Searching for a TDP-43 therapy

Genetic therapies against TDP-43 aggregates had been proposed for ALS disease.

While this is certainly feasible with the current state of art (see my book), it would certainly be sold at a cost similar to Zolgensma, because of the lack of competition in the field.

The classical drug which complies with the "Pfizer rule of five" is a peptide, so a peptide that would remove TDP-43 would be desirable. Peptides are low cost and easy to procure.

Improving an already published peptide

Several scientific articles [0] are suggesting that a peptide might help in ALS by removing TDP-43 from mitochondria.


While a patent was written in 2016 (and still valid for 2019) the main scientist lost faith in it as it is very toxic.

The proposed peptide is formed by assembling a M1 section from TDP-43 with a TAT peptide.
* The M1 section is: FPGACGL
* The M3 section is: GFGFV
* Tat is a regulatory protein that drastically enhances the efficiency of viral transcription, for example in HIV with a transition to the most dangerous form of AIDS (T-tropic).

Bioinformatics tools suggest that its biological function as a neurotoxin which blocks acetylcholine receptors.

An improved peptide

If we use a peptide made simply with the M1 section followed by the M3 section.

This peptide, FPGACGLGFGFV, is predicted by bioinformatics tools has not being a toxin, and not an antigen.

Therefore it seems safe (but more tests are needed), while retaining the two sections that are told as helping in removing the TDP-43 from mitochondria.

However a Blast test shows it is very close to other human proteins so it certainly have side effects. It should also be designed to able to traverse the blood-brain barrier, as well as entering cells.

Conclusion: While a lot of work is still necessary to design a peptide without side effects, some interesting results have already being achieved: A peptide with a design similar to peptides able to remove TDP-43 from mitochondriais designed. This peptide is probably not toxic, but probably with side effects. Its effectiveness to traverse the BBB was not tested with bioinformatic tools.

Tool for testing neurotoxin function:
Tool for testing immunogenicity:
Blast on human genome:

[0] Including: The Inhibition of TDP-43 Mitochondrial Localization Blocks Its Neuronal Toxicity Article in Nature medicine · June 2016 DOI: 10.1038/nm.4130


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.

There is many questions about how TDP-43 can be deleterious in ALS disease. Normally TDP-43 is involved in many repairing or protecting scenarios. In 2013 scientists proposed that misplaced TDP-43 was killing mitochondria, by disturbing their fission/fusion processes (mitochondria are very dynamic structures). However this is not the scientific consensus.

A scientific article published on October 30, 2019 about Alzheimer's disease confirms the effect of a peptide against the aggregation of TDP-4 in mitochondria. This peptide and others were already described in a 2016 ALS publication.

The molecular mechanisms by which TDP-43 contributes to the pathology of ALS remained elusive. In the 2016 article, the authors wrote that they found that TDP-43 accumulated in neuronal mitochondria in subjects with ALS or frontotemporal dementia. Neurodegenerative diseases are characterized by cytoplasmic localization of TDP-43 in granule types. The 2016 study directly linked the toxicity of TDP-43 to mitochondrial metabolism and proposed targeting the mitochondrial localization of TDP-43 as a promising therapeutic approach for ALS.

The authors of the 2019 study (Gao et al.), They, demonstrate that one of the two mitochondrial TDP-43 inhibitory peptides of the 2016 article, when administered late in the course of the disease, may attenuate the development and progression of cerebral neuronal loss and behavioral deficits in the 5XFAD transgenic mouse model in Alzheimer's disease.

If this peptide is effective against TDP-43 proteininopathies, it is a real breakthrough because a peptide is something that is easy to produce at a low cost.

In neurodegenerative diseases, TDP-43 is localized in the cytoplasm as well as in mitochondria that may be free in the cytoplasm or anchored in the endoplasmic reticulum, where it gives it the "raw" appearance of the endoplasmic reticulum.

TDP-43 or truncated forms of TDP-43 may be present inside or outside the mitochondria. The portion of the total length of TDP-43 within the mitochondria can bind to the subunits encoding the mitochondria-mediated messenger RNA (mRNA), whereas the truncated TDP-43 lacks the locating sequence. mitochondrial M1 is limited to the inner membrane space no effect on ND3 / 6 expression or mitochondrial function.

The mitochondrial localization of TDP-43 is dependent on its M1 motif, the deletion of which suppresses its mitochondrial accumulation with no significant effect on its half-life, dimerization, functional binding to mRNA targets and cytosolic TDP-43 expression. , nuclear or total. The PM1 synthesized peptide (YGRKKRRQRRRAQFPGACGL) in which the M1 motif was fused to the TAT peptide (GRKKRRQRRR), competitively inhibits the mitochondrial localization of TDP-43 and suppresses the TDP-43 induced toxicity on mitochondria and non-influencing neurons. on TDP-43 expression neurons

The authors used PM1, a peptidic inhibitor derived from TDP-43, as a continuous injection, to specifically reduce its expression in mitochondria. PM1 abolished TDP-43 protein kinetics, reversed neuronal loss, and reduced neuroinflammation in aged 5XFAD mice long after symptom onset. Since the amyloid plaque load was not attenuated or prevented by PM1, the authors' results clearly indicate that TDP-43 in mitochondria does not affect the pathology of Aβ.

