‐43 show impaired insulin‐mediated glucose uptake.

Does muscles kill motor neurons? In ALS, muscles are supposed to die from inactivity as motor neurons do not anymore activate them. A publication on MedRxiv proposes that it is actually the other way round: Muscles kill motor neurons. After all it is well known that many ALS patients were having intense sport activities. And an ALS-like phenotype was observed in mice when exogenous human mutant SOD1 expression was restricted to the skeletal muscle.

The authors of the pre-print, Laura Le Gall, Stephanie Duguez, Pierre Francois Pradat and colleagues, recall that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. So they hypothesized that muscle vesicles may be involved in ALS pathology.

An accumulation of multivesicular bodies was observed in muscle biopsies of 27 sporadic ALS patients.

Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing.

Compared to vesicles from healthy control myotubes, when administered to healthy motor neurons the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. enter image description here

This article may revolutionize the understanding of ALS' etiology.

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This book retraces the main achievements of ALS research over the last 30 years, presents the drugs under clinical trial, as well as ongoing research on future treatments likely to be able stop the disease in a few years and to provide a complete cure in a decade or two.

The insulin signaling pathway plays a crucial role in regulating the growth and metabolism of neurons. Deregulation of IGF-R signaling has been linked to a variety of neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's disease.

However, the role of insulin signaling in C9orf72 ALS / FTD is not yet clear. A positive correlation of the incidence of ALS with early-onset type 1 diabetes has been reported, and a decrease in insulin and IGF-1 in the blood and cerebrospinal fluid of patients with ALS, although the relevance of these findings to disease progression is unclear.

Intrathecal administration of IGF-1 improved motor performance, delayed disease onset, and prolonged survival in the SOD1G93A mouse model of ALS. However, three clinical trials of IGF-1 administered subcutaneously in ALS have reported conflicting results. The contradictory result of these trials may be due to insufficient administration of drugs to the brain and spinal cord and the fact that ALS has heterogeneous genetic risk factors.

Atilano, Isaacs, Partridge, and colleagues have shown in a recent article that insulin/IGF signaling is reduced in C9orf72 fly models using adult brain RNA sequencing. They further demonstrated that activation of insulin/IGF signaling can attenuate several neurodegenerative phenotypes in flies expressing expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR.

Poly-GR levels are reduced when components of the insulin/IGF signaling pathway are genetically activated in diseased flies, suggesting a rescue mechanism. This effect on poly-GR levels was confirmed in a mammalian cell model. Modulation of insulin signaling in mammalian cells also lowers poly-GR levels. Remarkably, the systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Their data suggest that modulation of insulin/IGF signaling may be an effective therapeutic approach against C9orf72 ALS/FTD.

PTEN acts as a tumor suppressor gene through the action of its protein phosphatase product. This phosphatase is involved in the regulation of the cell cycle, preventing cells from growing and dividing too quickly. It is a target of many anticancer drugs. Pten reduction has also been reported to reduce the toxicity of C9orf72 repeats expressed in a mammalian cell line, again in agreement with the results described by scientists and the potential therapeutic benefit of modulating this pathway. It is also interesting to note that the process of brain aging has been linked to decreased insulin signaling as well as impaired insulin binding, which may partly explain why aging is a problem. risk factor for the disease.

The authors found that intrathoracic insulin administration prolonged the survival of flies expressing G4C2 repeats. Although robust, the lengthening of the lifespan was relatively modest, which could be explained by the transient nature of the insulin treatment. Insulin and IGF-1 ligands have already been tested in neurodegenerative diseases.

Overall, the researchers' study suggests that modulation of the insulin / IGF signaling pathway could be an effective therapeutic intervention against hexanucleotide repeat extension associated with C9orf72 neurodegenerative diseases, with InR being a genetic modifier. It will be interesting in the future to study the need for downstream effectors of insulin signaling in the rescue of toxicity. It is important to note that in the Drosophila, there is a single insulin-like system that has the dual function of insulin / IGF signaling; thus, the mechanism of toxicity described in the researchers' model could also be linked to IGFs. Therefore, it will be important to test whether treatment with insulin or IGF can save the survival of other C9orf72 ALS / FTD vertebrate model organisms.

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This book retraces the main achievements of ALS research over the last 30 years, presents the drugs under clinical trial, as well as ongoing research on future treatments likely to be able stop the disease in a few years and to provide a complete cure in a decade or two.

