Sense, Missense or Nonsense - Interpreting Genetic Research in Autism (TCF4, TSC2 , Shank3 and Wnt)

Some clever autism researchers pin their hopes on genetics, while some equally clever ones are not convinced.

One big problem is that genetic testing is still not very rigorous, it is fine if you know what you are looking for, like a specific single gene defect, but if it is a case of find any possible defect in any of the 700+ autism genes it can be hopeless.

Most of the single gene types of autism can be diagnosed based on known physical differences and then that specific gene can be analyzed to confirm the diagnosis.

Today’s post includes some recent examples from the research, and they highlight what is often lacking - some common sense.

There are numerous known single gene conditions that lead to a cascade of dysfunctions that can result in behaviors people associate with autism.  However in most of these single gene conditions, like Fragile X or Pitt-Hopkins, there is a wide spectrum, from mildly affected to severely affected.

There are various different ways in which a gene can be disturbed and so within a single gene condition there can be a variety of sub-dysfunctions.  A perfect example was recently forwarded to me, a study showing how a partial deletion of the Pitt Hopkins gene (TCF4) produced no physical features of the syndrome, but did unfortunately produce intellectual disability.

The study goes on to suggest that “screening for mutations in TCF4 could be considered in the investigation of NSID (non-syndromic intellectual disability)”

Partial deletion of TCF4 in three generation family with non-syndromic intellectual disability, without features of Pitt-Hopkins syndrome

This all matters because one day when therapies for Pitt Hopkins are available, they would very likely be effective on the cognitive impairment of those with undiagnosed partial-Pitt Hopkins.

Another reader sent me links to the studies showing:-

Rapamycin reverses impaired social interaction in mouse models of tuberous sclerosis complex.

Reversal of learning deficits in a Tsc2+/- mouse model of tuberous sclerosis.

But isn’t that Tuberous sclerosis (TSC) extremely rare? like Pitt Hopkins.  Is it really relevant?

Tuberous sclerosis (TSC)  is indeed a rare multisystem genetic disease that causes benign tumors to grow in the brain and on other vital organs such as the kidneys, heart, eyes, lungs, and skin. A combination of symptoms may include seizures, intellectual disability, developmental delay, behavioral problems, skin abnormalities, and lung and kidney disease. TSC is caused by a mutation of either of two genes, TSC1 and TSC2, 

About 60% of people with TSC have autism (biased to TSC2 mutations) and many have epilepsy.

How rare is TSC?  According to research between seven and 12 cases per 100,000, with more than half of these cases undetected.  

Call it 0.01%, rare indeed.

How rare is partial TSC?  What is partial TSC?  That is just my name for what happens when you have just a minor missense mutation, you have a mutation in TSC2 but have none of the characteristic traits of tuberous sclerosis, except autism.

In a recent study of children with autism 20% has a missense mutation of TSC2. 

Not so rare after all.

Mutations in tuberous sclerosis gene may be rife in autism

Mutations in TSC2, a gene typically associated with a syndrome called tuberous sclerosis, are found in many children with autism, suggests a genetic analysis presented yesterday at the 2016 International Meeting for Autism Research in Baltimore.

The findings support the theory that autism results from multiple ‘hits’ to the genome.

Tuberous sclerosis is characterized by benign tumors and skin growths called macules. Autism symptoms show up in about half of all people with tuberous sclerosis, perhaps due to abnormal wiring of neurons in the brain. Tuberous sclerosis is thought to result from mutations in either of two genes: TSC1 or TSC2.

The new analysis finds that mutations in TSC2 can also be silent, as far as symptoms of the syndrome go: Researchers found the missense mutations in 18 of 87 people with autism, none of whom have any of the characteristic traits of tuberous sclerosis.

“They had no macules, no seizure history,” says senior researcher Louisa Kalsner, assistant professor of pediatrics and neurology at the University of Connecticut School of Medicine in Farmington, who presented the results. “We were surprised.”

The researchers stumbled across the finding while searching for genetic variants that could account for signs of autism in children with no known cause of the condition. They performed genetic testing on blood samples from 87 children with autism.

Combined risk:

To see whether silent TSC2 mutations are equally prevalent in the general population, the researchers scanned data from 53,599 people in the Exome Aggregation Consortium database. They found the mutation in 10 percent of the individuals.

The researchers looked more closely at the children with autism, comparing the 18 children who have the mutation with the 69 who do not.

Children with TSC2 mutations were diagnosed about 10 months earlier than those without a mutation, suggesting the TSC2 mutations increase the severity of autism features. But in her small sample, Kalsner says, the groups show no differences in autism severity or cognitive skills. The researchers also found that 6 of the 18 children with TSC2 mutations are girls, compared with 12 of 69 children who don’t have the mutation.

TSC2 variants may combine with other genetic variants to increase the risk of autism. “We don’t think TSC is the sole cause of autism in these kids, but there’s a significant chance that it increases their risk,” Kalsner says.

Effects of Combining Rapamycin and Resveratrol on Apoptosis and Growth of TSC2-Deficient Xenograft Tumors

"hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) is a consequence of tuberous sclerosis complex (TSC) 1/2 inactivation."

"the combination of rapamycin and resveratrol may be an effective clinical strategy for treatment of diseases with mTORC1 hyperactivation."

So for the 20% of autism with partial TSC, so-called Rapalogs and other mTOR inhibitors could be helpful, but Rapalogs all have side effects.

One interesting option that arose in my earlier post on Type 3 diabetes and intranasal insulin is Metformin. The common drug used for type 2 diabetes.

 

Metformin regulates mTORC1 signaling (but so does insulin).

