Wednesday, 21 October 2015

Biomarkers in Autism

This post has been sitting unfinished for a while, so I decided to publish it before I forget all about it

The two papers discussed today really confirm much of what we have already established in this blog, but they are very useful as a recap and for those with limited time.

The first paper is extremely comprehensive and, if you go through it very slowly, really tells you much of what you need to know about the biology of autism.  It is some wonder that so few clinicians are aware of these findings.


Autism spectrum disorders (ASDs) are complex, heterogeneous disorders caused by an interaction between genetic vulnerability and environmental factors. In an effort to better target the underlying roots of ASD for diagnosis and treatment, efforts to identify reliable biomarkers in genetics, neuroimaging, gene expression, and measures of the body’s metabolism are growing. For this article, we review the published studies of potential biomarkers in autism and conclude that while there is increasing promise of finding biomarkers that can help us target treatment, there are none with enough evidence to support routine clinical use unless medical illness is suspected. Promising biomarkers include those for mitochondrial function, oxidative stress, and immune function. Genetic clusters are also suggesting the potential for useful biomarkers.

Here are the key parts; I do suggest you read the full text of the paper.

Metabolic Biomarkers

There are no autism-defining, metabolic biomarkers, but examining the biomarkers of pathways associated with ASD can point to potentially treatable metabolic abnormalities and provide a baseline that can be tracked over time. Each child may have different metabolic pathologies related to SNPs, nutrient deficiencies, and toxic exposures. Examples of metabolic disorders that can lead to an autistic-like presentation include phenylketonuria (PKU) (37), disorders of purine metabolism (38), biotinidase deficiency (39), cerebral folate deficiency (40), creatine deficiency (41), and excess propionic acid (which is produced by Clostridium) (42, 43).

A recent review assessed the research on physiological abnormalities associated with ASD (44). The authors identified four main mechanisms that have been increasingly studied during the past decade: immunologic/inflammation, oxidative stress, environmental toxicants, and mitochondrial abnormalities. In addition, there is accumulating research on the lipid, GI systems, microglial activation, and the microbiome, and how these can also contribute to generating biomarkers associated with ASD.

The brain is highly vulnerable to oxidative stress (51), particularly in children (52) during the early part of development (47). As environmental events and metabolic imbalances affect oxidative stress and methylation, they also can affect the expression of genes.

Several studies have detected altered levels of a large collection of substances in body-based fluids from ASD subjects compared to controls (e.g., serum, whole-blood, and CSF) (53). These findings encompass either of two main disease-provoking mechanisms: a CNS disorder that is being detected peripherally [e.g., serotonin and its metabolites, sulfate (54), low platelet levels of gamma-aminobutyric acid (GABA) (55), low oxytocin (which affects social affiliation) (56), and low vitamin D levels (57, 58)] or a systemic abnormality that has repercussions in the brain (59).

Oxidative stress markers

Oxidative stress can be detected by studying antioxidant status, antioxidant enzymes, lipid peroxidation, and protein/DNA oxidation, all of which have been found to be elevated in children with autism (Table (Table2).2). Different subgroups of children with ASD have different redox abnormalities, which may arise from various sources

Measurements of antioxidant status include measurement of glutathione, the primary antioxidant in the protection against oxidative stress, neuroinflammation, and mitochondrial damage (68, 69). Glutathione is instrumental in regulating detoxification pathways and modulates the production of precursors to advanced glycation end products (AGEs) (70). Measuring reduced glutathione, oxidized glutathione, or the ratio of reduced glutathione to oxidized glutathione helps determine the patient’s oxidation status. In many patients with ASD, the ratio of reduced glutathione to oxidized glutathione is decreased, indicating a poor oxidation status

The enzyme glutathione peroxidase has been used as a marker and is typically reduced. There are mixed results concerning the enzyme levels of superoxide dismutase (SOD) (72). Other markers for glutathione inadequacy include alpha hydroxybutyrate, pyroglutamate, and sulfate, which can be assessed in an organic acid test. Lipid peroxidation refers to the oxidative degradation of cell membranes. There is a significant correlation between the severity autism and urinary lipid peroxidation products (67), which are increased in patients with ASD

Plasma F2t-Isoprostanes (F2-IsoPs) are the most sensitive indicator of redox dysfunction and are considered by some to be the gold standard measure of oxidative stress (73). They are increased in patients with ASD and are even higher when accompanied by gastrointestinal dysfunction (73).