Chronic administration of the PM1 peptide significantly attenuated TDP-43 protein kinetics, mitochondrial abnormalities, microgliosis, and even neuronal loss, but was without effect on amyloid plaque load in 12-month-old 5XFAD mice well after the onset of symptoms. PM1 also improved cognitive and motor functions in 12-month-old 5XFAD mice and completely prevented the development of mild cognitive impairment in 6-month-old 5XFAD mice.

Beyond its involvement in Alzheimer's disease, this article corroborates the 2016 article on ALS and therefore offers hope that a continuous (insulin pump-like) delivery of a low-cost peptide could to be very beneficial for ALS.


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 and cancer

There is a persistent mystery about the causative mechanisms of ALS. The intense work over the last two decades on SOD1 has not helped to conclusively understand its link with the disease. Many SOD1 mutations produce very similar ALS phenotypes. But these mutations have not prevented neurons from functioning properly for several decades, so it is difficult to invoke them to explain the onset of the disease. Even though there is less scientific work on FUS or TDP-43, as their discovery is more recent, the mystery is also complete on how a non-mutated and mis-located TDP-43 protein in the cytoplasm could kill a neuron. The only obvious case is that of C9orf72, where the dipeptide repeats, clearly could not produce functional protein. However, even in this case, it is unclear why ALS only occurs at an advanced age.

PARP is involved in DNA repair

There is a troubling link between cancer and ALS, for example, there is an inverse relationship between the onset of cancer and the onset of ALS. ALS medications also have anti-cancer properties. So, perhaps it's not surprising that they can share a fundamental cause: defects in DNA repair mechanisms. Poly-ADP-ribose polymerases (PARPs) are involved in DNA repair, as are FUS or TDP-43.

During DNA damage or cellular stress, ** PARP ** is activated, resulting in an increase in the amount of poly-ADP-ribose and a decrease in the amount of NAD +.

enter image description here

The poor localization of FUS and TDP-43 in the cytoplasm inhibits the mechanism of DNA repair

FUS and TDP-43 both play a role in the treatment of RNA, including splicing, transcription and transport. The involvement of FUS and TDP-43 in the response to cell genome damage has recently been discovered. In healthy neurons, FUS protects the genome by facilitating dependent recruitment of ** PARP-1 **. The authors report that TDP-43 is an essential component of the end-junction-mediated double-stranded DNA (DNA) repair pathway (NHEJ). TDP-43 is rapidly recruited to double-stranded DNA sites to stably interact with DDR and NHEJ factors, acting in particular as a scaffold for recruitment of the isolating XRCC4-DNA ligase 4 complex at DSB sites. Indeed, the presence of fragmentation of TDP-43 and its aggregation in ALS samples is strongly correlated with the presence of ** PARP-1 ** and cleaved caspase-3.

During apoptosis, PARP moves to the cytoplasm

Caspases are a family of cysteine ​​proteases that play an essential role in programmed cell death. This protease cleaves ** PARP-1 ** into two fragments, leaving it completely inactive to limit the production of poly-ADP-ribose. One of its fragments migrates from the nucleus to the cytoplasm and is considered a target of autoimmunity. At the beginning of 2019, dysregulation of PARylation was found to contribute to the pathogenesis of ALS by promoting protein aggregation.

Although PARylation occurs primarily on PARP proteins, the association of PAR with ALS-related granules has been observed.

Causal chain of ALS

The results of the scientists thus link the pathology of TDP-43 to altered repair of DSB and persistent DDR signaling in motor neuron diseases, and suggest that targeted therapies on double-stranded DNA repair could improve genome instability induced by the toxicity of TDP-43 in motor neuron diseases.

In summary the mechanism causing TDP-43 ALS would be:

  • Mutations of FUS or TDP-43 would render DNA repair ineffective.
  • The intervention of PARP would repair this DNA and relocate TDP-43 in granules in the cytoplasm.
  • This would further aggravate the problems of DNA repair.

A possible therapeutic mechanism

These new findings provide insight into how a DNA repair defect may be associated with FUS and / or TDP-43 neurodegeneration, and raises the question of whether the resolution of DNA ligation problems would be a pathway. promising for the development of neuroprotective treatments.

So mechanisms that would alleviate the burden of PARP (which is different from inhibiting it), would improve the pathology.


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.

What is interleukin 6?

