“Autism treatments proposed by clinical studies and human genetics are complementary” & the NSAID Ponstan as a Novel Autism Therapy

Today’s post was not my idea at all, it was the author of one of the papers who has drawn my attention to the subject.

Genetic studies are complicated and are not the sort of thing I would have chosen to read, let alone write about, before starting this blog. 

The optimal time to initiate pharmacological 

intervention in Autism?

However, much of the complex subject matter has now already been covered, step by step, in earlier posts. Regular readers should not feel put off.

It is perhaps easier to think about ion channel dysfunctions, or channelopathies.  Some of the key genetic dysfunctions produce these channelopathies.  There are many posts in this blog about channelopathies, partly because many therapies already exist to treat them.

Then we have the complex signaling pathways which are often the subject of cancer research, but we have seen that certain ones like RAS and PTEN are key to conditions like some autism and some MR/ID.

So it is not a big leap therefore to consider the findings of a statistical reassessment of the existing genome-wide association studies (GWAS).  As is often the case in medical science, it is the acronyms/abbreviations, like GWAS, that make it look more complex than it really is.

If you only ever read one paper about the genetics of autism, I suggest you make it this one.

Fortunately, the conclusion from the genetic study really fits nicely with the clinical studies reviewed on this blog and even my own first-hand experience of investigating and treating my n=1 case of autism.

Knut, the Biometrician

It was Knut who left a brief comment on this blog and, after a little digging, I was very surprised how much a statistician/biometrician could figure out about autism, from re-analyzing the existing genome-wide association studies (GWAS).

I think the Simons Foundation could save themselves a decade or two by giving him a call.

The Research

For those wanting the science-lite version, there is a short article reviewing the research in lay terms:-

Biostatistics provides clues to understanding autism: an interview with Dr Knut M. Wittkowski

“Hence, modulation of ion channels in children at the age of about 12 months, when the first symptoms of autism can be detected, may prevent progression to the more severe end of the spectrum.” .

The actual research paper is here:-

You may find it heavy going and I have highlighted some key parts.

A novel computational biostatistics approach implies impaired dephosphorylationof growth factor receptors as associated with severity of autism

  

“Despite evidence for a likely involvement of de novo and environmental or epigenetic risk factors, including maternal antibodies or stress during pregnancy  and paternal age, we contend that coding variations contribute substantially to the heritability of ASD and can be successfully detected and assembled into connected pathways with GWAS—if the experimental design, the primary outcome, the statistical methods used, and the decision rules applied were better targeted toward the particulars of non-randomized studies of common diseases.”

The data comes from the Autism Genome Project (AGP), and there are two sets of data AGPI and AGPPII.

The third data set is for Childhood Absence Epilepsy (CAE)

What I would call Classic Autism, others call severe autism or autistic disorder; Knut calls it Strict Definition Autism (SDA).  HFA is high functioning autism, much of which is Asperger’s Syndrome.

“Study design We aimed at risk factors specific to strict definition autism (SDA) by comparing case subpopulations meeting the definition of SDA and milder cases with ASD (excluding SDA), for which we here use the term ‘highfunctioning autism’ (HFA). To reduce variance, we included only subjects of European ancestry genotyped on the more frequently used platform in either stage. In AGP II, we also excluded female cases because of confounding between chip platform and disease severity. The total number of subjects included (m: male/f: female) was 547/98 (SDA) and 358/68 (HFA) in AGP I and 375 (SDA) and 201 (HFA) in AGP II.

Overall, the results (see Supplementary Figure 1 for a Manhattan plot) are highly consistent with previously proposed aspects of the etiology of ASD. The clusters of genes implicated in both of the independent stages (Figure 2a/b) consistently overlap with our published CAE results (Figure 2c), confirming the involvement of ion channels (top right) and signaling downstream of RAS (bottom left), with two noticeable additional gene clusters in ASD. Both stages implicate several genes involved in deactivation of growth factor (GF) receptors (Figure 2a/b, top left) as ASD-specific risk factors and chloride (Cl − ) signaling, either through Ca2+ activated Cl− channels

Click to enlarge the figure 

A new term is PTPR (protein tyrosine phosphatases receptor), just to confuse us it is also called RPTP.

