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Spicy News

Audio Podcast (192kbps MP3)
(video at bottom of text)

Welcome back to the Cripple Command Center or C-to-the-3 podcast on ericvalor.org. My name is Eric Valor and I will be your host and head nerd in charge. In this episode I will report on a recent webinar given by CIRM, the California Institute of Regenerative Medicine, and a particular clinical trial discussed therein. I will also report on a new potential treatment target for ALS found using CRISPR, a new proposed treatment for SOD1-linked ALS, a new open-label trial for a form of curcumin, and a new stem cell model to assess possible treatments for neurodegenerative diseases such as ALS.

The first item up for bids is the new CRISPR study. Researchers at Stanford University have used CRISPR/Cas9 gene editing technology to gain insight into the genetic basis of ALS. The team, led by Doctor Aaron Gitler, used the technology to sift through the entire human genome to find genes that help neurons defend against toxic protein aggregation. In ALS it is known that proteins inside the motor neurons clump together, depriving the cell of the beneficial function of these proteins as well as choking the machinery which normally breaks down malformed or deficient proteins for recycling by the cell. The Stanford team used CRISPR to sequentially alter genes which either help cells cope with protein aggregation or enhance the toxicity. They ceased the function of each gene one by one in what’s called a “genetic knock-out” and evaluated the effect. That way they can identity potential drug targets for future therapies. You can read more about this in the Stanford News Center website. You can read the paper in Nature Genetics.

Our next item is the CIRM webinar and the upcoming clinical trial described therein. The webinar was held on Facebook. It was an “Ask The Experts” format where information is presented and then questions are asked by people watching the webinar online. The presentation was made by Doctors Clive Svendsen, Bob Baloh, and Ralph Kern. First, a little information was shared first about the NurOwn therapy in trial by Brainstorm. The Phase 3 trial is enrolling 300 people with enrollment expected to finish by early 2019. The protocol is repeat injections of the person’s own mesenchymal stromal cells which reduce local inflammation and secrete trophic factors which help the motor neurons heal and grow.

The juiciest part of the webinar was the presentation of the new Phase 1 clinical trial being prepared by Cedars-Sinai in Los Angeles. This trial is similar to the old Neuralstem trial using human fetal brain-derived neural progenitors which are multi-potent stem cells which can only become cells of neural lineage (neurons, astrocytes, microglia, etc.). In contrast to the Neuralstem product where the cells mostly became interneurons these cells become astrocytes which would replace the native astrocytes which become toxic in ALS. The cells are transduced using GDNF (glial-derived neurotrophic factor).

Cedars also uses a new method called “chopping” (which when you read the paper on the method should really be called “slicing”) to expand the cell line into a pharmaceutically-relevant number. A clump of neural cells called a neurosphere is repeatedly sliced up and the parts allowed to expand. The usual method is to break apart the neurosphere by hand and individually plate the cells. Chopping is much faster and increases yield by allowing the cells to be cultured in 3D rather than in 2D. This means the cells have more contact with others which promotes health and growth. Instead of having cells just on the left and right and on top and bottom, they also have cells above and below. Like penguins grouping together tightly in a snowstorm the cells fare much better in a 3D arrangement as in the body. Like the Neuralstem trial the Cedars trial uses a laminectomy to inject the cells into the spine but uses a new rig which is significantly less invasive. The patients undergo a year of immunosuppression to ensure the body doesn’t reject the implants. After that the cells are expected to survive for life.

Next up is the new clinical trial for the spice curcumin being put together by my friend and ALS Untangled colleague Doctor Richard Bedlack. The trial is called ROAR for Replication Of ALS Reversals. ROAR is open-label with no placebo and is designed to have faster enrollment with much better retention, and wide inclusion criteria like no 2-year cutoff or many of the other exclusions found in usual trials. That’s not to say that the usual trial design is “bad” but ROAR is looking for a huge unmistakable signal: A reversal of ALS such as regaining use of limbs or getting out of a wheelchair (the latter being a more extreme example). The trial will also have few/no in-person visits. ROAR will use a particular product called Theracumin which is a highly concentrated form of curcumin which is readily absorbed, properly metabolized, and is well-tolerated even in high doses. Trial participation for each person is 6 months. The real-time results will be available on Patients Like Me and the full protocol will be made available on www.alsreversals.org should anyone not in the trial want to follow along.