'Metformin activates AMPK by inhibiting oxidative phosphorylation, which in turn negatively regulates mTORC1 signaling via activation of TSC2 and inhibitory phosphorylation of raptor. In parallel, metformin inhibits mTORC1 signaling by suppressing the activity of the Rag GTPases and upregulating REDD1."

Source:  Rapalogs and mTOR inhibitors as anti-aging therapeutics

Clearly you could also just use intranasal insulin.  It might be less potent but should have less side effects because it acting only within the CNS (Metfornin would be given orally).

The Shank protein and the Wnt protein family

Mutations in a gene called Shank3 occur in about 0.5 percent of people with autism.  
But what about partial Shank3 dysfunction?

Shank proteins also play a role in synapse formation and dendritic spine maturation.

Mutations in this gene are associated with autism spectrum disorder. This gene is often missing in patients with 22q13.3 deletion syndrome

Researchers at MIT have just shown, for the first time, that loss of Shank3 affects a well-known set of proteins that comprise the Wnt signaling pathway.  Without Shank3, Wnt signaling is impaired and the synapses do not fully mature.

Neuroscientists illuminate role of autism-linked gene

“The finding raises the possibility of treating autism with drugs that promote Wnt signaling, if the same connection is found in humans”

I have news for MIT, people already do use drugs that promote Wnt signaling, FRAX486 and Ivermectin for example.  All without any genetic testing, most likely.

Reactivating Shank3, or just promote Wnt signaling

The study below showed that in mice, aspects of autism were reversible by reactivating the Shank3 gene.  You might expect that in humans with a partial Shank3 dysfunction you might jump forward to the Wnt signaling pathway and intervene there.

Mouse study offers promise of reversing autism symptoms

One reader of this blog finds FRAX486 very helpful and to be without harmful side effects.  FRAX 486 was recently acquired by Roche and is sitting over there on a shelf gathering dust.

Where from here?

I think we should continue to look at the single gene syndromes but realize that very many more people may be partially affected by them.

Today’s genetic testing gives many false negatives, unless people know what they are looking for; so many dysfunctions go unnoticed.

This area of science is far from mature and there may be many things undetected in the 97% of the genome that is usually ignored that affect expression of the 3% that is the exome.

So best not to expect all the answers, just yet, from genetic testing; maybe in another 50 years.

Understanding and treating multiple-hit-autism, which is the majority of all autism, will require more detailed consideration of which signaling pathways have been disturbed by these hits.  There are 700 autism genes but there a far fewer signaling pathways, so it is not a gargantuan task.  For now a few people are figuring this out at home.   Good for them.

I hope someone does trials of metformin and intranasal insulin in autism.  Intranasal insulin looks very interesting and I was surprised to see in those earlier posts is apparently without side effects.

The odd thing is that metformin is indeed being trialed in autism, but not for its effect on autism, but its possible effect in countering the obesity caused by the usual psychiatric drugs widely prescribed in the US to people with autism.

My suggestion would be to ban the use of drugs like Risperdal, Abilify, Seroquel, Zyprexa etc.

Vanderbilt enrolling children with autism in medication-related weight gain study

Here are details of the trial.

Treatment of Overweight Induced by Antipsychotic Medication in Young People With Autism Spectrum Disorders (ASD) (MET)

Metformin will be dispensed in a liquid suspension of 100 mg/mL. For children 6-9 years of age, metformin will be started at 250 mg at their evening meal for 1 week, followed by the addition of a 250 mg dose at breakfast for 1 week. At the Week 2 visit, if metformin is well-tolerated, the dose will be increased to 500 mg twice daily. For children from 10-17 years of age, metformin will be started at 250 mg at their evening meal for 1 week, followed by the addition of a 250 mg dose at breakfast for 1 week. At the Week 2 visit, if metformin is well-tolerated, the dose will be increased to 500 mg twice daily. At the Week 4 visit, if metformin is well-tolerated, the dose will be increased to 850 mg twice daily.

Dendritic Spines in Autism – Why, and potentially how, to modify them

This blog is getting rather more detailed than I had anticipated.  

Today’s post is about something very complex, but not fully understood by anyone, so I will be somewhat superficial in my coverage.  Just click on the links to learn more detail.

There are two words that may be new to you – Morphology and Dendritic Spines.

Morph in Twin Decks

Morphology, in biology, the study of the size, shape, and structure of animals, plants, and microorganisms and of the relationships of the parts comprising them.

For today it is really could be thought of as the variability in size and shape of something.

A dendritic spine is a small protrusion from a neuron's dendrite that typically receives input from a single synapse. Dendritic spines serve as a storage site for synaptic strength and help transmit electrical signals to the neuron's cell body. Most spines have a bulbous head (the spine head), and a thin neck that connects the head of the spine to the shaft of the dendrite. The dendrites of a single neuron can contain hundreds to thousands of spines. In addition to spines providing an anatomical substrate for memory storage and synaptic transmission, they may also serve to increase the number of possible contacts between neurons.

Now we combine our two new words and have a better summary of what this post is about:

Morphology of dendritic spines and mental disease

It turns out that shape of dendritic spines may play a key role in mental disease, including autism.

The shape is not fixed and live imaging studies have revealed that spines are remarkably dynamic, changing size and shape over timescales of seconds to minutes and of hours to days.

The shape is important as it impacts on function, malformations lead to dysfunctions that can affect a myriad of brain functions.

Here are some variations in the shape of dendritic spines.

In case you are thinking this is all rather abstract, let’s jump forward to a patent for a possible new treatment for autism.

Afraxis Patent

Methods for treating autism 

  

SUMMARY OF THE INVENTION

Described herein are p21 -activated kinase (PA ) inhibitors that alleviate, ameliorate, delay onset of, inhibit progression of, or reduce the severity of at least one of the symptoms associated with autism.