Decreased levels of major antioxidant serum proteins transferrin (iron-binding protein) and ceruloplasmin (copper binding protein) have been observed in patients with ASD. The levels of reduction in these proteins correlate with loss of previously acquired language (47) although there are mixed reviews of the significance of this (66).

Plasma 3-chlortyrosine (3CT), a measure of reactive nitrogen species and myeloperoxidase activity, is an established biomarker of chronic inflammatory response. Plasma 3CT levels reportedly increased with age for those with ASD and mitochondrial dysfunction but not for those with ASD without mitochondrial dysfunction (65).

3-Nitrotyrosine (3NT) is a plasma measure of chronic immune activation and is a biomarker of oxidative protein damage and neuron death. This measure correlates with several measures of cognitive function, development, and behavior for subjects with ASD and mitochondrial dysfunction but not for subjects with ASD without a mitochondrial dysfunction (65).

Mitochondrial dysfunction markers

Mitochondrial dysfunction is marked by impaired energy production. Some children with ASD are reported to have a spectrum of mitochondrial dysfunction of differing severity (44) (Table (Table3).3). Mitochondrial dysfunction, most likely an early event in neurodegeneration (76), is one of the more common dysfunctions found in autism (77) and is more common than in typical controls (78). There is no reliable biomarker to identify all cases of mitochondrial dysfunction (79). It is possible that up to 80% of the mitochondrial dysfunction in patients with both ASD and a mitochondrial disorder are acquired rather than inherited (44).

Mitochondrial dysfunction can be a downstream consequence of many proposed factors including dysreactive immunity and altered calcium (Ca2+) signaling (80), increased nitric oxide and peroxynitrite (68), propionyl CoA (81), malnutrition (82), vitamin B6 or iron deficiencies (83), toxic metals (83), elevated nitric acid (84, 85), oxidative stress (86), exposure to environmental toxicants, such as heavy metals (8789), chemicals (90), polychlorinated biphenyls (PCBs) (91), pesticides (92, 93), persistent organic pollutants (POPs) (94), and radiofrequency radiation (95). Other sources of mitochondrial distress include medications such as valproic acid (VPA), which inhibits oxidative phosphorylation (96) and neuroleptics (97, 98).

Markers of mitochondrial dysfunction include lactate, pyruvate and lactate-to-pyruvate ratio, carnitine (free and total), quantitative plasma amino acids, ubiquinone, ammonia, CD, AST, ALT, CO2 glucose, and creatine kinase (CK) (44). Many studies of ASD report elevations in lactate and pyruvate, others report a decrease in carnitine, while others report abnormal alanine in ASD patients (44) or elevations in aspartate aminotransferase and serum CK (99). Increases in lactate are not specific and may only occur during illness, after exercise or struggling during a blood draw (100).

Rossignol and Frye (44) recommend a mitochondrial function screening algorithm. This includes fasting morning labs of lactate, pyruvate, carnitine (free and total), acyl carnitine panel, quantitative plasma amino acids, ubiquinone, ammonia, CK, AST/ALT, CO2, and glucose (44). The interpretation of such a panel and the indications for specific treatments has not yet been established.


The methylation pathway provides methyl groups for many functions, including the methylation of genes, which can result in the epigenetic changes of turning genes on and off (Table (Table4).4). This transfer occurs when S-adenosylmethionine (SAM) donates a methyl group and is transformed to S-adenosylhomocysteine (SAH). SAH can be transferred to homocysteine, which can either be re-methylated to methionine or be transferred by the sulfuration pathway to cysteine to create glutathione. With increased oxidative stress, SAH might be diverted away from the methylation pathway to the sulfuration pathway in order to make more glutathione. This will result in less methionine and less methylation ability.

A marker of methylation dysfunction is decreased SAM/SAH ratio in patients with ASD. Fasting plasma methionine decreases since through SAM it is the main methyl donor. Fasting plasma cysteine, a sulfur containing amino acid is the rate-limiting step in the production of glutathione and is significantly decreased. Plasma sulfate is decreased, which may impair detoxification pathways. Homocysteine is generally increased, but the studies are mixed (66). Vitamin B12 and folate are required for the methylation pathway. The MTHFR genetic SNP is reported to heavily influence the methylation pathway (66).