Interleukin 6 (IL6) is a potent pleiotropic cytokine that regulates cell growth and differentiation and plays an important role in the immune response. Deregulated IL6 production is implicated in the pathogenesis of many diseases, such as multiple myeloma, autoimmune diseases and prostate cancer. In addition to other functions, interleukin 6 (IL-6) is involved in the development of immunological and inflammatory reactions. Autoimmune diseases such as rheumatoid arthritis are associated with abnormally high levels of IL-6.

enter image description here

How does it work?

IL-6 had previously been classified as a proinflammatory cytokine, but the anti-inflammatory (beneficial) effects of myokines in general of interleukin-6 of muscle origin are now recognized. So we have a cytokine that can have two modes, one beneficial, the other deleterious, how is that possible?

The explanation could be that the signaling pathways upstream and downstream of IL-6 differ markedly between myocytes and macrophages. It appears that unlike IL-6 signaling in macrophages, which depends on activation of the NFκB signaling pathway, intramuscular IL-6 expression is regulated by a signaling cascade network, including Ca2 + / NFAT and glycogen / p38 MAPK pathways.

IL6 has 2 signaling paradigms: IL6 signaling and IL6 signaling. Although conventional IL6 signaling occurs via IL6 receptors bound to the membrane, IL6 retransformation is induced by a systemic and localized increase in the extracellular soluble IL6 receptor (sIL6R). generated by proteolytic cleavage, called "shedding," of the receptor from the cell surface. These soluble receptors can be activated by IL6 and activate signaling cascades. Thus, IL6 trans-signaling activates the IL6 signaling pathways in cells that do not express the IL6 receptor.

Are there different reactions to IL6 in humans?

In humans, there are at least two alleles for the IL6 receptor (Asp358Ala, A / C, rs2228145), the A allele (Asp358) being the main allele and the C allele (Ala358), the variant allele. The expression of the IL6 receptor (IL6R) is favored by the C allele. In individuals with IL6R allele, increased receptor expression improves both localized and systemic IL6 transsignalization in the presence of IL6. This allele is associated with certain diseases such as asthma.

Why would IL6 have an interest in treating ALS?

Perhaps because a patient with ALS was reported to have had a remission in 2014 by consuming lunasin, a soy peptide, researchers have wondered whether IL6 transsignalization could play a role. potential in ALS.

How did the scientists proceed?

IL6 and sIL6R levels were measured in samples in a cohort of patients with ALS and compared to healthy patients. Their results suggest that the IL6R C allele influences IL6 signaling in the central nervous system of patients with ALS. In a second cohort of ALS subjects with more definite clinical data, the presence of the IL6R C allele was associated with a more rapid progression of the disease. These results suggest that identifying patients with the IL6R C allele may provide useful information for predicting disease progression and identifying those who may benefit most from IL6R blocking therapies.

What happened in 2014

ALS experts will recall that in 2014 Mike McDuff, who has ALS, experienced dramatic improvements in speech, swallowing and strength in ALS. lunasin.

Dr. Bedlack from SLA Duke Clinic confirmed that Mike McDuff's symptoms had actually improved dramatically. A clinical trial was then conducted to evaluate the interest of lunasin in the case of ALS. Fifty people with ALS were put on the diet containing exactly the lunasin Mike McDuff had followed and were followed for one year. The clinical trial was completed in September 2017. Unfortunately, there is no evidence that lunasin slowed, stopped, or reversed ALS in clinical trial participants. Gastrointestinal adverse events were more frequent than expected in the trial participants, including cases of constipation severe enough to warrant hospitalization.


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.

I did published a book on ALS research:

Caveats: I am not a doctor, nor a scientist and English is not my mother-tongue.

Here are some take home points:

  • Scientists are obsessed by SOD1 (2% of all ALS cases) as a model for ALS. However there is overwhelming evidence this is a fruitless pursuit.

  • There are nearly no treatments:

    • For all pALS, a very imperfect treatment is Nurown, but it exists!
    • For SOD1 pALS (2% of all cases), there are two treatments that are in clinical trials.
    • For the other (98%) pALS there are no drugs in the pharmaceutical pipeline. However for most pALS (TDP-43 / 95% of all cases) there are genetic therapies that have recently been published by scientists, but if no one tries to defend them, it will take another 10 years before they are marketed.
  • The ALS research is bizarre, scientists often contradict colleagues but nobody seems to care. The consensus still cites theories that have been disproved since decades, like glutamate excitotoxicity. ALS is certainly not one homogeneous disease, but it is still treated as such by scientists. Animal models of ALS have little value in translation of drugs to humans, but moreover often ALS research is done on insects (that have an exoskeleton), or even on unicellular organisms. There is no formalism anywhere, little effort to falsify any thesis.