It was found in 2009 that toxicity of SOD1 is secondary to a gain of toxic function rather than a loss of enzymatic function of the SOD1 enzyme, thus reducing levels of the mutant protein was predicted to slow progression in SOD1-linked ALS. Tofersen (formerly known as BIIB067), is an antisense oligonucleotide designed for adults with a confirmed superoxide dismutase 1 (SOD1) genetic mutation, a subtype of familial ALS that makes up 2 percent of all ALS cases. 

As reducing levels of the mutant protein is predicted to slow progression in SOD1-linked ALS, this antisense oligonucleotide mediates the degradation of superoxide dismutase 1 (SOD1) messenger RNA. As there is less SOD1 mRNA, there is less production of the SOD1 protein. This is not without risks in itself, SOD1’s role is to protect against anti-oxidants which are the normal by-product of cellular respiration.

Tofersen was developed in a long time partnership between Biogen and Ionis Pharmaceuticals.  With the time doses went from 3mg up to 100mg. They conducted a phase 1-2 ascending-dose trial evaluating Tofersen in adults with ALS due to SOD1 mutations. In each dose cohort (20, 40, 60, or 100 mg), participants were randomly assigned in a 3:1 ratio to receive five doses of Tofersen or placebo, administered intrathecally for 12 weeks.  The results were presented in July 2020.

The results were encouraging, but as for any ALS trial they are anything but stellar. Lumbar puncture-related adverse events were observed in most participants. Among participants who received Tofersen, one died from pulmonary embolus on day 137, and one from respiratory failure on day 152; one participant in the placebo group died from respiratory failure on day 52.

48 participants received all five planned doses. The technical indicators where better for participants having the higher doses. Patients taking the highest dose of Tofersen saw their ALS Functional Rating Scale score decrease by an average of 1.2 points, while the placebo group saw their score decrease by an average of 5.63 points.

Toby Ferguson the principal investigator acknowledged: “What we saw is a decline in ALS function, breathing and strength that was much greater in the placebo groups than in the treated groups. That said, if I were critical, I could poke holes in any one piece of those data, but we also have the data that SOD1 is being reduced.”

ALS is a difficult field in which drugs that show early promise can turn out to offer no benefit, or pose “unjustified risks” to patients in later studies. Biogen knows this as well as anyone, having seen its last ALS effort, dexpramipexole, fail in phase 3. Since then, the company has switched its focus from drugs with specific mechanisms, like anti-inflammatories, to medicines that target genetic mutations. Biogen strategy is  to learn more about the biology of the disease from looking at genetically defined populations (SOD1, C9orf72, ATXN2, XPO1) and then apply those findings to the broader population.

Despite those mixed results and as for other ALS drugs (Nurown, Cu(II)ATSM, Masitinib, AMX0035), there is an online petition for a compassionate access. Nearly 70,000 people have signed this online petition asking Biogen to provide an experimental ALS drug to a patient requesting it under compassionate use, but Biogen disagrees.

At the heart of Biogen’s denial is the question of broader access. The fastest way to get Tofersen to as many patients as possible would be regulatory approval, wrote Biogen chief medical officer, Maha Radhakrishnan, M.D., in a March 17 letter to an ALS patient, Stockman Mauriello that she shared with WCNC. That, of course, requires the completion of clinical trials.

“Providing individual access to Tofersen at this time could jeopardize access to Tofersen for hundreds of SOD1-ALS patients by impeding our ability to complete the study that will determine whether Tofersen is efficacious and safe and to seek subsequent regulatory approvals as quickly as possible,” Radhakrishnan wrote.

It's a moral position, it would not be fair to study participants who were randomized to placebo if Biogen were to offer the drug to patients not participating in the study.

However Biogen will provide early access to Tofersen only after study participants are no longer taking placebo.

“We cannot overlook these patients when considering questions of broader access, and cannot keep them on placebo while at the same time offering Tofersen to those outside of our study,” Sandrock wrote. “Offering Tofersen outside of the study would risk failing to complete the study and risk failing to obtain access for all SOD1-ALS patients".

“These patients agreed to participate in our study acknowledging the risk that they may not receive Tofersen, and with hope that Tofersen could be shown to work and be approved for all patients,” Sandrock wrote.

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