Immune dysregulation

Cytokine evaluation

Chronic inflammation and microglia cell activation is present in autopsied brains of people with ASD (101, 102) (Table (Table5).5). Factors that increase the risk of activating brain microglia include traumatic brain injury (TBI) (103) reactive oxygen species (104) and a dysfunctional blood brain barrier (105). The blood brain barrier can be compromised by oxidative stress (106), acutely stressful situations (107), elevated homocysteine (108), diabetes (109), and hyperglycemia (110). Cytokines can pass through a permeable blood brain barrier and start this process (111). Hence, cytokines can serve as a marker of the immune dysregulation, which can further complicate ASD.

Autoimmunity and maternal antibodies

Autoimmune autistic disorder is proposed as a major subset of autism (118), and autoimmunity may play a role in the pathogenesis of language and social developmental abnormalities in a subset of children with these disorders (119). There are many autoantibodies found in the nervous system of children with ASD who have a high level of brain antibodies (120, 121). These can be measured as biomarkers in this subset of ASD patients. The anti ganglioside M1 antibodies (122), antineuronal antibodies (123), and serum anti-nuclear antibodies (123, 124) correlate with the severity of autism. Other autoantibodies postulated to play a pathological role in autism include: anti neuron-axon filament protein (anti-NAFP) and glial fibrillary acidic protein (anti-GFAP) (125), antibodies to brain endothelial cells and nuclei (119), antibodies against myelin basic protein (126, 127), and anti myelin associated glycoprotein, an index for autoimmunity in the brain (128). BDNF antibodies were found higher in ASD (129), and low BDNF levels may be involved in the pathophysiology of ASD (130).

Antibodies in patients with autism are found to cells in the caudate nucleus (131), cerebellum (132, 133), hypothalamus and thalamus (121), the cingulate gyrus (134), and to cerebral folate receptors (135). Children with cerebellar autoantibodies had lower adaptive and cognitive function as well as increased aberrant behaviors compared to children without these antibodies (132).

Mother’s immune status

Research studies indicate an association between viral or bacterial infections in expectant mothers and their ASD offspring (136, 137). Maternal antibodies cross the underdeveloped blood brain barrier of the fetus (138) leading to impaired fetal neurodevelopment and long-term neurodegeneration, neurobehavioral, and cognitive difficulties (139).


When the gut becomes inflamed, it breaks down and becomes permeable, sometimes referred to as dysbiosis. Dysbiosis is reported to be an upstream contributing factor to autoimmune conditions and inflammation. Markers under consideration include circulating antibodies against tight junction proteins, LPS, actomyosin (145) calprotectin (146), and lactoferrin (147). Dysbiosis was found in 25.6% of patients with ASD (148). It is proposed to have a direct effect on the brain as it is a hypothesized source of inflammation (149151) and autoimmunity (152, 153), possibly through molecular mimicry (154). Diet is one source of dysbiosis (155).

Amino acids and neuropeptides

Platelet hyperserotonemia is considered one of the most consistent neuromodulator findings in patients with ASD (Table (Table6).6). As for other neuropeptides, a recent review reported approximately 15 components that are altered in ASD compared to controls (53). Among them, interesting research has been done on glutamate, GABA, BDNF, and dopamine and noradrenaline systems. A recent study reported a positive correlation between severity of clinical symptoms and plasma GABA levels in patients with ASD, supporting the idea of a disrupted GABAergic system (156).


Fatty acid analysis

Abnormal fatty acid metabolism may play a role in the pathogenesis of ASD and may suggest some metabolic or dietary abnormalities in the regressive form of autism (42, 157). There is evidence of a relationship between changes in brain lipid profiles and the occurrence of ASD-like behaviors using a rodent model of autism (42). Hyperactivity in patients was inversely related to the fluidity of the erythrocyte membrane and membrane polyunsaturated fatty acid (PUFA) levels (158). Imbalances of membrane fatty acid composition and PUFA loss can affect ion channels and opiate, adrenergic, insulin receptors (159) and the modulation of (Na + K)-ATPase activity (160). Analysis of red blood cell membrane fatty acids is a very sensitive indicator of tissue status and may reflect the brain fatty acid composition (161).
Seventeen percent of children with ASD manifest biomarkers of abnormal mitochondrial fatty acid metabolism, the majority of which are not accounted for by genetic mechanisms (162). Patients with ASD had reduced percentages of highly unsaturated fatty acids (163) and an increase in ω6/ω3 ratio (158).