What can you expect to find in this book:

  • A brief description of ALS and its common variants (PLS, PMA, etc): ~7 pages

  • A description of the cell in general, from an ALS point of view : ~15 pages

  • A strong focus on the neuronal cells, again with ALS in mind: ~34 pages

  • The main themes in ALS research (dying forward, excitotoxicity, virus, etc): ~40 pages

  • Main achievements of ALS research (SOD1, TDP-43, discovery, etc): ~113 pages

  • A focus on clinical trials and 28 drugs: ~37 pages

  • Different kind of therapies (MSC, ASO, etc): ~20 pages

  • A possible new therapy for ALS (if only a company had the will to investigate it!): ~20 pages

  • Futures therapies that are researched now (creating or grafting new neurons): ~17 pages

This is not an easy read, so I tried to explain terms, provide a large section on the neuronal cell at the beginning, and wrote 276 footnotes.

There are no speculations, nor pseudo scientific babble. I am not overly kind either with ALS scientists, clearly they can do much better.

Jean-Pierre Le Rouzic

My book on ALS research

Extracellular mitochondria and their impact on neurons

Mitochondria are frequently exchanged between cells and must change their shape accordingly to suit their environment. "Most scientists believe that mitochondria outside cells must have come from dead or dying cells," said Mochly-Rosen, who has just published an article in Nature Neuroscience. "But we found a lot of highly effective mitochondria in the culture broth, as well as some that were damaged, and the glial cells that release them seem very alive."

As recently discovered, even healthy cells regularly release mitochondria into their immediate environment.

An enzyme that destroys mitochondria

An enzyme called Drp1 that facilitates mitochondrial fission can become overactive because aggregates of neurotoxic proteins such as those associated with Alzheimer's, Parkinson's or Huntington's disease, or amyotrophic lateral sclerosis.

A fragment of protein that specifically blocks mitochondrial fission

About seven years ago, the Mochly-Rosen team designed a protein fragment, called the P110 peptide, that specifically blocks Drp1-induced mitochondrial fission when it occurs at an excessive rate, as it is the case when a cell is damaged.

Mitochondria and immune system

The relationship between mitochondria and eukaryotes has been critical to the success of metazoan life on Earth. Cellular colonization by ancestral α-proteobacteria more than a billion years ago provides benefits in terms of energy production and oxygen utilization. However, host cells needed to recognize and protect their increasingly essential endosymbioli while simultaneously identifying and repelling phylogenetically related pathogenic bacterial invaders. As a result, mitochondria have become immunologically preferred.

Nevertheless, misidentification of extracellular mitochondrial DNA, damaged mitochondria, or other damage-related molecular structures (DAMP) as a bacterium can trigger innate (sterile) immune mechanisms that in turn contribute to mitochondrial dysfunction. the spread of pathology in acute and chronic inflammatory diseases.

Loss of the immune privileged state is correlated with mitochondria damaged by microglia

Their results showed that the loss of the immune privileged state of extracellular mitochondria was correlated with an increased release of mitochondria damaged by microglia, and that the extracellular mitochondria damaged directly contributed to the spread of the disease by acting as the innate immune response by targeting adjacent astrocytes. and neurons.

An increase in Drp1 - Fis1 - mediated mitochondrial fission in activated microglia triggers the formation of fragmented and damaged mitochondria that are released from these cells, thereby inducing an innate immune response.

Fragmented mitochondria are biomarkers of neurodegeneration

Clinical and experimental studies have identified fragmented mitochondria in the biofluids of patients with subarachnoid hemorrhage and stroke patients, suggesting that their presence in the extracellular space is a biomarker of neurodegeneration and neurodegeneration. the severity of the disease. Their data showed a causal role of dysfunctional extracellular mitochondria in the propagation of neurodegenerative signals from microglia. Innate immune responses in neurodegenerative diseases begin early in the pathogenesis of these diseases and are associated with minimal, if any, infiltration of immune cells derived from blood in the brain. Resident brain cells, microglia and astrocytes, trigger this sterile immune response, contributing to neuronal dysfunction and degeneration.

P110 peptide reduces the release of damaged mitochondria from microglia

The authors have previously reported that neurons harbor neurotoxic proteins. Their data showed that the Drp1-Fis1 inhibitory peptide P110 reduces mitochondrial fission and subsequent release of damaged mitochondria from microglia, thereby inhibiting astrocyte activation and protecting neurons from innate immune attacks.

A vicious circle leads to neurodegeneration

Their data suggest instead that a relay of glie-neuron-to-glia signaling plays an important role in neurodegeneration. By fueling the vicious circle, neurotoxic protein-induced neuronal death generates additional cellular debris and debris (DAMP), as well as dysfunctional mitochondria released by microglia expressing neurotoxic proteins, exacerbate astrocyte activation. and chronic pathogenic inflammation.

Thus, neuronal cell death and the final phenotype of the disease occur via the activation of the innate immune response as well as via the direct effects of neurotoxic protein-induced cell death.