Receptor Protein Tyrosine Phosphatases in Nervous System Development

 

For example, the receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ) are expressed primarily in the nervous system and mediate cell adhesion and signaling events during development.

In an earlier post I highlighted the numerous dysfunctions in growth factors (GF) in autism.  Knut is highlighting here the effect of PTPR on growth factors.  Later it is suggested that this cascade of GF dysfunctions could be halted, pharmacologically if it was identified very early.  But, as Courchesne from UC San Diego noted, by the time people have been identified as having autism, around three years old, the accelerated brain growth has already run its course.

You would need to intervene around one year old.

Broad evidence for involvement of PTPRs One of the most striking observations is the involvement of at least five PTPRs in ASD (Figure 2, 10 o’clock position). PTPRs (Table 1e) regulate GF signaling through reversible protein tyrosine dephosphorylation.72 PTPRT (90th/20th, 8.57) was implicated in ASD by a deletion73 (Table S2 AU018704) and a somatic mutation

It was my post pondering the reasons for the positive effect of potassium supplementation that drew Knut’s attention to this blog.  Now we move on to Knut’s ideas on potassium and chloride channels.

K+ and Cl− ion channels as drug targets

Aside from PTPRs (Figure 2, 10 o’clock) as a risk factor for protracted GF signaling, our results suggest a second functional cluster of genes, involved in Cl− transport and signaling, as specific to ASD (Table 1f). In AGP I, the CaCCs ANO4 and ANO7 scored 1st and 70th, respectively. In AGP II, the lysosome membrane H+ /Cl- exchange transporter CLCN7 scored 21st, followed by CAMK2A, which regulates ion channels, including anoctamins82 (55th), and LRRC7 (densin-180), which regulates CAMK2A83 (Figure 2a/b, 2 o’clock). The role of the anoctamins in pathophysiology is not well understood, except that CaCC activity in some neurons is predicted to be excitatory84 and to have a role in neuropathic pain or nerve regeneration. More recently, CaCCs have also been suggested as involved in ‘neurite (re)growth’. Finally, we compared the HFA and SDA cases as separate groups against all parental controls in the larger AGP I population. Overall, the level of significance is lower and the enrichment is less pronounced, especially for the SDA cases (Supplementary Figure 9), as expected when cases and some controls are related. For the HFA cases (Figure 4, and Supplementary Figure 8), however, a second anoctamin, ANO2, located on the other arm of chromosome 12, competes with ANO4 (Figure 1, left), for the most significant gene among the result. Hence, drugs targeting anoctamins might have broader benefits for the treatment of ASD than in preventing progression to more severe forms of autism. ANO2 and ANO6 are associated with panic disorder and major depressive disorder, respectively. ANO3, ANO4, ANO8 and ANO10, but not ANO1, are also expressed in neuronal tissue.86 As ‘druggable channels’, anoctamins ‘may be ideal pharmacological targets to control physiological function or to correct defects in diseases’.  Few drugs, however, target individual anoctamins or even exclusively CaCCs. Cl− channel blockers such as fenamates, for instance, may decrease neuronal excitability primarily by activating Ca2+-dependent outward rectifying K+ channels.

Here is a follow-up paper with consideration of the possible next steps.

Early Pharmacological Intervention in Autism: Wide-Locus GWAS Leading to Novel Treatment Options

Gene gene environment behavior development interaction at the core of autism:

Here, we combine a recent wide-locus approach with novel decision strategies fine-tuned to GWAS. With these methodological advances, mechanistically related clusters of genes and novel treatment options, including prevention of more severe forms of ASD, can now be suggested from studies of a few hundred narrowly defined cases only.

(Nonsyndromic) autism starts with largely unknown prenatal events (♂: age, ♀: virus/stress ...)

• Mutations in growth factor regulators (PTPRs) lead to neuronal overgrowth (brain sizes).

• Mutations in K+/Cl− channels cause Ca2+ mediated over excitation of neurons (“intense world”).

• Stressful environments (urbanization) contribute to epistatic interaction (increasing prevalence).

• This GGE interaction causes “migraine-like” experiences during the “stranger anxiety” period where children learn verbal/social skills, leading to behavioral maladaptation (“tune-out”).