Why curcumin? Because it’s generally regarded as safe and is inexpensive to obtain. There are some 12,000 published papers studying curcumin’s effects. Curcumin has powerful antioxidant effects, can reduce protein aggregation in cells, can induce beneficial gene activation, and can alter the gut microbiome, the vast population of bacteria in the stomach and intestines, in ways that are beneficial to health including the brain and nervous system (seriously!). In mouse neuroblastoma cells transfected with mutant TDP43, curcumin improved excitotoxicity, improvd mitochondrial function, and reduced oxidative stress and protein aggregation. Unfortunately these studies have not been independently replicated. You can learn more about curcumin and prior data as relates to ALS in the ALS Untangled report on the subject and by watching the video presentation Dr. Bedlack gave on the ROAR trial.

Alrighty, what do we have next? Ah, yes, the new stem cell model meant to assess potential treatments for neurodegenerative diseases like ALS. The model is specifically for Alexander disease (AxD) because of its relatively simple pathology. But it’s relevant to Alzheimer’s, Parkinson’s, and ALS because it focuses in on malfunctioning astrocytes. In AxD, astrocytes with a mutation in glial fibrillary acidic protein (GFAP) secrete a protein called CHI3L1, a marker of neuroinflammation that suppresses neural development-related processes, including myelination. The CHI3L1 suppresses cells that are precursors to oligodendrocytes, the cells that wrap the neural axons in a fatty substance called myelin. Myelin acts like the plastic insulation of electrical wires and performs much the same function. The idea is that by understanding more about astrocytes that would reveal clues about how to treat diseases like Alzheimer’s and ALS.

And that’s all for this episode of the C-to-the-Three Podcast on ericvalor.org. I am, and always will be, Eric Valor. Until next time, breathe easy.

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Guest Blog: Me!

SRG Research News – First A Little Background

As most of you know, I started SciOpen Research Group as a way for me to be able to fire actual bullets in the battle against ALS (well, actually metaphorical, but you get the idea). Our first project failed to extend life in the classic ALS mouse model so we retained the money raised to conduct the planned second part of that experiment. We had another project already in the research pipeline waiting to take the next step in development. For two years SRG was working on creating a novel molecule which would treat the desired pathway without becoming toxic like the reference molecule does at therapeutic doses. Suddenly we had the opportunity to collaborate with researchers already investigating the same pathway, albeit in different conditions (watch the video announcement), with their own library of candidate molecules.

Our collaboration’s first phase is to create a novel transgenic mouse species which represents a 100% drug efficacy in order to be a proof of concept. The project should run through the last half of 2016. As you will see below, a study was recently published which shows that SRG is definitely onto something. Our target protein is significantly elevated in human patients, and that targeting it brings positive results. The study is great indirect support of our project’s goal.

And now, the guest blog featuring myself!

Good News For Our Latest Project!

A recent report published in Science magazine strongly suggests that SciOpen Research Group is onto something with its currently ongoing study of necroptosis in ALS. Necroptosis is a “cousin” of apoptosis. In contrast to apoptosis, which happens regularly in the body, necroptosis is a form of programmed cell death which happens under inflammatory conditions and in which the components of the dead cell spill into the extracellular space. The spilling of the cellular components trigger a response in which immune cells are recruited to the area. Necroptosis is known to be a driver of both genetic ALS and sporadic ALS.

The subject study is not a direct support, in that it was looking at how the optineurin protein contributes to ALS. However, the results showed significant increase of the MLKL protein in human patients and that elimination of the RIPK3 protein or inhibition of RIPK1 had modest but nevertheless positive effects on survival of the SOD1 mice (along with positive biological evidence). This suggests that SRG is on the right track with its MLKL study. We believe that acting on MLKL will have a stronger effect without disrupting other cellular functions which depend on RIPK3 and/or RIPK31 (MLKL is involved only in necroptosis).