Biomedical Interventions

There are no published studies of interventions for ASD that use neuroimaging or genetic biomarkers in a prospective manner to guide treatment. Biomedical interventions based on body fluid/product biomarkers have been used in a small but growing numbers of well designed, published studies. Several recent reviews summarize these.

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If you have managed to digest all of that information, here is another very interesting paper.

The researchers are, as so often, from Johns Hopkins.  This time they propose an idea to simplify the understanding of the bewildering number of autism sub-types.

I have frequently commented in this blog that in many identified underlying dysfunctions, being hyper (too much) or hypo (two little) causes the same effect, i.e. autism.

They split autism into:-

·        hyper-active pro-growth signaling pathways (e.g. big heads)
·        hypo-active pro-growth signaling pathways  (e.g. small heads)

So the first question is whether the patient is type A or type B.

It is definitely a step forward in simplifying what is going on, so that one day a clinician, without being a Nobel Laureate, could treat autism without just using trial and error.  If the clinician had also read, and understood, the first paper, he/she really would be able to help the patient.

The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD) presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions.

Friday, 16 October 2015

It’s not Autism, it’s Sotos Syndrome – and more about GABA therapies

I recently returned from a 25 year class reunion; of the 200 or so class members about 120 turned up. Of the 200 we know that at least 5 have a son with autism and at least one has a nephew with autism.  So I had my first ever “autism lunch” discussing all those tricky issues we are left to deal with.

What was immediately apparent was how different each child’s “autism” was and that none of them were the autism-lite variants that are now being so widely diagnosed in older children. or even adults .  Of the six, two are non-verbal, one is institutionalized, yet one talks a lot.  Three sets of parents are big ABA fans and one child did not respond to ABA.

You may be wondering about that high incidence of autism.  This was not a gathering of science boffins or mathematicians; this was at a business school.  One thing is obvious, you can correlate some autism incidence with educational level.  You can connect all sorts of measures of IQ to autism, from having a math prodigy in the family, to having professors at Ivy league type Universities, particularly in Mathematics.  It does appear to be true that the so-called clever genes are also associated with some types of autism.

I presume that if my science-only university organized such events the incidence of autism would be even higher.

On the way back home we met an acquaintance at the airport, who was telling us all about his son with Sotos Syndrome.  "It is not autism", we were informed, but then I am not quite sure what is.  When you look it up, many of the symptoms look just like autism.  In fact, it is a single gene dysfunction that leads to gigantism and various elements of autism.

This brings me to the painting above of Peter the Wild Boy; it is not me I should point out.  The above Peter was a German boy who came to live in England in the 18th Century; he was non-verbal and is now thought to have had Pitt Hopkins Syndrome.  Like Sotos, this is another very rare single gene disorder.

We have already come across Rett Syndrome, which for some reason is treated as autism.

Fragile X is thought of as a syndrome where autism can be comorbid.

Timothy Syndrome is fortunately extremely rare, but I have already drawn on it in my own research into autism.

There are also autism related disorders involving multiple genes.

Prader–Willi syndrome  is a rare genetic disorder in which seven genes (or some subset thereof) on chromosome 15 (q 11–13) are deleted or unexpressed (chromosome 15q partial deletion) on the paternal chromosome.  If the maternally derived genetic material from the same region is affected instead, the sister Angelman Syndrome is the result.

The most frequent disorder caused by known multiple gene overexpression is Down Syndrome.  We saw in earlier post that DS is caused by the presence of all or part of a third copy of chromosome 21.  This results in over-expression of some 300 genes.

Why So Many Syndromes

Even before the days of genetic testing, these syndromes had been identified.  How could that be?  Each syndrome is marked by clear physical differences.

These physical differences where used to identify those affected.

Within autism too, sometimes there are physical differences.  Big heads, small heads, slim stature or heavy stature, advanced bone age or retarded bone age.

So many syndromes , but no therapies

Many of the rare syndromes have their own foundations funding research, mainly on the basis that if there is a known genetic dysfunction there should be matching therapy somewhere.