Activation of the innate immune response and neuronal protein-induced neuronal cell death in neurodegenerative disease models are both dependent on excessive Drp1-Fis1-induced mitochondrial fragmentation.

The minimal amount of damaged mitochondria required for the propagation of neuronal cell death is also unknown, and the transfer of functional mitochondria between microglia and astrocytes and between glia and neurons plays a role in physiological conditions. However, researchers know that extracellular mitochondria are essential for mediating this pathological pathology from cell to cell.

The ratio of damaged mitochondria to functional mitochondria in the extracellular medium determines the fate of neurons. Although damaged extracellular mitochondria are deleterious, functional mitochondrial transfer is protective, as previously demonstrated, for example in a murine model of acute lung injury and in a stroke model. The question of whether extracellular mitochondria damaged enter the neurons, as suggested for functional mitochondria in a previous study, has not yet been determined.

It is not the amount of extracellular mitochondria but rather the ratio of damaged mitochondria to functional mitochondria in the extracellular environment that governs the outcome of neurons and is determined by the extent of pathological fission in the microglia donor.

A slow path to developing a drug

Their data suggest that selective inhibition of pathological mitochondrial fission in microglia (mediated by Drp1 - Fis1) without affecting mitochondrial physiologic fission reduces the propagation of neuronal injury by two mechanisms

First, P110 reduced activation of the innate immune response in microglia and astrocytes and cytokine-induced neuronal cell death induced by extracellular and dysfunctional mitochondria.

Second, the inhibition of pathological mitochondrial fission by P110 in donor microglia contributed to neuronal cell survival by increasing the ratio of healthy mitochondria to damaged ones released by donor cells, thereby protecting neurons.

Suppression of DrP1 - Fis1 mediated mitochondrial fission is an easily translatable approach to interrupting this pathogenic microglia-to-astrocyte-to-neuron mitochondrial pathology, and promoting the transfer of healthy mitochondria to neurons.

However, they consider that any means of normalizing the balance between healthy and damaged mitochondria within the neuronal environment, for example by removing damaged and fragmented mitochondria with specific antibodies or by introducing healthy mitochondria, could also provide neuronal protection in neurodegenerative diseases.

Article from Nature Neuroscience: Fragmented mitochondria released from microglia trigger A1 astrocytic response and propagate inflammatory neurodegeneration


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.

Study of Edaravone in ALS Korean patients.

- Posted in English by

Purpose of this study

Edaravone was approved as a therapeutic drug against ALS in June 2015 in Japan and by the Korean Ministry of Food and Drug Safety in December 2015.

In this observational study, on ALS patients in the Korean population, patients treated with edaravone showed modest results on ALSFRS-R and lung function tests. Several previous studies on edaravone also reported quite low results in the treatment of ALS.

Results of this new study on Edaravone

The phase 3 clinical trial on edaravone showed an average decrease in ALSFRS-R of 5.01 points in 6 months in the treated group, and an average decrease of 7.50 points in 6 months in the control group.

The patients involved in this study showed an average decrease of 5.75 point.

The initial characteristics of ALS patients included in this study had an average ALSFRS-R of 34.25 and an average CVF of 75%, reflecting a more advanced stage of ALS patients in this new study compared to patients in the recent trial. phase 3 clinical trial.

It should be noted that a recent study of advanced ALS patients with a FVC of less than 60% showed no benefit of edaravone, reflecting the importance of early intervention in the treatment of ALS patients. The study in Korean patients also showed some efficacy in ALS patients with a mean CVF score of 75%.

In the present study, the results also indicate that the reduction is not limited to a specific area, but also relates to different areas of ALSFRS-R.

Castillo-Viguera et al. have suggested that removal of more than 20% of ALSFRS-R is clinically significant; The phase 3 clinical trial on edaravone had shown a 33% decrease in progression, but the present study showed only a slower progression of 23% after 6 months.

Adverse effects of Edaravone

Edaravone is known to cause frequent side effects, in up to 84% of patients. The most common side effects are bruising, constipation, contact dermatitis, dysphagia, eczema and inflammation of the upper respiratory tract (in order of decreasing frequency); 16% of patients experienced serious adverse events. In the present study, two patients presented with eczema and pruritus, which were well tolerated with oral antihistamine and steroidal therapy. Transient leukopenia should also be noted in a patient who has recovered after a few days of initial treatment. No deaths were encountered during the follow-up period.

The limitations of the present study are as follows. The study was observational, with no control group for comparison. The small number of patients recruited must be taken into account in the evaluation of the results.


This is a study of Korean patients on the open-label study of edaravone in patients with ALS. The treatment was well tolerated without significant adverse events. Consistent with previous studies in Japan, the United States and Europe, the present study shows that the treatment was well tolerated and showed only a slight improvement at a later stage of ALS.

The study is available here:


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.