This study is YOUR study. It would not be position without your support. SciOpen Research Group is the world’s first fully functional “guerilla biotech”. We function only with your support and study pathways other research organizations either miss or ignore. And we can do it for much less because we are purely volunteer and have no overhead. 100% of your donations go directly to research. To support us you can make a tax-deductible donation (USA residents only) by going to our Donations page, purchase some SRG Gear, and/or go shopping on Amazon Smile and name SciOpen Research Group as your charity of choice (we are a registered and approved nonprofit under IRS 501c3). We work on ALS for you, the ALS Community, because we are part of the ALS Community. Help us continue our novel research into eradicating ALS.

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Guest Blog: Me!

Good News For Our Latest Project!

A recent report published in Science magazine strongly suggests that SciOpen Research Group is onto something with its currently ongoing study of necroptosis in ALS. Necroptosis is a “cousin” of apoptosis. In contrast to apoptosis, which happens regularly in the body, necroptosis is a form of programmed cell death which happens under inflammatory conditions and in which the components of the dead cell spill into the extracellular space. The spilling of the cellular components trigger a response in which immune cells are recruited to the area. Necroptosis is known to be a driver of both genetic ALS and sporadic ALS.

The subject study is not a direct support, in that it was looking at how the optineurin protein contributes to ALS. However, the results showed significant increase of the MLKL protein in human patients and that elimination of the RIPK3 protein or inhibition of RIPK1 had modest but nevertheless positive effects on survival of the SOD1 mice (along with positive biological evidence). This suggests that SRG is on the right track with its MLKL study. We believe that acting on MLKL will have a stronger effect without disrupting other cellular functions which depend on RIPK3 and/or RIPK31 (MLKL is involved only in necroptosis).

This study is YOUR study. It would not be position without your support. SciOpen Research Group is the world’s first fully functional “guerilla biotech”. We function only with your support and study pathways other research organizations either miss or ignore. And we can do it for much less because we are purely volunteer and have no overhead. 100% of your donations go directly to research. To support us you can make a tax-deductible donation (USA residents only) by going to our Donations page, purchase some SRG Gear, and/or go shopping on Amazon Smile and name SciOpen Research Group as your charity of choice (we are a registered and approved nonprofit under IRS 501c3). We work on ALS for you, the ALS Community, because we are part of the ALS Community. Help us continue our novel research into eradicating ALS.

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Of Mice And Me

As many of my readers know, about two years ago I came across a study investigating a novel molecule for the treatment of Alzheimer’s. The molecule, J147, is a synthetic derivative of curcumin. Curcumin and other similar molecules have long been under study for neurodegenerative diseases. Unfortunately curcuminoids have rather poor bioavailability, meaning they are quickly excreted from the body and require high amounts to have a therapeutic value. Like curcumin, J147 is “orally available” (meaning it is introduced to the body by eating it) but is more than 100X as potent. This means a much smaller quantity is necessary for therapeutic effect. So far, we haven’t found a toxic dose of J147. Work on toxicity is ongoing.

In the Alzheimer’s study J147 had remarkable results in that model. The pathways acted upon were quite relevant to ALS. These include potent antioxidant effects, significant reduction of microglia activation and migration, and reduction of heat-shock protein expression which indicates a shift back toward cellular homeostasis. More recent data (unpublished) indicates an effect in reducing astrocyte activation, which is sufficient to rapidly kill even healthy motor neurons.

Unfortunately, because J147 is pleiotropic, pharmaceutical companies weren’t interested. The current research paradigm is to focus on single molecular targets. For diseases with a single mechanisms, that’s a fine method of attack. But ALS has quite a few things going on simultaneously. All prior single-target treatments have failed and the current growing opinion is that successful treatment would require a cocktail of drugs. Better to have a single pleiotropic substance than a mixture of chemicals with uncertain interactions.