Claims  

WHAT IS CLAIMED IS:

1. A method for treating autism comprising administering to an individual in need thereof a therapeutically effective amount of a p21 -activated kinase (PAK) inhibitor.

2. The method of claim 1, wherein the PAK inhibitor modulates dendritic spine morphology or synaptic function.

3. The method of claim 2, wherein the PAK inhibitor modulates dendritic spine density.

4. The method of claim 2 or 3, wherein the PAK inhibitor modulates dendritic spine length.

5. The method of any of claims 1-4, wherein the PAK inhibitor modulates dendritic spine neck diameter.

6. The method of any one of claims 1-5, wherein the PAK inhibitor modulates dendritic spine head volume.

7. The method of any one of claims 1-6, wherein the PAK inhibitor modulates dendritic spine head diameter.

8. The method of claim 1 or 2, wherein the PAK inhibitor modulates the ratio of the number of mature dendritic spines to the number of immature dendritic spines.

9. The method of claim 1 or 2, wherein the PAK inhibitor modulates the ratio of the dendritic spine head diameter to dendritic spine length.

10. The method of claim 1 or 2, wherein the PAK inhibitor modulates synaptic function.

Etc …

Of course, plenty of patents turn out to be worthless nonsense, but I think the people at Afraxis do know what they are doing; time will tell.

Morphology or Number of Dendritic Spines?

The PAK1 researchers and others believe the morphology (shape) of the dendritic spines is the problem, others believe the problem is that there are just too many of them.

Research has shown that a particular gene (NrCAM) can increase/decrease the number of dendritic spines.

Studies at University of North Carolina showed that knocking out the NrCAM gene caused mice to exhibit the same sorts of social behaviors associated with autism in humans.

Researchers from Columbia University found an overabundance of the protein MTOR in mice bred to develop a rare form of autism. By using a drug to limit MTOR in mice, the Columbia researchers were able to decrease the number of dendritic spines and thus prune the overabundance of synaptic connections during adolescence. As a result, the social behaviors associated with autism were decreased. However, the drug (Rapamycin) used to limit MTOR can cause serious side effects.

Children with Autism Have Extra Synapses in Brain

Dr. Tang measured synapse density in a small section of tissue in each brain by counting the number of tiny spines that branch from these cortical neurons; each spine connects with another neuron via a synapse.

By late childhood, she found, spine density had dropped by about half in the control brains, but by only 16 percent in the brains from autism patients.

“It’s the first time that anyone has looked for, and seen, a lack of pruning during development of children with autism,” Dr. Sulzer said, “although lower numbers of synapses in some brain areas have been detected in brains from older patients and in mice with autistic-like behaviors.”

Using mouse models of autism, the researchers traced the pruning defect to a protein called mTOR. When mTOR is overactive, they found, brain cells lose much of their “self-eating” ability. And without this ability, the brains of the mice were pruned poorly and contained excess synapses. “While people usually think of learning as requiring formation of new synapses, “Dr. Sulzer says, “the removal of inappropriate synapses may be just as important.”

“What’s remarkable about the findings,” said Dr. Sulzer, “is that hundreds of genes have been linked to autism, but almost all of our human subjects had overactive mTOR and decreased autophagy, and all appear to have a lack of normal synaptic pruning. This says that many, perhaps the majority, of genes may converge onto this mTOR/autophagy pathway, the same way that many tributaries all lead into the Mississippi River. Overactive mTOR and reduced autophagy, by blocking normal synaptic pruning that may underlie learning appropriate behavior, may be a unifying feature of autism.”

Maness, a member of the UNC Neuroscience Center and the Carolina Institute for Developmental Disabilities, also said that there are likely many other proteins downstream of NrCAM that depend on the protein to maintain the proper amount of dendritic spines. Decreasing NrCAM could allow for an increase in the levels of some of these proteins, thus kick starting the creation of dendritic spines.

Knocking out the gene NrCAM increases the number of dendritic spines  

   

Gene linked to increased dendritic spines -- asignpost of autism

  

The view from Japan

RIKEN is a large research institute in Japan, with an annual budget of US$760 million.  Their Brain Science Institute (BSI) has a mission to produce innovative research and technology leading to scientific discoveries of the brain.  So RIKEN  BSI is like MIT just for the brain.

Science does tend to stratify by geography.  Just as we saw that NGF (Nerve Growth Factor) is the preserve of the Italians, when it comes to PAK it is the Japanese.

As you can see below the Japanese are firmly behind PAK1. 

 Pak1 regulates dendritic branching and spine formation.

Abstract

The serine/threonine kinase p21-activated kinase 1 (Pak1) modulates actin and microtubule dynamics. The neuronal functions of Pak1, despite its abundant expression in the brain, have not yet been fully delineated. Previously, we reported that Pak1 mediates initiation of dendrite formation. In the present study, the role of Pak1 in dendritogenesis, spine formation and maintenance was examined in detail. Overexpression of constitutively active-Pak1 in immature cortical neurons increased not only the number of the primary branching on apical dendrites but also the number of basal dendrites. In contrast, introduction of dominant negative-Pak caused a reduction in both of these morphological features. The length and the number of secondary apical branch points of dendrites were not significantly different in cultured neurons expressing these mutant forms, suggesting that Pak1 plays a role in dendritogenesis. Pak1 also plays a role in the formation and maintenance of spines, as evidenced by the altered spine morphology, resulting from overexpression of mutant forms of Pak1 in immature and mature hippocampal neurons. Thus, our results provide further evidence of the key role of Pak1 in the regulation of dendritogenesis, dendritic arborization, the spine formation, and maintenance.

SHANK3 and Dendritic Spines

Mutations of the SHANK3 gene are known to cause autism. 

Researchers in France found that SHANK3 mutations lead to modification of dendritic spine morphology and they identified the mechanism.