An inhibitor of RPK1 has been tested for safety in healthy people

Why take an interest in RPK1?

Serine / threonine protein kinase 1 (RIPK1) interacting with receptors is an intracellular protein involved in the regulation of inflammation and cell death. RIPK1 is activated in response to several inflammatory stimuli, including tumor necrosis factor alpha (TNF-α) signaling by the TNF 1 receptor. When activated, RIPK1 elicits multiple cellular responses, including cytokine release, microglial activation, and necroptosis, a regulated form of cell death.

The early role of RIPK1 in this signaling cascade led to the hypothesis that inhibition of RIPK1 signaling could be beneficial in diseases characterized by excess cell death and inflammation such as amyotrophic lateral sclerosis (ALS).

Indeed, inhibition of RIPK1 activity has been shown to protect against necroptotic cell death in vitro over a range of cell death models (see below).

In animal models of diseases ranging from ulcerative colitis to multiple sclerosis, inhibition of this pathway protects against pathology and cell death. These non-clinical findings, coupled with observations of increased activity of RIPK1 in human diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and multiple sclerosis, suggest that inhibition of RIPK1 could be beneficial in many different chronic diseases.

What problems are there with RPK1 inhibitors?

Inhibitors of RIPK1 are currently being evaluated as treatments for systemic inflammatory diseases, including inflammatory bowel disease and psoriasis, but there is no evidence that previously studied inhibitors in humans enter the system. central nervous system (CNS). To evaluate the potential for inhibition of RIPK1 as a therapeutic for chronic neurodegenerative diseases, it is necessary to study the pharmacokinetics (PK), pharmacodynamics (PD) and safety profile of a molecule capable of entering in the CNS at effective concentrations.

DNL104 is a selective inhibitor of CNS penetrable RIPK1 activity developed by Denali Therapeutics as a potential treatment for neurodegenerative disease. Denali, a CNS biotechnology company, is made up of veterans from Genentech, and joined the RIK1 program in 2016 with the acquisition of Incro Pharmaceuticals. Sanofi paid $ 125 million (€ 110 million) by the end of 2018 for participation in two developing RIPK1 inhibitors in Denali. The agreement covers small molecules designed to treat several neurodegenerative and systemic inflammatory diseases.

What is the current knowledge on the subject?

Inhibition of phosphorylation of RIP K 1 shows protection against pathology and inflammation in vitro and in animals, induced by various challenges, including in animal models with CNS disease (AD and ALS).

What question did this study address?

The safety, tolerability, pharmacokinetic, and pharmacodynamic effects of the CNS-penetrating RIP1 kinase inhibitor D NL104 were tested in randomized, placebo-controlled, increasing dose placebo-controlled trials.

What does this study add to our knowledge?

The results show that DNL104 inhibits phosphorylation of RIPK1 in healthy healthy volunteers with no effect on central nervous system safety, but liver toxicity issues have been raised in the multiple-dose-escalation study, in which 37.5 % of subjects (6 subjects) developed high liver function tests. related to the drug, of which 50% (3 subjects) were classified in the category inducing a drug-induced liver injury (DILI).

Why focus on necroptosis?

In 2014, we knew for a long time that the origin of ALS was not in motor neurons, but in other cells. But 8 years after the discovery of TDP-43 and 3 years after the discovery of C9orf72, most knowledge about the mechanisms of motor neuron degeneration in ALS still came from studies on SOD1-type mouse models. A clear conclusion from these studies is that non-neuronal cells play a critical role in the neurodegeneration related to SOD1 mutations. Indeed, the presence of healthy glial cells significantly delayed the onset of motor neuron degeneration, increasing the life without disease by 50%.

Since the work of the Jean-Pierre Julien Group in 2005, it has been suggested several times that interneurons, myelinating Schwann cells of the peripheral nervous system and endothelial cells of the vascular system could be at the origin of ALS. But other studies have suggested instead that astrocytes could cause spontaneous degeneration of motor neurons. For example, in 2003, researchers led by Don Cleveland of the University of California at San Diego involved astrocytes in motor neuron death, showing that administering SOD1 to these non-neuronal cells still resulted in motor neuron disease.

Agnostic research on the cause of ALS

Usually when a scientist decides to set up an experiment, he wants to test a hypothesis. The hypothesis itself is based on a model of the disease. A new trend in biology is to do research without having a preconceived idea (the model of the disease). It is believed that this is a difficult way to achieve results that could not have been achieved by conventional procedures.

In order to determine whether astrocytes from sALS patients can kill motoneurons independently without being exposed to SOD1, the Przedborski group decides to study the mix of different types of cells after they have been exposed to ALS, without prejudging of what causes ALS. For that they decide to design "their" in-vitro model of ALS. This well-cited article (100 times), however, contradicts many other studies.

