The over-activated immune system, Kv1.3, ASD-IE, Acacetin and sloppy science

One of the people I have met during my investigation into autism, recently pointed out to me that much scientific research cannot be trusted.  He forwarded a study to me showing just how many researchers admit is omitting data that did not suit what they were trying to prove.  I replied that I made a point of checking the credentials of the lead author.  He then replied that it is not the lead researcher who collects the data, he has a little army of PhD students doing this and nobody is checking them.

The study showed it was the younger researchers, eager to prove themselves that were the most likely to “fiddle” the results.  The problem is that by the time you become an “older researcher” you are not the one collecting the data.

Doubts about Sprouts 

One of the people who I keep forgetting to add to my Dean’s list is John Gargus; he is a professor at University of California at Irvine and Director of the Center for Autism Research and Treatment at UCI.  He is also a specialist in the complex field of ion channels and channelopathies.

He was asked to comment about the Johns Hopkins broccoli/Sulforaphane autism trial.

Trial sprouts doubts about broccoli extract for autism

Cruciferous caution: 

Some independent researchers have similar reservations, noting that the control group showed an unusually small placebo response.“You always see a 20 to 25 percent improvement in placebo,” says John Jay Gargus, director of the Center for Autism Research and Translation at the University of California, Irvine. For example, the placebo effect plagued trials of the gut hormone secretin and antidepressants for autism.
“It’s stunning that they’ve managed to have found a placebo that doesn’t give the placebo effect that we see in every other neuropsychiatric drug trial,” Gargus says.

Now as regular readers will know, at least in Monty, aged 11 with ASD, broccoli sprout powder and we assume the Sulforaphane produced by it, does have a near immediate effect.

But as Gargus says, you will always have some people with the placebo appearing to improve.  In an old post I looked at the placebo effect in autism.  It seems that the more involved the trial and hence the more 1:1 attention the child gets, the more the placebo appears to make things better.  In fact it was not the placebo, it was the 1:1 attention that improved the autism.

So, just as we have to be cautious of the placebo effect, we have to be cautious of sloppy science/scientists.  When financial interests are involved you need to be even more cautious.

The other thing I have learnt to be cautious of, is scientists who have spent many years in one very narrow field, often trying to prove their initial hypothesis to be correct.  Their eyes are then closed to everything else.

Autism Flare-ups, Summertime raging and GI issues

We have investigated in depth the fact that in some people with autism their immune system appears to be over-activated, as the result of an allergic response.  What then happens is that their autism “flares-up” and therapies that previously worked, seem to stop doing so.

The conclusion was that the allergy had caused mast cell activation and this triggered the release of pro-inflammatory chemicals (IL-6, histamine etc).  The solution was:-

·        Avoid the allergen (a type of food, or even airborne pollen)
·        Use mast cell stabilizers to minimize degranulation; even common H1 anti-histamines are partially effective
·        Inhibit the potassium ion channel Kv1.3, which seems to mediate the resulting “over-activation” of the immune response.

The good news is that it really does work and not just in Monty.  The bad news is that the optimal therapy uses a prescription drug (Verapamil).

While trawling through the research on novel anti-oxidants, I stumbled upon something that may help those people who cannot access Verapamil.

There is a flavonoid called Acacetin, which is found in asplenioid ferns.  This flavonoid has long been has used for its anti-inflammatory and immunomodulatory effects.  Now it has been shown to block Kv1.3 channels and inhibits human T cell activation.  This is one of the effects of Verapamil (there are others).  Acacetin has also been shown to have anti-cancer properties in prostate cancer cells.

Acacetin Blocks Kv1.3 Channels and Inhibits Human T Cell Activation

Acacetin (5,7-dihydroxy-4'-methoxyflavone)exhibits in vitro and in vivo anticancer activity through the suppression of NF-κB/Akt signaling in prostate cancer cells

Remember the odd therapy used to block Kv1.3, those TSO parasites, I mentioned in previous posts.  My favorite is this one:-

Immunomodulatory Therapy in Autism - Potassium ChannelKv1.3, Parasitic Worms, and their ShK–related peptides

Acacetin is available as a supplement.  So if you think blocking Kv1.3 might help and cannot access Verapamil or TSO, there are other options.

Indeed, for completeness, there at least two other Kv1.3 blockers that are available.  One is progesterone, the hormone and the other is Curcumin.

  

A nongenomic mechanism for progesterone-mediated immunosuppression: inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes.  Curcumin serves as a human kv1.3 blocker to inhibit effector memory T lymphocyte activities.

  

You may recall that Progesterone was found to be highly neuro-protective and for this reason was trialed for use in the ER, immediately after a traumatic brain injury.  It was shown to save lives.  In autism, we previously found that some people, at the high functioning end, find they feel better when they apply progesterone cream, i.e. transdermal route.