SHANK3 mutations identified in autism lead to modification of dendritic spinemorphology via an actin-dependent mechanism

You may recall in my earlier posts on growth factors that it was this type of autism that responded to treatment with IGF1.

SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients

If you take a broader look at today’s subject you will see that various growth factors are indeed closely involved.  Here is some comment from Wayman Lab at Washington State University:- 

"Not surprisingly, abnormalities in dendritic arborization and spinogenesis, which diminish neuronal connectivity, are a common feature of the cognitively compromised aging brain as well as numerous forms of mental retardation including Fragile X, Fetal alcohol, Downs and Retts syndromes.

It is clear that changes in synaptic activity and neurotropic factors (e.g., BDNF) are effective initiators of the remodeling process and result in long-term alterations in dendrite and spine structure. What is not known are the molecular mechanisms that underlie how they stimulate dendritic spine formation."

Take your pick

So it looks like three different methods may exist to potentially modify dendritic spine numbers and morphology:-

1.   PAK1

Much work is ongoing regarding PAK1.  It is my current favorite.

For those interested here is a recent study using FRAX486 on Fragile X mice.

Rescue of Fragile X Syndrome phenotypes in Fmr1 KO mice by the small molecule PAK inhibitor FRAX486

Abnormal dendritic spines are a common feature in FXS, idiopathic autism, and intellectual disability. Thus, this neuroanatomical abnormality may contribute to disease symptoms and severity. Here we take a hypothesis-driven, mechanism-based approach to the search for an effective therapy for FXS. We hypothesize that a treatment that rescues the dendritic spine defect may also ameliorate behavioral symptoms. Thus, we targeted a protein that regulates spines through modulation of actin cytoskeleton dynamics: p21-activated kinase (PAK). In a healthy brain, PAK and FMRP - the protein product of fmr1 - antagonize one another to regulate spine number and shape. Inhibition of PAK with a strategy utilizing mouse genetics reverses spine abnormalities as well as cognitive and behavioral symptoms in fmr1 KO mice, as we demonstrated in our previous publication. This discovery highlights PAK as a potential target for drug discovery research. In this thesis work, we build on this finding to test whether the small molecule FRAX486 - selected for its ability to inhibit PAK - can rescue behavioral, morphological, and physiological phenotypes in fmr1 KO mice. Our results demonstrate that seizures and behavioral abnormalities such as hyperactivity, repetitive movements, and habituation to a novel environment can all be rescued by FRAX486. Moreover, FRAX486 reverses spine phenotypes in adult mice, thereby supporting the hypothesis that a drug treatment which reverses the spine abnormalities can also treat neurological and behavioral symptoms.

2. mTOR

In spite of its noted toxicity, Rapamycin, is about to be tested in a clinical trial on a rare type of autism called TSC:-

Rapalogues for Autism Phenotype in TSC: A Feasibility Study (RAPT)

Funnily enough the trial is taking place at the Kennedy Krieger Institute.

When commenting on the use of Bumetanide for autism, I recall the President of the Institute was quoted as saying:-

"So many things cure cancer in mice and rats, and so many things cure all kinds of things and then when we give them to humans they have adverse effects and don't fix the problems we thought they could fix," says Gary Goldstein, president and CEO of the Kennedy Krieger Institute, a Baltimore-based clinic and research center. "I wouldn't give it to my child, I can tell you that."

I found it a little odd that he gave the green light to trialing Rapamycin in children, given the long list of very nasty side effects.

  

3.  NrCAM 

Manesslab at UNC is clearly the centre for research into finding therapeutic agents surrounding NrCAM.  It looks like this is still some way from trials in humans.

“Too many spines and too many excitatory connections that are not pruned between early childhood and adolescence could be one of the chief problems underlying autism. Our goal is to understand the molecular mechanisms involved in pruning and find promising targets for therapeutic agents.”

Conclusion

It should not be surprising that multiple pathways may have the same therapeutic benefit on dendritic spines.  We only need one to be safe and effective.

The link back to human growth factors is interesting since we know these are disturbed in autism and other mental conditions, but the dysfunction varies by sub-type.  In fact, Nerve Growth Factor (NGF) would likely be an effective therapy for dementia and perhaps even Retts syndrome.

In the next post we will learn some more interesting things about growth factor anomalies in autism.  It turns out that something called Akt, also known as protein kinase B (PKB), may be behind them all. A related protein called protein kinase C (PKC), is known to affect the morphology of dendritic spines. There is also protein kinase A (PKA).  Both PKA and PKB have been shown to have reduced activity in regressive autism, this will also be covered later. 

PAK inhibitors not just for Cancer, Alzheimer’s and Neurofibromatosis, but also for Autism, Schizophrenia, Fragile X and Shank 3

You might be wondering, what does a time bomb have to do with all the above conditions.  The answer is a substance in the human body called PAK1.  PAK1 appears to have no useful bodily function, after birth, but it appears to be behind very many dysfunctions in the body.  One scientist suggested that it is there to ensure that we do not live forever.

PAK1 is at the centre of a very expensive effort to develop effective cancer drugs; since the majority of cancers, for males or females, involve PAK1.  If you can block or inhibit PAK1, you can stop tumour growth in many types of cancer.  It turns out that PAK1 is also involved in Alzheimer’s, Huntington's Disease, Neurofibromatosis, Autism, Schizophrenia, Fragile X and Shank 3.

Cancer drugs are big business and budgets seem to be almost limitless.  The good thing is that as long as the PAK1 inhibitor can cross the blood brain barrier (BBB), what works for cancer, is likely to have an effect in all the mentioned brain conditions, including autism.