In April, 2013, I created SciOpen Research Group in order to have an entity capable of negotiating research and licensing of novel molecules with the promise of treatment of ALS. J147 is our first project. In the early summer of 2013, SRG applied to Prize4Life for access to their colony of G93A transgenic research mice at Jackson Laboratories. Our research proposal for J147 was accepted and we were given granted sufficient animal numbers to properly conduct our study. We received the mice and started the experiment at the end of January.

We are very excited to have commenced our first research program and demonstrate that guerrilla biotechs can perform quality science. To that end, we created a crowdfunding campaign on Indiegogo to obtain funding for the next step of the experiment – microscopic tissue examination. This will tell us exactly what J147 did to help the motor neurons in the mice.

Please donate if you can. All donations are tax-deductible. If you cannot donate please spread word about SRG and our need for funding this new and exciting research.

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Pot Luck

An article appeared on social media about a group of parents using cannabidiol (CBD) for their children’s epilepsy. Unlike the usual reports of people using marijuana and subjectively reporting “improvements”, this group of patient advocates went and filed an Investigational New Drug (IND) with the FDA. Don’t get me wrong – I support the medical (and recreational) use of marijuana, but heretofore the real scientific data available has been extremely thin. Rather than going on Silk Road to get a bunch of medicine then post wonderful stories on social media, this group created a real clinical trial in cooperation with FDA and a company named GW Pharmaceuticals which supplied a pure oil formulation of CBD. This is a very important development in patient-driven access to investigational drugs. Far better than the usual DIY projects (even the handful started by yours truly), this type of project can deliver real, verifiable, and scientifically-accepted results.

The body contains cannabinoid receptors both in the CNS and periphery. The most well-known cannabinoid ligand is THC (a CB1 agonist) which is responsible for the euphoric psychoactive effect in marijuana. Both natural and synthetic cannabinoids long been of interest in treating disease. What’s of most interest in medicine are the anti-inflammatory effects of CB2 agonists such as cannabidiol or CBD. Endogenous CB2 receptors are upregulated in the spinal cords of SOD1 transgenic mice. CBD agonists show symptomatic improvement in several inflammatory diseases. There is evidence that CB2 receptors are upregulated in response to the inflammatory microglial activation in ALS. Several studies have shown that CB2 agonists have a beneficial effect in transgenic SOD1 mice. This data shows that more work, perhaps in in human patients, is warranted.

Alternative medicine is very popular in the ALS Community because, frankly, there is nothing currently available proven to extend the lives of PALS. Unfortunately most experiments are done without adequate objective observation and recording of data. Instead all that is reported are vague descriptions of improvement, skewing any rational perception of the particular alternative medicine. This causes more desperate patients to attempt the alternative with the same lack of adequate reporting.

This post, however, is not about calling for an IND for CBD (which would nevertheless be a good idea). The point here is to spotlight that a group of patients and/or advocates got together to do an experiment outside of an institutional clinical trial. They led the way and did it themselves while preserving the valuable objective data. They created their own hope in a seemingly hopeless situation. This is the ultimate expression of DIY Medicine, done properly and openly. Any other method is a waste of time, money, and health.

There is actually much more opportunity than just experiments with speculative alternative medicine. Hope exists for the approximately 60% of living PALS who don’t qualify for clinical trials. That hope is the FDA Expanded Access Program (EAP). PALS should request EAPs for those investigational treatments which have passed the Phase 2 endpoint requirements of safety and suggested efficacy. Furthermore, they should support efforts to bring EAPs to the ALS Community. Living, even for the healthy, requires hope. We, the ALS Community, like everything else we have accomplished, must create our own hope by being pioneers and responsible citizen scientists.

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Carpe Fragments

In the developing embryo, motor neurons develop and nearly half preferentially die prior to birth (Henderson, et al., 1997, “Hepatocyte growth factor (HGF/SF) is a muscle-derived survival factor for a subpopulation of embryonic motoneurons”). As shown in Forger, et al., 2001 (“Blockade of Endogenous Neurotrophic Factors Prevents the Androgenic Rescue of Rat Spinal Motoneurons”), loss of muscular targets also leads to post-natal motor neuron degeneration. Post-natal mice engineered to have degenerated muscle spindles exhibit ataxia and resting tremors, indicating a decrease in proprioception due to loss of sensory-motor synapses (Frank, et al., 2002, “Muscle Spindle-Derived Neurotrophin 3 Regulates Synaptic Connectivity between Muscle Sensory and Motor Neurons”).