Diane Re and Virginia Le Verche isolate astrocytes derived from post mortem motor cortex and spinal cord tissue from six SALS patients and 15 controls. They realize that after one month of culture, astrocytes have dominated other cultures. The researchers then mixed these astrocytes with motor neurons derived from human embryonic stem cells. While neurons thrived when co-occurring with non-sALS control astrocytes, their number began to fall after only four days of culturing with sALS astrocytes. All of this clearly shows that astrocytes from SALS patients specifically kill motor neurons, unlike control astrocytes.

However, other types of neurons than the motoneurons were resistant to the deleterious signals delivered by sALS astrocytes, and the fibroblasts of sALS patients also did not destroy the motoneurons, indicating that the toxic relationship was astrocyte-specific. and SALS motor neurons. To determine the role of SOD1 the researchers inhibited the expression of this protein in astrocytes using four small hairpin RNAs. The treatment failed to protect the motor neurons. The decrease in TDP-43 expression in astrocytes did not save them either.

Controversial research

These results contradict a study conducted by a team of Brian Kaspar, who found that astrocytes derived from neural progenitor cells taken from sALS patients needed SOD1 to destroy motor neurons, even though sALS patients showed no evidence of mutation of this gene (Haidet-Phillips et al., 2011). But in 2014, in the same issue as the publication of the Przedborski group, the Haidet-Phillips group publishes an article1 that is very similar to that of the Przedborski group, except that it incriminates NF-κB and therefore a mechanism for apoptosis rather than necroptosis, but in any case SOD1 is no longer supposed to be the primary cause of ALS.

For this team the inactivation of SOD1 in human astrocytes of patients with SALS does not preserve the motor neurons. How ALS astrocytes become toxic remains completely obscure. No known ALS-related mutations were identified in their samples and yet the toxic phenotype persisted even after several passages of adult astrocytes in culture. The authors suggest that necroptosis is the dominant mode of cell death in their in vitro model of sALS.

In 2019 it is difficult to say who is right between all these contradictory studies. Apoptosis and necroptosis are major mechanisms of cell death that usually result in opposite immune responses. Apoptotic death usually leads to immunologically silent responses, while death by necroptosis releases molecules that promote inflammation, a process called necrosis.


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 drugs that may enter a phase III trial in 2019


Arimoclomol is an experimental drug developed by CytRx Corporation. Arimoclomol is believed to function by stimulating a normal cellular protein repair pathway through the activation of molecular chaperones. Since damaged proteins, called aggregates, are thought to play a role in many diseases, CytRx believes that arimoclomol could treat a broad range of diseases.

Heat shock transcription factor 1 (HSF1), a central coordinator of the chaperone response, helps cells managing proteins that are misfolded or aggregated. HSF1, which is activated during times of stress, switches on multiple genes, including the gene encoding the disaggregase Hsp40.


NurOwn is an experimental cell-based therapy by BrainStorm Cell Therapeutics that contains autologous cultured mesenchymal bone marrow stromal cells secreting neurotrophic factors including BDNF, GDNF and HGF, as a possible treatment for patients with ALS.

Brainstorm’s NurOwn therapy consists of bone marrow stem cells taken from each individual person, differentiated into cells that make neuroprotective growth factors, and infused back into muscle or the spinal cord.

Brainstorm’s therapeutic differs from that of the better-known Neuralstem, Inc., of Rockville, Maryland, which is transfusing neural stem cells from fetal tissue into the spinal cords of people with ALS.

Though it has great potential for clinical applications, the differentiation of MSCs is precisely regulated and coordinated by mechanical and molecular signals from the extracellular environment and involves complex pathways at the transcriptional and post-transcriptional levels that remain largely unexplored.

MSC-NTF cells are Mesenchymal Stromal Cells (MSC) induced to express high levels of neurotrophic factors (NTFs) using a culture-medium based approach.


ODM-109 aims in part, to improve breathing in ALS by improving the performance of muscles in the diaphragm. The drug candidate, also known as oral levosimendan, increases the force of contraction of certain muscles by boosting the calcium sensitivity of troponin C.

The drug candidate is an oral formulation of levosimendan. An intravenous formulation of levosimendan, marketed under the name Simdax, is clinically approved in some countries for the treatment of acute heart failure. Levosimendan's positive inotropic and vasodilator effects are tied to its abilities to increase calcium sensitivity and open ATP-sensitive potassium positive ion (K+) channels (mitoKATPchannels)

Levosimendan favourably affects mitochondrial adenosine triphosphate synthesis, conferring cardioprotection and possible neuronal protection during ischemic insults. In a model of spinal cord injury, levosimendan has been reported to attenuate neurologic motor dysfunction. This finding is supported by the fact that the selective mitoKATPchannel opener, diazoxide, is an effective neuroprotectant, as has been demonstrated in an ischemia reperfusion study in rats.