What is odd, is that in the rare condition of Neurofibromatosis Type 1, which in mild cases might be considered autism with spots, families with the condition are widely aware of PAK1 and are not waiting for drugs to be commercialized.  They are using naturally available PAK inhibitors, like a particular kind of Propolis from New Zealand.  It seems that NF-1, along with PANDAS and PANS, is thought of as a disease to be treated, whereas the much more common, autism, still is not.  Odd isn’t it?

Many of the researchers looking at PAK are Japanese and this in itself is interesting.  Japanese medicine, like Russian medicine, is a world of its own; indeed Russian researchers are also heavily involved in PAK research.   So many clever minds are engaged in this effort.

There are as yet no commercially available PAK1 drugs, but there are many experimental ones.

One problem I have observed is that there are three very similar types of PAK - PAK1, PAK2 and PAK3.  The new drugs seem to inhibit all the three, to greater or lesser extents.  The problem I have seen is that PAK2 is actually good for you.  Blocking PAK1 and PAK2 in mice might work wonders, but in humans this might not be true.  It appears we need PAK1-specific drugs, that do not affect PAK2.

PAK Research in Detail

Since even Wikipedia does not cover the science of PAK in any depth, neither will I.  I have found an excellent collection of research from 2013 that will tell the scientists among you, everything there is to know.  It is available as book or electronically, if you look on google for a minute or two you may find a free ebook version.

PAKs, RAC/CDC42 (p21)-activated Kinases, 1st Edition

Towards the Cure of Cancer and Other PAK-dependent Diseases

 It is very readable and if you are interested in cancer or Alzheimer’s it should also be of interest.

In my post I will just look at the treatment possibilities and research that shows it should be effective.

I will look at a wide range of conditions related to autism, namely:-

• Schizophrenia  (adult-onset autism)
• Neurofibromatosis Type 1 (autism with spots)
• Fragile X (autism with low muscle tone and MR)
• Shank 3
• Mental Retardation (MR)

 

 Mast cells will also make another guest appearance.  

I have already suggested in early posts that following rare genetic conditions may not lead us anywhere in our search for effective autism therapies;  however, when you have three of them, plus schizophrenia, then we have to take note.

As a bonus we have another Nobel Laureate, this time Susumu Tonegawa from MIT.  He works at MIT’s Picower Center for Learning and Memory, along with Mike Bear, who we have previously covered in relation to both Arbaclofen and mGluR5.  Tonegawa suffered his own tragedy when his teenage son committed suicide in his dorm room at MIT.

p21 activated kinases (PAKs) and PAK inhibitors

PAKs are not somethings you are likely to heard of, even the ever up to date Wikipedia has virtually nothing to say on the subject; I guess we must be at the cutting edge.

PAKs are a family of enzymes in the body.  They are implicated in many biological processes, one of which is cancer.  The chemicals that reduce the activity of these enzymes are called PAK inhibitors 

We are interested in Group 1 PAKs that is to say PAK1, PAK2 and PAK3; in particular we want to find PAK1 inhibitors.

To date a lot of money has been spent looking for drugs that are effective PAK inhibitors, but also safe for humans 

The Role of PAK1 in Brain Dysfunction

PAK1 appears to play a central role in lost brain cell function in  Schizophrenia, Fragile X, Shank 3 and  Neurofibromatosis Type 1 (NF-1).  Different scientists are involved in these different areas and their explanation of what is going on does vary.  But in effect they all found (in their mouse models) that by inhibiting PAK, they could restore lost brain function.

There is now a research drug called FRAX486 that looks particularly effective and this is the drug used in the trials I will detail later.

The problem is that research drugs take decades to become approved human drugs and I do not want to wait decades.  So the choice is either to use the research drug or find another PAK-inhibitor.  I opt for the latter.

Note on Mast Cells

Regular readers will have noticed how I believe mast cells play a surprisingly important role in autism.  Here is a link and a summary from a paper showing how PAK2 plays a role in stabilizing mast cells, whereas PAK1 plays an opposing role in making them degranulate.  When this happens histamines, IL-6 and other inflammatory agents are released.  So PAK2 does some good.

Pak2 Kinase Restrains Mast Cel lFcϵRI Receptor Signalling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

 

 The Research Studies

Susumu Tonegawa at MIT is one of the clever scientists pursuing PAK inhibitors;  he is looking at Fragile-X and now, it appears autism.  I think he is the clear expert in this field.

Having established its role in many cancers,  next came its role NF-1, Shank-3, Fragile-X and most recently schizophrenia.  Since schizophrenia is very common and clearly overlaps mainstream autism, we will start there.

Experimental Drug FRAX486 Reverses Schizophrenia In Mice

"A new study shows that one of a class of compounds known as PAK inhibitors, appears to have reversed behaviors associated with schizophrenia and restored some lost brain cell function in adolescent mice with a rodent version of the mental illness. The researchers at Johns Hopkins found that the compound FRAX486 appears to halt an out-of-control biological “pruning” process in the schizophrenic brain during which important neural connections are unnecessarily destroyed."

 Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by the small-molecule PAK inhibitor FRAX486.

Moreover, this PAK inhibitor—which we call FRAX486—also rescues seizures and behavioral abnormalities such as hyperactivity and repetitive movements, thereby supporting the hypothesis that a drug treatment that reverses the spine abnormalities can also treat neurological and behavioral symptoms. Finally, a single administration of FRAX486 is sufficient to rescue all of these phenotypes in adult Fmr1 KO mice, demonstrating the potential for rapid, postdiagnostic therapy in adults with FXS.

PAKs inhibitors ameliorateschizophrenia-associated dendritic spine deterioration in vitro and in vivoduring late adolescence

Significance

Drug discovery in psychiatry has been limited to chemical modifications of compounds originally discovered serendipitously. Therefore, more mechanism-oriented strategies of drug discovery for mental disorders are awaited. Schizophrenia is a devastating mental disorder with synaptic disconnectivity involved in its pathophysiology. In this study, we studied a biological pathway underlying synaptic disturbance and examined whether p21-activated kinase inhibitors ameliorate the pathology in vitro and in vivo. The beneficial effects of these inhibitors reported here may provide us with an opportunity for drug discovery in major mental illnesses with synaptic disturbance.