One interesting factor seems to suggest a link with testosterone in preserving motor neurons, which could be a possible explanation for the statistically higher numbers of men affected in middle-age or above, and that of women in post-menopause, when hormone levels experience radical shift. Indeed, Cilliary Neurotrophic Factor, a potent motor neuron trophic factor, is regulated by gonadal hormones (Forger, et al., 1998, “Ciliary Neurotrophic Factor Receptor in Spinal Motoneurons is Regulated by Gonadal Hormones”).

Leaving aside the question of hormone levels, there is much evidence that muscle-derived neurotrophic factors are necessary for the health and survival of the motor neurons. One in particular, Motoneuronotrophic Factor 1 (MNTF1), appears essential to this critical process. Experiments in Wobbler mice show that motor neuron disease increases as MNTF1 levels decrease (http://www.ncbi.nlm.nih.gov/pubmed/10453487). MNTF1 was first described in the early 90s, and the human form was successfully cloned as an artificial protein. Various fragments were extracted and shown to have neurotrophic effect.

Two overlapping domains of a 33 amino acid fragment of MNTF1, dubbed the Fred and Wilma domains, are sufficient to stimulate motor neuroprotection in a manner similar to the whole 33 amino acid MNTF1 fragment. The Fred domain is sufficient to direct selective reinnervation of muscle targets by motor neurons in vivo in a manner similar to the 33 amino acid MNTF1 fragment. A recombinant protein containing the Fred domain maintained motoneuron viability, increased neurite outgrowth, reduced motoneuron cell death/apoptosis and supported the growth and spreading of motoneurons into giant, active neurons with extended growth cone-containing axons.

For those curious about the amino acids in each domain, please refer to the image below:

Genervon has patented these fragments and is using them in a Phase 2-A clinical trial in ALS.

From the above it is quite possible that at least some forms of ALS are caused by a sort of a muscular dystrophy (not to be confused with the distinct condition by that name). It therefore stands to reason that there is reason for hope that some will benefit. The standard caveat of basic and preclinical research often not translating to human trials obviously applies. However, we are entering an exciting time where extremely potent shots are being taken at more fundamental aspects of ALS. One or a combination seem likely to have the effect we have been waiting for.

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The Mito-Can’t-Rias

One of the earliest observations in ALS is a weakening of the energy-producing organelles of the cell called mitochondria. The mitochondria are found to only output about half of the energy as normal while producing more waste in the form of Reactive Oxygen Species (ROS). Think of your car engine horribly out of tune where you don’t get your normal acceleration and nasty black smoke pours out of your tailpipe which causes you to fail smog inspection, only this smoke causes your car to rust away from the inside out. In addition to reduced energy output, mitochondria in neurodegenerative diseases also don’t repair themselves well and have, in previous research, suffered from axonal transport problems which appeared linked to the “die-back” of the axon and detachment from the neuromuscular junction (NMJ).

However, a recent study that used live imaging of mouse motor neurons showed that axonal transport was independent of mitochondrial density and axonal degeneration. In mice with different SOD1 mutations, axonal transport was affected differently (or not at all). The motor neurons of the mice bred to have the SOD1 G93A mutation, which are used in the majority of studies because they are the model most understood, showed extensive axonal transport problems early on while G85R motor neurons showed very little even up to end-stage. Even mice bred to overexpress (multiple copies of the gene) human wild-type (non-mutant) SOD1 showed axonal transport issues without motor neuron degeneration. The “code” for the mutations refer to nucleotide transpositions at numbered locations in the SOD1 gene that creates the SOD1 protein. Below you can see movies of transport in the various types of neurons relative to controls.