Curcumin has been used for centuries as a drug.

I have not tried them, but I will continue to use Verapamil.  Acacetin, Progesterone and Curcumin share some, but not all of each other’s effects.

Progesterone, in common with Verapamil, affects both potassium and calcium channels.

There are many different potassium and calcium channels and you would hope to find a selective channel blocker and hence affect only the ones you need to.

ASD-IS  (Inflammatory Subtype)

I came across a promising study on Paul Whiteley’s blog.  It is a study of a sub-type of autism characterized by fluctuating behavioral symptoms following immune insults.  In the trial group the children all had GI problems, some had enterocolitis or esophagitis.  The entire group had been noticed by teachers/therapists to lose cognitive skills following immune insults.

Cytokine profilesby peripheral blood monocytes are associated with changes in behavioral symptoms following immune insults in a subset of ASD subjects: an inflammatory subtype?

Regular readers of this blog will see lots of familiar points.  This appears to be exactly the same thing as my “over-activated immune response”.

Now this study comprised children who had Non-IgE mediated allergies.  This does matter because classic allergies are called IgE-mediated and they result in little cells called mast cells getting activated and then releasing IL-6 and histamine in the blood supply.

From Wikipedia we have a summary:-

Conditions caused by food allergies are classified into 3 groups according to the mechanism of the allergic response:

1.     IgE-mediated (classic) – the most common type, occurs shortly after eating and may involve anaphalaxis.

2.     Non-IgE mediated – characterized by an immune response not involving immunoglobulin E; may occur some hours after eating, complicating diagnosis.

3.     IgE and/or non-IgE-mediated – a hybrid of the above two types.

Treating allergy is a “fuzzy” area and, depending on which country you live in, some aspects are seen as science and others pseudo-science. 

Perhaps we should see it as an important, but emerging field of science.

I am not an allergist/immunologist, so I have to look things up.

Since in the trial the children had Non-IgE mediated allergies, we can then look to see whether mast cell activation is relevant.

NON-IgE MEDIATED FOOD ALLERGY 

Mast cell and eosinophil activation is an important component of the non-IgE-mediated response

The authors of the autism study believe that the research subjects with allergy did not have mast cell activation, because they had NON-IgE mediated allergies.

Since I am not an allergist, all I can say is the author of the above paper from the Royal Free & University College School of Medicine in London thinks that mast cell activation is an important component of the non-IgE-mediated response.

Anyway, make your own mind up and continue to see what the study found.

The study looked at children with autism and allergy, whose autism flares up and affects (impairs) their cognitive function.  This group is ASD-IS (Inflammatory Subtype)

ASD-IS children: ASD-IS children are defined as those with a history of fluctuating behavioral symptoms following immune insults (mainly microbial infection). Symptoms must have been documented by individuals other than parents, such as teachers/therapists, a minimum of three times. In addition, a history of persistent GI symptoms, often diagnosed as non-IgE mediated food allergy (NFA - see next section for diagnostic criteria), is required. Among the ASD-IS subjects, 14/24 subjects were diagnosed with food protein induced enterocolitis syndrome (FPIES), a severe form of NFA, prior to enrollment in this study, and two ASD-IS subjects were diagnosed with eosinophilic esophagitis (EoE) on the basis of biopsy results. These ASD-IS subjects reported to have had loss of once-acquired cognitive skills based on the reports of teachers, therapists and/or previous records of developmental assessment.

We defined ‘flares’ as worsening behavioral symptoms following immune insults, despite the resolution of acute conditions such as viral syndrome (that is, the resolution of other infectious symptoms if associated with a microbial infection, lack of fever, and no other acute physical symptoms associated with immune insults). Most of the immune insults in this study were clinically judged to be microbial infection (mainly viral syndrome). In ASD-IS children, we obtained samples at least once in the ‘flare’ and ‘non-flare’ states. Changes in behavioral symptoms by parental reports were confirmed by reports from

teachers and other caregivers.

The authors have an entirely different hypothesis to mine.

But I find their data remarkably similar to what I see being caused by a pollen allergy in my son - summertime autism flare-up and regression.  This is why there were so many posts about the inflammatory cytokine IL-6 and how to minimize it.

Have the authors stumbled upon exactly the same phenomenon as I did?  I very much think so.

I have shared my therapy with the authors, but they think that Non-IgE mediated allergies have nothing to do with mast cell activation.   That sounds odd to me.

GERD/Reflux, Autism, Head Banging and mGlu5

This brief post addresses one further issue as to why people with autism can often suffer from various nasty gastrointestinal (GI) problems. 

First a recap.

Mast Cell Activation

We have already seen that some people’s GI problems are caused by mast cell activation/degranulation.  These cells are activated by allergens (certain foods in this case) and then they release histamine and other pro-inflammatory agents like IL-6.  Degranulation of mast cells can itself cause pain, but the main problem is the resulting damage/inflammation caused by the IL-6 and histamine.