Abstract

Drug discovery in psychiatry has been limited to chemical modifications of compounds originally discovered serendipitously. Therefore, more mechanism-oriented strategies of drug discovery for mental disorders are awaited. Schizophrenia is a devastating mental disorder with synaptic disconnectivity involved in its pathophysiology. Reduction in the dendritic spine density is a major alteration that has been reproducibly reported in the cerebral cortex of patients with schizophrenia. Disrupted-in-Schizophrenia-1 (DISC1), a factor that influences endophenotypes underlying schizophrenia and several other neuropsychiatric disorders, has a regulatory role in the postsynaptic density in association with the NMDA-type glutamate receptor, Kalirin-7, and Rac1. Prolonged knockdown of DISC1 leads to synaptic deterioration, reminiscent of the synaptic pathology of schizophrenia. Thus, we tested the effects of novel inhibitors to p21-activated kinases (PAKs), major targets of Rac1, on synaptic deterioration elicited by knockdown expression of DISC1. These compounds not only significantly ameliorated the synaptic deterioration triggered by DISC1 knockdown but also partially reversed the size of deteriorated synapses in culture. One of these PAK inhibitors prevented progressive synaptic deterioration in adolescence as shown by in vivo two-photon imaging and ameliorated a behavioral deficit in prepulse inhibition in adulthood in a DISC1 knockdown mouse model. The efficacy of PAK inhibitors may have implications in drug discovery for schizophrenia and related neuropsychiatric disorders in general.

There are many other neuropsychiatric disorders with synaptic changes that might benefit from these compounds. The Tonegawa laboratory previously published that PAK inhibition and knockout are protective against synaptic deterioration in an animal model for Fragile X syndrome (38, 39). In addition, several lines of evidence have suggested the involvement of PAKs in Alzheimer’s disease and mental retardation (40⇓⇓–43). Studies that aim to identify rare variants associated with neuropsychiatric disorders may further reveal PAK family genes as genetic factors. Thus, consideration of these compounds in many other neuropsychiatric disorders may also be an important subject in future studies.

As far as we are aware, PAKs are regarded as therapeutic targets in cancer and immune/allergy-related conditions. Although this question requires careful consideration, we expect minimal adverse effects of PAK inhibitors when we target neuropsychiatric disorders.

This is an interesting patent that was granted on the basis of using PAK1 inhibitors to treat social learning disorders

Patent - Treatments for social learning disorders

 

Abstract

The use of Pak1 inhibitors to treat social or learning disabilities is disclosed. In one embodiment patients exhibiting social or learning disabilities as well as abnormally low NF1 activity are administered PAK inhibitors to treat the social or learning disabilities. Reductions in PAK activity have been found to ameliorate the effects of aberrant neurofibromatosis type 1 activity.

Applicants have demonstrated that defects in NF1 gene leads to deficiencies in learning including for example, deficiencies in social learning. The NF1 gene encodes neurofibromin, which negatively regulates Ras GTPase activation, and thereby reduces the strength and duration of Ras signal transduction. P21-activated kinase (Pak1) is a downstream effector regulated by the Rho family of GTPases that mediate diverse cellular functions including cytoskeletal dynamics, vesicular transport, and gene expression.

Applicants have discovered that the deficit in social learning associated with Nf1+/− mice is rescued by deletion of the Pak1 gene. Accordingly, applicants anticipate that patients having defective NF1 activity can be treated with PAK inhibitors (e.g., a Pak1 inhibitor) to treat learning disabilities and other symptoms or conditions resulting from deficient Nf1 activity. In accordance with one embodiment a method for treating an NF1 deficiency (i.e., decreased NF1 gene expression, decreased NF1 protein product, or decreases functionality of the NF1 protein product relative to the native NF1 gene product) associated learning disability is provided. In one embodiment the method comprises the steps of identifying a patient with defective NF1 activity and administering to said patient a pharmaceutical composition comprising an effective amount of a PAK inhibitor

Neurofibromatosis

In case you do not know, neurofibromatosis (NF1) is one of the most common single gene disorders.  It is associated with skin conditions of widely varying magnitude, but surprisingly many autistic-like neurobehavioral developmental disorders are present.  It seems that NF1 is highly comorbid with autism and ADHD.  A recent survey showed half of parents reported autistic behaviours, far higher than the literature had suggested.  Since only 20% of cases have physical complications, it would seem highly likely that many cases are misdiagnosed as autism.

Behaviour in children with Neurofibromatosis Type 1: where does autism fit in?

 

 NEUROFIBROMATOSIS TYPE 1 (NF1) A GUIDE FOR ADULTS AND FAMILIES WHO HAVE A DIAGNOSIS OF NF1

Neurofibromatosis is considered a treatable medical condition, even in countries that do not regard autism as treatable.  In the United Kingdom there are two clinical centres for the condition, and in Germany it seems that Hamburg is the clinical centre of excellence.

A clue to the therapy of neurofibromatosis type 2: NF2/merlin is a PAK1 inhibitor.

Pak1 regulates multiple c-Kit mediated Ras-MAPKgain-in-function phenotypes in Nf1+/− mast cells

 

Mental Retardation (MR)

I have already mentioned in previous posts that some types of mental retardation may indeed by treatable, this was based on my observation that certain drugs can produce cognitive improvement in autism.

So it was a nice surprise to find in the literature that PAK3 has been shown to be involved in some types of MR.  That would imply PAK3 inhibitors might have some effect on MR.