• The lack of verbal/social stimuli causes “patches of disorganization” (Stoner 2014, NEJM) as a form of developmental maladaptation when underutilized brain areas are permanently “pruned”. The PTPRs point to a short window of opportunity (WoO) for pharmacological intervention:

• Treatment has to begin as early as possible, while neurons are still growing (12 months of age. Broad support for the proposed unifying etiology and the 2nd year of life as the WoO:

• Regression (“loss of language”) seen in some children >12 mos of age.

• “Patches of disorganization” in >2 yr old brains.

• Romanian orphans developed “quasi-autism” when placed into foster care at >24 mos of age. 

• Hearing impairment leading to intellectual disability when diagnosed >24 mos of age.

 A rational drug target: treating either of two epistatic risk factors suffices:

• Blocking growth factors (Gleevac, ...) is unacceptable in children merely at risk of ASD.

• Ion channel modulators have been used in small children for arthritis and seizures.

Here is a response to Knut’s first paper from a professor at the UCLA medical school who suggests the combination of the specific NSAID and bumetanide. 

The professor would better understand the mechanism of action of bumetanide in autism if he read Ben Ari’s research more thoroughly, or even this blog.

  

Autism treatments proposed byclinical studies and human genetics are complementary

  

The article by Wittkowski et al.¹ reports results of human genetic studies that suggest that a nonsteroidal anti-inflammatory drug (NSAID) given for a few months from the time of the first symptoms might help some children who are at risk of developing more severe forms of atrial septal defect.

While the authors mention the recent article by Lemonnier et al.,² which reported that a clinical study of the diuretic Bumetanide was partially effective in children with milder forms of autism, they seem to have overlooked that these two treatments may well be complementary, leading to sequential interventions, each targeting specific risks related to well-defined stages in the development of brain and social interactions.

Since abnormal brain development in autistic disorder goes through different stages from infancy to childhood, targeting different developmental stages with different treatment interventions may well be necessary to foster continued normalization of brain growth.

Bumetanide is known to block inward chloride transporters, yet the relation of this mechanism to the etiology of autism is unknown. Wittkowski et al. identified mutations in calcium-activated (outward) chloride channels as associated with autistic disorder, suggesting loss-of-function mutations in anoctamins as one of the risk factors for autism. This provides a testable hypothesis for the mechanism by which Bumetanide alleviates symptoms of autism. For example, mouse models could test whether Bumetanide ameliorates a stress-induced phenotype caused by a knockout/down in ANO2 and/or ANO4.

A second cluster of genes identified receptor protein tyrosine phosphatases, which downregulate growth factors. These findings support the notion that successful treatment should start as early as possible,³ while neuronal development still takes place.

The rationale for combining these two treatments rests on the fact that Bumetanide is contraindicated in infancy because it is known to interfere with neuronal development when used long term. In contrast, the NSAID proposed in the second study has been given for decades to children with juvenile idiopathic arthritis from 6 months of age on, with no adverse effects on brain development. It is known to modulate chloride channels (see above) as well as potassium channels.⁴

In conclusion, I wish to extend their hypothesis based on the synergy of the two treatment approaches: (1) early treatment with NSAID can reduce early maladaptive behaviors that cause abnormal pruning of neurons in the cortical areas; (2) these children could subsequently benefit from Bumetanide, which would compensate for the primary ion channel defect, but could not reverse the secondary effect of abnormal pruning.

This hypothesis allows for a novel two-way interaction between behavior and molecular events. Traditionally, one assumes that molecular events determine behavior. The new hypothesis, based on human genetics, also allows for symptoms (such as the absence of social interactions, delayed speech onset and language development) during certain sensitive periods to change molecular events (pruning of neurons in areas required for normal development).

Therapeutic implications from the genetic analysis

Some of the therapies that Knut is proposing, based on the genetic analysis, have already been reviewed in this blog.  Some have not.  A few therapeutic ideas in this blog actually target genes Knut has identified, but not highlighted a therapy.

I will just review the drugs and genes that the above study highlights.

Benzodiazepines

Low dose clonazepam fits in this category.  We have the work of Professor Catterall to support its use.  At higher doses, benzodiazepines have different effects but use is associated with various troubling side effects.

Bumetanide

Bumetanide is at the core of my suggested therapy for classic autism or what Knut calls SDA (strict definition autism).  We have Ben-Ari to thank for this

Fenamates (ANO 2/4/7 & KCNMA1)

Here Knut is trying to target the ion channels expressed by the genes ANO 2/4/7 & KCNMA1. 