WT-SOD1

G93A-SOD1

WT-SOD1

G85R-SOD1

WT-SOD1

WT-SOD1 (human, overexpressed)

I have previously posted about the role of mitochondria in ALS and discussed various implications. I have also posted about the possibility of a role of SOD1 in both FALS and SALS as well as the possibility of SOD1 as a prion in the propagation of ALS. In all the mutant SOD1, mitochondria show dysfunction early. Mitochondria also have “communication” with the Schwann cells at the NMJ which is an early event in the destruction of the NMJ and commencement of the axonal die-back.

Nothing is as simple as we would like, and ALS exemplifies this axiom. What would seem apparently causal might merely suggest another, as yet undiscovered, mechanism which may underlie other related observed effects. And sometimes what appears to be a related effect may just be something separate. Regardless of any doubt cast on the role of axonal transport in ALS, the mitochondrial dysfunction with which it is associated still appears strongly implicated. The cause of that remains under investigation.

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Assaultrocytes

In my post Prion, Garth! I had talked about mutant or misfolded SOD1 (mSOD1) spreading like a prion and infecting neighboring cells. A recently released study from Johns Hopkins shows for the first time in vivo that astrocytes expressing mutant mSOD1 damage the motor neurons they are supposed to protect. In the study, the authors transplanted SOD1G93A glial-restricted precursor cells—glial progenitors capable of differentiating into astrocytes—into the cervical spinal cord of rats to reveal how mutant astrocytes influence WT motor neurons and other cells types (microglia and astrocytes) in an in vivo setting. The G93A mutation of SOD1 is the most studied version of that particular genetic mutation responsible for many inherited (or familial) ALS cases.

This is the strongest evidence to date that “assaultrocytes” are primary culprits in ALS. It also further suggests that even some sporadic ALS can be caused by misfolded SOD1 which escapes proteasomal degradation and gets into the extracellular space. It also suggests that targeting mSOD1 and stem cell implantation of astrocytes can be viable treatment methods.

EDIT: Here is a more in-depth article by Amber Dance at the ALZ Forum.

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Prion, Garth

in a previous post, I had discussed the possibility of misfolded SOD1 aggregating with TDP43 resulting in depletion of TDP43 from the nucleus which caused disruption of cellular processes and cell death. Further, misfolded SOD1 induces glial activation seen in ALS which poisons otherwise healthy neurons.

A new study explores the mechanism behind this prion-like behavior, and further indicates that extracellular misfolded SOD1 can be a cause of even sporadic ALS. It also points to a molecular target which could halt progression cold.

The study was done at the Brain Research Centre based at the University of British Columbia and the Vancouver Coastal Health Research Institute, in collaboration with researchers at the University of Alberta. The research was supported in part by Amorfix Life Sciences which has a “vaccine” against misfolded SOD1 already in development.

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Axon-Axoff

Why do axons go downhill? Gravity has nothing to do with it…

The linked study, though not specifically aimed at ALS, does seem to tie together a few “hot topics” in the pathology: axonopathy, oxidative damage, and mitochondrial dysfunction. With many plausible theories of damage, trying to link some together may provide more clues as to cause and/or (more importantly) ways to effectively treat the disease. Part of the tie to ALS in this study is the fact that mice bred to lack the SOD1 gene exhibit a similar disease process as mice bred with a copy of the malformed SOD1 gene which is a known cause of familial ALS. Reinserting the gene in the “knockout” mice, but only in the mitochondria, rescued the mice from disease.

Mitochondria are the power plants of the cells and as a consequence also produce molecules which can cause cellular damage (ROS, the “industrial waste” of cellular energy production). Anything which disrupts the ability to clear out this waste can cause damage to the cell. Damage to mitochondria impacts their ability to provide energy to the cell. Motor neurons have high energy requirements and due to their extremely long axons have mitochondria operating at large distance from the cell body. There are currently a few clinical trials going into Phase III aimed at mitochondrial support.

While the study doesn’t provide an answer (except that this problem is “way beyond eating blueberries”), it does provide some important clues for further investigation.