Since MR is highly comorbid with autism, perhaps PAK3 is also involved in autism. 

  

PAK1 AS A THERAPEUTIC TARGET

Importance of the field

P21-activated kinases (PAKs) are involved in multiple signal transduction pathways in mammalian cells. PAKs, and PAK1 in particular, play a role in such disorders as cancer, mental retardation and allergy. Cell motility, survival and proliferation, the organization and function of cytoskeleton and extracellular matrix, transcription and translation are among the processes affected by PAK1.

8. PAK1 in neurological and mental disorders

PAK3 in clearly involved in some neurodegenerative disorders and variants of mental retardation and plays a special role in synapse formation and plasticity in hippocampus. However, the involvement of PAK1 in these processes is less clear-cut. For example, both PAK1 and PAK3 were reduced in the hippocampus affected by Alzheimer disease, yet only PAK3 was affected in some other areas of the diseased brain. However, this reported loss of the PAKs from the cytosol appears to be accompanied by re-localization of PAKs to the membrano-cytoskeletal fractions, where they appear to be active. Using staining for drebnin and reduction in dendrites as indicators, Dr. Cole’s group has observed that a dominant-negative form of PAK1 sensitizes, while the wild type form protects from some effects of beta-amyloid oligomers in cultured primary neurons. However, in both cases it is hard to rule out that ectopically expressed PAK1 in some of these experiments acted as a surrogate for the highly homologous PAK3.

Dominant-negative PAK1, which, potentially, inhibits other PAK isoforms as well, upon expression in mouse forebrain affected synapse morphology and consolidation of long-term memory, but rescued some defects of a mouse model of Fragile X syndrome.

In case of Huntington’s disease, PAK1 specifically co-localizes with huntingtin inclusions in the affected brain146. In tissue culture models, interference with PAK1 function modestly decrease the formation of aggregates by mutant huntingtin, while the constitutively active PAK1 enhances the aggregation. Accordingly, similar activity was reported for PAK1 regulator α-PIX. The matter is complicated, however, by the observation that kinase activity of PAK1 is dispensable for this phenomenon. Overall, it appears that pathological changes in the brain could be associated both with elevated and reduced function of PAKs and the specific role of PAK1 in these processes may be variable as well.

PAK signalling in neuronal physiology.

Group I p21-activated kinases are a family of key effectors of Rac1 and Cdc42 and they regulate many aspects of cellular function, such as cytoskeleton dynamics, cell movement and cell migration, cell proliferation and differentiation, and gene expression. The three genes PAK1/2/3 are expressed in brain and recent evidence indicates their crucial roles in neuronal cell fate, in axonal guidance and neuronal polarisation, and in neuronal migration. Moreover they are implicated in neurodegenerative diseases and play an important role in synaptic plasticity, with PAK3 being specifically involved in mental retardation. The main goal of this review is to describe the molecular mechanisms that govern the different functions of group I PAK in neuronal signalling and to discuss the specific functions of each isoform.

SHANK-3

The SHANK3 gene is a member of the Shank gene family. Shank proteins are multidomain scaffold proteins of the postsynaptic density that connect neurotransmitter receptors, ion channels, and other membrane proteins to the actin cytoskeleton and G-protein-coupled signaling pathways.  Mutations of the SHANK3 gene are known to be associated with autism.  

It is complex, but it appears that the reducing effect of Shank3 knockdown on NMDARs and F-actin is blocked by PAK1 inhibitors

Shank3 Deficiency Induces NMDA Receptor Hypofunction via an Actin-Dependent Mechanism

Shank3, which encodes a scaffolding protein at glutamatergic synapses, is a genetic risk factor for autism. In this study, we examined the impact of Shank3 deficiency on the NMDA-type glutamate receptor, a key player in cognition and mental illnesses. We found that knockdown of Shank3 with a small interfering RNA (siRNA) caused a significant reduction of NMDAR-mediated ionic or synaptic current, as well as the surface expression of NR1 subunits, in rat cortical cultures. The effect of Shank3 siRNA on NMDAR currents was blocked by an actin stabilizer, and was occluded by an actin destabilizer, suggesting the involvement of actin cytoskeleton. Since actin dynamics is regulated by the GTPase Rac1 and downstream effector p21-activated kinase (PAK), we further examined Shank3 regulation of NMDARs when Rac1 or PAK was manipulated. We found that the reducing effect of Shank3 siRNA on NMDAR currents was mimicked and occluded by specific inhibitors for Rac1 or PAK, and was blocked by constitutively active Rac1 or PAK. Immuno cytochemical data showed a strong reduction of F-actin clusters after Shank3 knockdown, which was occluded by a PAK inhibitor. Inhibiting cofilin, the primary downstream target of PAK and a major actin depolymerizing factor, prevented Shank3 siRNA from reducing NMDAR currents and F-actin clusters. Together, these results suggest that Shank3 deficiency induces NMDAR hypofunction by interfering with the Rac1/PAK/cofilin/actin signaling, leading to the loss of NMDARmembrane delivery or stability. It provides a potential mechanism for the role of Shank3 in cognitive deficit in autism.

PAK, p21-activated kinase, is the key downstream effector of Rac1, which stimulates spine synapse formation and neurite outgrowth by facilitating actin filament assembly. Different mutations in the PAK genes have been identified in mental retardation cases. Mice expressing a forebrain-specific dominantnegative form of PAK show fewer dendritic spines, altered spine morphology, and changes in synaptic strength.  Shank proteins have been shown to form a complex with PAK and overexpression of Shank in cultured neurons promotes synaptic accumulation of PAK. Consistently, we have found that Shank3 knockdown leads to reduced PAK1 activity. Moreover, inhibiting PAK1 decreases the basal NMDAR current, and the reducing effect of Shank3 knockdown on NMDARs and F-actin is occluded by PAK1 inhibitors and blocked by constitutively active PAK1. These data suggest that Rac1/PAK1- mediated actin dynamics is important for NMDAR membrane delivery/maintenance and its regulation by Shank3.