The effective therapy is a mast cell stabilizer.  These include Verapamil (better known as a calcium channel blocker), Cromolyn Sodium, Ketotifen, Azelastine and to a lesser extent most anti-histamines like Claritin, Zyrtec etc.  Quercetin, the flavonoid, also has an effect.

Pancreatic Dysfunction

We also saw that L-type calcium channel (Cav1.2) dysfunction in the pancreas may disrupt the production of certain digestive enzymes.  The lack of these enzymes will disrupt the digestive process and likely affects other processes elsewhere in the body.  Verapamil blocks the Cav1.2 channel.

Ulcerative Colitis

We saw that inflammation and colitis, as diagnosed by an endoscopy, is another comorbidity of autism; this may be in part caused by the mast cell degranulation, but it does fit with the broader hypothesis of the over-activated immune system.  We saw how the potassium ion channel Kv1.3 was the mechanism behind some useful immuno-suppressive therapies, including those TSO parasites.  For those who are skeptical, here is another recent study, I just found:-

Expression of T-cell KV1.3 potassium channel correlates with pro-inflammatory cytokines and diseaseactivity in ulcerative colitis.

  

Kv1.3 should then be a target to treat ulcerative colitis and, I believe, autism itself. Some Kv1.3 blockers exist today; one is Verapamil, another is Curcumin, for those who prefer supplements to drugs.

Curcumin Serves as a Human Kv1.3 Blocker to Inhibit Effector Memory T Lymphocyte Activities

Before I forget to write this down somewhere, it appears that Kv1.3 can also be modulated by PKA and PKC, which decrease its activity. 

Dual Regulation of the n Type K+ Channel in Jurkat T  lymphocytes by Protein Kinases A and C*

We have already come across protein kinase B (PKB) and there will be a post soon of PKA, PKB and PKC.  This all links back to oxidative stress, neuroinflammation and even those dendritic spines.

  

Reflux

Today’s post is about reflux, sometimes known as gastroesophageal reflux disease (GERD) or gastro-oesophageal reflux disease (GORD).  Reflux is when the acid from the stomach rises through the esophagus/oesophagus to the mouth.

Many adults suffer from reflux from time to time and there are many OTC and prescription drug treatments. It can cause pain and discomfort, and would be particularly troubling if you could neither verbalize, nor understand your symptoms.

Why this post?

You may wonder why I have jumped from broccoli (the previous post) to reflux.  There is a reason.

I was recently listening to a conversation between doctors about a head-banging child and then came “it’s not autism; he’s got reflux, that is why he was banging his head.”

That sounded very odd to me.

It turns out many people with autism suffer from reflux, so you could say it is a comorbidity.  But why might that be?

mGlu5 receptors and disease

In an earlier, rather complicated, post I introduced the glutamate receptor, mGlu5.  This receptor is at the centre of research into Fragile X at MIT.  Fragile X is the most common single gene cause of autism.  It has been shown that mGlu5 dysfunction appears in many types of autism and indeed schizophrenia (adult-onset autism).

   

I then chanced upon a recent paper on mGLu5 and came across this section:-

Through contributions to synaptic plasticity, mGlu5 receptors have been implicated in neuronal processes such as learning and memory as well as disorders including Fragile X Syndrome (FXS), tuberous sclerosis, autism, epilepsy, schizophrenia, anxiety, neuropathic pain, addiction, Alzheimer’s disease, Parkinson’s disease, L-DOPA-induced dyskinesias, and gastroesophageal reflux disease

That was quite a surprise, but yet another good lesson of why the comorbidities should all be carefully researched.

 

The full paper, for anyone with time on their hands is:- 

Location-dependent signaling of the Group 1 metabotropic glutamate receptor, mGlu5.

Conclusion

If you have autism, you may have an mGlu5 dysfunction.  This will become treatable once the needed PAMs (Positive Allosteric Modulators) and NAMs (Negative Allosteric Modulators) have been brought to market.  A great deal of research is ongoing.

In the meantime, mGlu5 dysfunction is quite possible elsewhere in the body.  mGlu5 dysfunction is associated with some very rare disorders, but the common ones are diabetes and reflux.

The head-banging boy very possibly had both autism and reflux; he did develop diabetes.

For more on autism and diabetes, a short, thought provoking, but technical, paper:-

Insulin signaling and autism

Interestingly, we saw earlier that Verapamil seems to offer protection against type 1 and 2 diabetes. This time it is its calcium channel blocking role that is the mechanism.

Verapamil drug may reverse diabetes related death of pancreatic beta cells

No big surprise that Verapamil is an ingredient of the autism Polypill.

Verapamil drug may reverse diabetes-related death of pancreatic beta cells

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.