·        ANO 2/4/7 are calcium activated chloride channels. (CACCs)

·        KCNMA1 is a calcium activated potassium channel.  KCNMA1 encodes the ion channel K_Ca1.1, otherwise known as BK (big potassium).  This was the subject of post that I never got round to publishing.

  

Fenamates are an important group of clinically used non-steroidal anti-inflammatory drugs (NSAIDs), but they have other effects beyond being anti-inflammatory.  They act as CaCC inhibitors and also stimulate BKCa channel activity.

  

Fenamates stimulate BKCachannel osteoblast-like MG-63 cells activity in the human.

 “The fenamates can stimulate BKCa channel activity in a manner that seems to be independent of the action of these drugs on the prostaglandin pathway”

Molecular and functional significance of Ca2+-activated Cl− channels in pulmonary arterial smooth muscle

“Of this “first generation” of CaCC inhibitors, NFA (a fenamate called niflumic acid)  is the most potent blocker of these channels and the compound most frequently used to investigate the physiological role of CaCCs”

Choice of Fenamate

There are several fenamate-type NSAIDs, but one is a very well used generic drug, Mefenamic acid known as Ponstan, Ponalar, Ponstyl, Ponstel and other generic names.  It is even available as a syrup for children.

 It is not available in all countries.

Gabapentin

Gabapentin is used primarily to treat seizures and neuropathic pain. It is also commonly prescribed for many off-label uses, such as treatment of anxiety disorders, insomnia, and bipolar disorder.

Some people with autism are prescribed Gabapentin.  Some people suffer side effects and others do not.

If you have a dysfunction of voltage operated calcium channels, Gabapentin should help.

Memantine

This is all about modifying NMDA receptors.  Memantine is but one method.

http://epiphanyasd.blogspot.rs/2015/06/altered-homeostasis-in-autism-cl-k-ca2.html

Minocycline

Minocycline is an antibiotic with several little known extra properties.  In autism, we looked at its ability to reduce microglial activation and so improve autism.  A clinical trial showed that it did not help autism.

Minocycline also affects MMP-9.  MMP-9 is an enzyme found to be associated with numerous pathological processes, including cancer, immunologic and cardiovascular diseases.

High MMP-9 activity levels in fragile X syndrome are lowered by minocycline.

 “ The results of this study suggest that, in humans, activity levels of MMP-9 are lowered by minocycline and that, in some cases, changes in MMP-9 activity are positively associated with improvement based on clinical measures.”

So if you are treating a case of Fragile-X, or partial "Fragile-X-like" autism, better take note.

Rapamycin

Rapamycin and mTOR was the subject of the following post:

mTOR – Indirect inhibition, the Holy Grail for Life Extension and Perhaps Some Autism

Both too much and too little mTOR can occur in autism.

Conclusion

My conclusion is probably different to yours.

For me, it seems that all the pieces really are fitting together and so this blog on the cause and treatment of classic autism will eventually cover the current scientific knowledge, in its entirety.  No complex areas are off limits, because in the end they are not as complex as they seem, when you lift the veil of jargon and acronyms.

From the all-important therapeutic perspective, new insights from today’s post are:-

·        Those with a dysfunction of voltage operated calcium channels might want to give Gabapentin (Neurontin) a try.

·        The fenamate-type NSAID mefenamic acid,  widely known as Ponstan, really should be tested, either at home, or in a clinical trial.

This statistical analysis is based on “all autism”, so any one person would be highly unlikely to have all the mentioned dysfunctions.  These are the most common genetic dysfunctions and many can both hypo and hyper, as in the case of NMDA dysfunctions and indeed mTOR. 

In Knut’s chart, I would add a green line pointing to RAS and PTEN with the word Atorvastatin.  Baclofen would point to the growth factors.  Verapamil would point in multiple places.

The motto of University of Tübingen, where Knut originally comes from, is Attempto !  The Latin for "I dare".

This might be a useful motto for readers of this blog, and also a good tittle for a book on treating autism.

Kvx.y-channelASD, Navx.y-channelASD, Cavx.y-channelASD and channelASD-channelepsy phenotype

Perugia is an ancient university city in Central Italy.  If you live in North America you may recall it in connection with a high profile murder trial.  You probably would not expect it to produce clever insights into autism.