Mast Cells

Mast cells are the cells that react will allergens and lead to the release of histamine and many other inflammatory agents like the cytokine IL-6.  It is shown that PAK1 plays a key role in mast cell degranulation and could therefore play a key role in treating allergies and asthma.

APak1-PP2A-ERM signaling axis mediates F-actin rearrangement and degranulation in mast cells.

Abstract

Mast cells coordinate allergy and allergic asthma and are crucial cellular targets in therapeutic approaches to inflammatory disease. Allergens cross-link immunoglobulin E bound at high-affinity receptors on the mast cell's surface, causing release of preformed cytoplasmic granules containing inflammatory molecules, including histamine, a principal effector of fatal septic shock. Both p21 activated kinase 1 (Pak1) and protein phosphatase 2A (PP2A) modulate mast cell degranulation, but the molecular mechanisms underpinning these observations and their potential interactions in common or disparate pathways are unknown. In this study, we use genetic and other approaches to show that Pak1's kinase-dependent interaction with PP2A potentiates PP2A's subunit assembly and activation. PP2A then dephosphorylates threonine 567 of Ezrin/Radixin/Moesin (ERM) molecules that have been shown to couple F-actin to the plasma membrane in other cell systems. In our study, the activity of this Pak1-PP2A-ERM axis correlates with impaired systemic histamine release in Pak1(-/-) mice and defective F-actin rearrangement and impaired degranulation in Ezrin disrupted (Mx1Cre(+)Ezrin(flox/flox)) primary mast cells. This heretofore unknown mechanism of mast cell degranulation provides novel therapeutic targets in allergy and asthma and may inform studies of kinase regulation of cytoskeletal dynamics in other cell lineages.

Where to find your PAK-inhibitor?

In the literature you will find that there are various different PAK inhibitors

 

Not surprising if you want to want to inhibit PAK1, PAK2 and PAK3, then FRAX486 is a good choice.

But where do you get FRAX486 from?

There is even a link for that.

Susumu Tonegawa, Afraxis and Roche

It looks like in about 2007 Tonegawa has created a start-up company called Afraxis to develop FRAX 486.  Having done further research and raised some venture capital they licensed their drug portfolio to the drug major, Roche, in 2013.

I hope this works out better for Tonegawa that Roche’s deal with his MIT colleague Mark Bear who also linked up his start-up Seaside Therapeutics with Roche.  That one did not end so well.

 

Avalon Ventures’ Afraxis Licenses Entire Drug Portfolio to Genentech

Roche’s Genentech has licensed global rights to develop and commercialise Afraxis’ entire portfolio of CNS compounds in a deal worth up to US$187.5 M. Afraxis’ lead programme targets PAK (p21-activated kinase) and has initially been focused on developing disease-modifying therapies for Fragile X syndrome, the most common inherited cause of mental retardation. Although not a sale, the deal will still provide an exit for Avalon Ventures, Afraxis’ sole shareholder, and follows the acquisition of Avalon-backed Zacharon Pharmaceuticals by BioMarin Pharmaceutical earlier in January 2013. For Roche, the deal supplements an already robust neuroscience pipeline.

 

Any other alternatives?

Fortunately another Japanese scientist, Hiroshi Maruta, has written a paper on all the possible PAK inhibitors available today for humans.

Effective neurofibromatosis therapeutics blocking the oncogenic

kinase PAK1

If you read his paper, he is pointing in the direction of the natural world and a special kind of propolis rich in CAPE (caffeic acid phenethyl ester) produced by bees in New Zealand.  His fall back is an old drug for humans and pets called ivermectin, which was found by chance to have a secondary affect as a PAK-inhibitor.

CAPE (caffeic acid phenethylester)-based propolis extract (Bio 30) suppresses the growth of humanneurofibromatosis (NF) tumor xenografts in mice.

It is a substance, CAPE, specific to the New Zealand bees that makes their propolis act as a PAK-inhibitor.  Regular propolis from your health food store is most likely made by the wrong type of bees.

So if you do not fancy waiting 15 years for Roche to commercialize Susumu Tonegawa’s clever discoveries from MIT which may or may not be effective in humans, you could stick with the clever Japanese and follow Hiroshi Maruta’s thinking and go down under to New Zealand.  During its long isolation, New Zealand developed a distinctive biodiversity of animal, fungal and plant life; most notable are the large number of unique bird species and by the sound of it some pretty special bees.

 

Can a bee product really be an effective drug? I definitely start as a sceptic, but the natural flavonoid Quercetin really does work, so why not Propolis?  Propolis has been used medicinally for more than a thousand years, but only the New Zealand one and one Brazil variety contain PAK inhibiting compounds. 

There is also an odd saying from Germany, that "bee keepers do not get cancer". Maybe there is something in this?

The problem with many of the other natural PAK-inhibitors is their bioavailability.  They may work in the test tube, but the human body does not absorb them enough for them to be effective.  Curcumin, Resveratrol, Honokiol (from Magnolia bark) all appear, but unless you can absorb them and they can cross the blood brain barrier (BBB) they will not work.

The NF-1 and NF-2 sufferers have zeroed in on the BIO30 Propolis as the realistic alternative.  I think they made the right choice.

Conclusion

The logical conclusion is to buy some BIO30 Propolis and give it a try.  I hope Susumu Tonegawa and Roche eventually make a commercially available drug, but  new drugs seem to take 15 years to bring to the market.  The existing drug, Ivermectin, really should be given a clinical trial in NF-1 or Fragile-X.