IONIS-SOD1Rx is a generation 2.0 antisense drug specifically designed to inhibit production of mutant superoxide dismutase (SOD1). SOD1 mutations account for approximately 20% of familial ALS cases.

This drug is the result of a collaboration between Biogen and IONIS Pharmaceuticals (formerly ISIS). A Phase III clinical trial is recruiting participants as of April 2019. Study completion is expected in May 2020.

Researchers are also considering antisense treatment for another genetic form of ALS caused by expansions in the C9ORF72 gene. Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. The antisense oligonucleotide works by targeting and attaching itself to the stretch of RNA with the mistake so that the protein cannot be formed,, so prevent accumulation of these outside the nucleus.

Part of Isis’ success stems from chemically modifying oligonucleotides to make them last longer in the body and bind more tightly to their target RNAs.

SOD1Rx, like Kynamro, includes 2′-O-methoxyethyl sugars on its backbone. This modification typifies Isis’ second generation of oligonucleotide chemistry, but the company has developed other options. For SOD1 antisense, they plan to make it more potent before starting further safety trials, probably with higher doses and longer treatment times.


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.

Split hand syndrome and ALS

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What is the split hand syndrome?

Many scientists subscribe to the “dying back” hypothesis, whereby degeneration begins at the neuromuscular junction when motor neurons retreat from the synapse. A few others, prefer the “dying forward” or upper motor neuron hypothesis. They believe ALS begins in the brain, before spreading to lower motor neurons. In medicine, the split hand syndrome is a neurological syndrome in which thumb hand muscles undergo mass loss, while the muscles on the side of the little finger are spared. This makes it difficult to grab small objects between thumb and forefinger. If there are no lesions affecting the branches of the ulnar nerve that are directed to the unused muscles, it is almost certain that the lesion is located in the anterior horn of the spinal cord at C8-T1.This area is often associated with ALS and is the place where higher motor neurons join lower motor neurons. This syndrome has been proposed as a relatively specific sign of amyotrophic lateral sclerosis, but it can also occur in other anterior horn disorders, such as spinal muscular atrophy, Charcot-Marie-Tooth disease, poliomyelitis and progressive muscle atrophy. The phenomenon is observed in more than half of ALS patients, and the underlying mechanism is not fully understood. To a certain extent, these characteristics can also be observed during normal aging. The term split-hand syndrome was coined for the first time in 1994 by a Cleveland Clinic researcher named Asa J. Wilbourn.

How does this syndrome relate to ALS?

Composite motor action potential (CMAP) is an electromyographic study (electrical study of muscle function). Several studies have shown a significant reduction in the amplitude of motor action potentials during low frequency repetitive nerve stimulation (RNS) of muscles involved in ALS.

The motor plate is a type of synapse that allows the transmission of a nerve message from a nerve fiber to a muscle fiber in the form of a chemical message by neurotransmitters that will bind to the specific receptors on the surface. muscle fibers. It is not known if the dysfunction of the motor plate is involved in the formation of the divided hand.

A study showing that neuromuscular junction degradation is linked to this syndrome

Dong Zhang, Yuying Zhao, Yan Chuanzhu, Lili Cao and Wei Li have studied the dysfunctions of the neuromuscular junction in different muscles of the hand in patients with ALS, to determine if these dysfunctions are related to the phenomenon of the divided hand. This clinical study at Shandong University's Qilu Hospital enrolled 51 ALS patients, 24 patients with myasthenia gravis had a decrease in RNS, and 20 patients with Lambert Eaton Myasthenia Gravis Syndrome (LEMS).

Who were the patients?

The mean age at onset of the 51 patients with ALS was 58 years old. This group included 23 women and 28 men. The evolution of their disease has varied from 5 to 24 months. Of these, 36 patients had upper limbs, 10 lower limbs and 5 patients had a bulbar form. Patients with myasthenia gravis included 9 men and 15 women, and the mean age was 44 years. The LEMS patients included 16 men and 4 women, and the average age was 59 years old.

What did this study find?

Among the fifty-one ALS patients, thirty-one patients had a split of the hand, 24 patients with the upper limb form and 6 patients with the lower limb form. There was no statistical difference in the frequency of hand splitting between the upper limb group and the lower limb group. There was no hand fracture in patients with bulbar-type ALS. This study showed that more than 60% of the hand muscles of ALS patients had a negative △ D similar to that of patients with myasthenia gravis, but significantly different from that of patients with LEMS, suggesting that Postsynaptic abnormalities could play a major role.


A dysfunction of neuromuscular transmission has been found in the hand muscles of patients with ALS, it is confirmed that the abductor pollicis brevis (short abductor muscle of the thumb) is involved in this syndrome. The dysfunction of the neuromuscular transmission of this muscle could be involved in the formation of the split hand phenomenon. While not a breakthrough, this study highlight a disease starting at the neuromuscular junction, not at the interface between upper neurons and lower neurons in the spine.


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.

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