In fact, Italy is a rare country outside the US that has leading autism researchers.

Today’s post is to draw your attention to very insightful paper about some of the ion channel dysfunctions in autism.  This paper is about those concerning potassium.

The nice touch was their suggestion that we could classify some people with ASD, some with epilepsy and some people with both, by their ion channel dysfunction.

So if like some people, you have a dysfunction of the L-type calcium channel Ca_v1.2, you would become:-

Ca_v1.2-channelASD

Somebody with Dravet Syndrome (epilepsy) with ASD, would become:-

Na_v1.1channelASD-channelepsy

The underlying assumption by the authors is that a type of single ion channel dysfunction, generally triggered by the underlying gene being dysfunctional, and may account for many cases of “autism”.

This is interesting, but I tend to believe that multiple ion channel dysfunctions (channelopathies) are present and in some cases the underlying gene itself is not the problem.  Ion channels and transporters are proteins and each type is indeed expressed by its gene, but the degree to which that gene is expressed is also determined by many other factors.  So over/under expression of, for example, an ion transporter might well not correspond to any genetic error.

We have already seen that in addition to ion channels there are also various types of ion transporter/exchangers. Two very important ones in autism are NKCC1 and KCC2, they determine the chloride concentration within brain cells.  Too many NKCC1 transporters and/or too few KCC2 transporters mean that the level of chloride is too high.  This then causes an ongoing dysfunction of the neurotransmitter GABA.  So in this case the route problem is not a dysfunction of the transporter rather there are just too many of them.

NKCC1 is also expressed in many regions of the brain during early development, but not in adulthood.^[5] This change in NKCC1 presence seems to be responsible for altering responses to the neurotransmitters GABA and glycine from excitatory to inhibitory, which was suggested to be important for early neuronal development. As long as NKCC1 transporters are predominantly active, internal chloride concentrations in neurons is raised in comparison with mature chloride concentrations, which is important for GABA and glycine responses, as respective ligand-gated anion channels are permeable to chloride. With higher internal chloride concentrations, outward driving force for this ions increases, and thus channel opening leads to chloride leaving the cell, thereby depolarizing it. Put another way, increasing internal chloride concentration increases the reversal potential for chloride, given by the Nernst equation. Later in development expression of NKCC1 is reduced, while expression of a KCC2 K-Cl cotransporter increased, thus bringing internal chloride concentration in neurons down to adult values

So we could call this common autism phenotype NKCC1 over expression.

Then the type of autism I am interested in would become:-

Ca_v1.2-channelASD with NKCC1 over expression

This assumes that no potassium or sodium channels are affected.

Back to Potassium Ion Channelopathies


Here is the paper from Perugia:-

Update on the implication of potassium channels in autism: K+ channel autism spectrum disorder

 .. a mounting body of evidence indicates that ion channel dysfunction may well enhance autism susceptibility also when other contributing alleles are coinherited.

Direct and indirect defects in K+  channels have been implicated in ASDs pathogenesis, likely altering crucial neural network processes in several brain areas including the cerebellum, a structure that emerges as critically involved in determining the core features of ASDs. Abnormal synaptic transmission and dendritic spine pathology play crucial roles in ASDs. Notably, the activity of many thousands synapses is controlled by a single astrocyte. Thus, aberrant astrocyte dependent synaptic functions and CNS development, induced by defective ion channels, represent an interesting causative hypotheses for ASDs

Kv4.2 – ChannelASD

The presence of Kv4.2 channels in hippocampus appears fundamental, mostly at early developmental stage when neuronal activity drives synaptic maturation and network refinement. At hippocampal synapses, the gradual reduction in GluN2B/GluN2A subunit ratio, during post-natal development, is correlated with AMPA expression and synaptic maturation. Ablation of Kv4.2 in mice abolished this phenomenon and resulted in a higher number of silent synapses in the adulthood.  Given the importance of Kv4.2 in brain development and functioning, defects of this channel have been unsurprisingly correlated with a broad spectrum of neurological disorders. Gene deletion in mice leads to increased susceptibility to convulsant stimuli  and truncating mutation of Kv4.2 in humans leads to temporal lobe epilepsy

Kv4.2 channel expression may also participate in establishing the conditions for the development of ASDs, given that Kv4.2 mRNA can bind to the fragile X mental retardation protein (FMRP), which is associated to fragile X syndrome (FXS), the most common monogenic cause of autism and inherited intellectual retardation

Kv7.3 – ChannelASD

KCNQ3 and KCNQ2 gene mutations segregate with various forms of Kv7.3/Kv7.2-channelepsies

  

KCa1.1 – ChannelASD

The calcium-activated K+ 230 (KCa) channels are highly conserved across species, and widely expressed in the human brain.

KCa1.1 loss-of function mutations likely alter pyramidal neurons excitability and result in impairment of neural networks in hippocampus, an area implicated in cognition, mood disorders and ASD. However, these mutations may also affect cerebellar PNs excitability, development, learning and memory processes, suggesting that KCa1.1 channels dysfunction may impact these crucial neurophysiological processes occurring within the cerebellum and result in the psychomotor development and cognition features of ASD

Recently, KCa1.1 channels have been implicated in ASD on a different ground, since their activity is regulated by FMRP, whose mutation produces FXS.

Notably, FMRP can also bind to Na+-activated K+ channel Slack  (i.e. KCa4.1), and thus regulate its activity.

Interestingly, intellectual disability only occurs in those patients who carry mutations in Slack channels, further suggesting a role for this channel type in both epilepsy and cognitive disorders

Inwardly-rectifying K+ channels

Inwardly-rectifying K+ (Kir) channels take their name from the greater conductance at potentials negative to EK, while at more positive values the outward flow of K+  ions is variably inhibited by cytoplasmic polyamines and Mg2+, by means of affinity dependent blockade

Kir2.1 – ChannelASD

Loss-of-function mutations in the KCNJ2 gene are responsible for the rare Andersen-Tawil syndrome a  condition characterized by long QT-syndrome, cardiac arrhythmia, skeletal abnormalities, periodic paralysis, mood disorders and seizures

genetically-induced Kir2.1 defects, beside causing SQT3 syndrome, may possibly result in functional impairment of neural networks where this channel type  resides and contribute to ASDs pathogenesis

I do think Kir2.1 is interesting because it seems to be related hypokalemic sensory overload, which if a key feature of many people’s ASD and indeed ADHD.

Interestingly, a reader with the above Andersen-Tawil syndrome and relatives with ASD, told me how many of them smoke and feel much better by doing so.

Direct block of inward rectifier potassium channels by nicotine.

Abstract

Nicotine has been shown to depolarize membrane potential and to lengthen action potential duration in isolated cardiac preparations. To investigate whether this is a consequence of direct interaction of nicotine with inward rectifier K(+) channels which are a key determinant of membrane potentials, we assessed the effects of nicotine on two cloned human inward rectifier K(+) channels, Kir2.1 and Kir2.2, expressed in Xenopus oocytes and the native inward rectifier K(+) current I(K1) in canine ventricular myocytes. Nicotine suppressed Kir2.1-expressed currents at varying potentials negative to -20 mV, with more pronounced effects on the outward current between -70 and -20 mV relative to the inward current at hyperpolarized potentials (below -70 mV). The inhibition was concentration dependent. For the outward currents recorded at -50 mV, the IC50 was 165 +/- 18 microM. Similar effects of nicotine were observed for Kir2.2. A more potent effect was seen with I(K1) in canine myocytes. Significant blockade ( approximately 60%) was found at a concentration as low as 0.5 microM and the IC50 was 4.0 +/- 0.4 microM. The effects in both oocytes and myocytes were partially reversible upon washout of nicotine. Antagonists of nicotinic receptors (mecamylamine, 100 microM), muscarinic receptors (atropine, 1 microM), and beta-adrenergic receptors (propranolol, 1 microM) all failed to restore the depressed currents, suggesting that nicotine acted directly on Kir channels, independent of catecholamine release. This property of nicotine may explain its membrane-depolarizing and action potential duration-prolonging effects in cardiac cells and may contribute in part to its ability to promote propensity for cardiac arrhythmias

Some people with ASD find nicotine patches helpful.  This could help for various reasons, but if they are Kir2.1 – ChannelASD, then likely it is blocking the misbehaving potassium channels.