As of today, there are no approved therapies for any of these syndromes.

The Futility of Genetic Research?

A great deal of autism research funding goes into looking for target genes.  The idea goes that once you know which gene is the problem you can work out how to correct it.  There are numerous scientific journal dedicated to this approach.

Since no progress has been made in treating known genetic conditions leading to “autism”, is all this research effort well directed?  Some clever researchers think it is not.

All I can do is make my observations from the side lines.

What do Down Syndrome, Autism and Pitt Hopkins Syndrome all have in common?

In at least some of those affected, they have the identical excitatory-inhibitory imbalance of GABA, that can be corrected by Bumetanide.

If you did whole exome genetic testing on the responders with these three conditions you would not find a common genetic dysfunction; and yet they respond to the same therapy.

I am actually all for continued genetic research, but those involved have got to understand its limitations, as well as its potential.

More on GABA

This post returns to the theme of the dysfunctional GABA neurotransmitter because the research indicates it is present in numerous of the above-mentioned conditions. 

·        Autism
·        Fragile X
·        Rett Syndrome
·        Down Syndrome
·        Neurofibromatosis type 1
·        Tourette syndrome
·        Schizophrenia
·        Tuberous sclerosis complex (TSC)
·        Prader-Willi syndrome
·        Angelman Syndrome

Based on feedback to me, we should add Pitt Hopkins Syndrome to the above list.

The GABA dysfunction is not the same in all the above conditions, but at least in some people, Bumetanide is effective in cases of autism, Down Syndrome and Pitt Hopkins Syndrome.  I suspect that since it works in mice with Fragile-X , it will work in at least some humans.

GABAA has already been covered in some depth in this blog, but I am always on the lookout for more on this subject, since interventions are highly effective.  It is complicated, but for those of you using Bumetanide, Low Dose Clonazepam, Oxytocin and some even Diamox, the paper below will be of interest.

Regular readers will know that in autism high levels of chloride Cl inside the neuron have been shown to make GABA excitatory rather than inhibitory.  This leads to neurons firing too frequently;  this results in effects ranging from anxiety to seizures and with reduced cognitive functioning.  Therapies revolve around reducing chloride levels, this can be done by restricting the flow in ,or by increasing the flow out.  The Na+/K+/Cl cotransporter NKCC1  imports Cl into the neuron.  By blocking this transporter using Bumetanide you can achieve lower Cl within the neuron, but with this drug you also affect NKCC2, an isoform present in the kidney, which is why Bumetanide is a diuretic.  Some experimental drugs are being tested that block NKCC1 without affecting NKCC2 and better cross the blood brain barrier. 

The interesting new approach is to restore Cl balance by increasing KCC2 expression at the plasma membrane.  This means increasing the number of transporters that carry  Cl  out of the neurons.

In the Modulation of GABAergic transmission paper there is no mention of acetazolamide (Diamox) which I suggested in my posts could also reduce Cl, but via the AE3 exchanger.  This would explain why Diamox can reduce seizures in some people.

The paper does mention oxytocin and it does occur to me that babies born via Cesarean/Caesarean section will completely miss this surge of the oxytocin hormone.  This oxytocin surge is suggested to be key to the GABA switch, which should occur soon after birth when GABA switches from excitatory to inhibitory.  In much autism this switch never takes place.

That would suggest that perhaps all babies born via Caesarean section should perhaps receive an artificial dose of oxytocin at birth.  This might then reduce the incidence of GABA dysfunctions in later life, which would include autism and some epilepsy.

Indeed, children born by Caesarean section (CS) are 20% more likely to develop autism.

Conclusions and Relevance  This study confirms previous findings that children born by CS are approximately 20% more likely to be diagnosed as having ASD. However, the association did not persist when using sibling controls, implying that this association is due to familial confounding by genetic and/or environmental factors.

So as not to repeat the vaccine/autism scare, the researchers do not say that Caesarean section leads to more autism, rather that the kinds of people who are born by Caesarean section already had an elevated risk of autism.  This is based on analysing sibling pairs, but I do not entirely buy into that argument.  They do not want to scare people from having a procedure that can be life-saving for mother and baby.

If you look at it rationally, you can see that the oxytocin surge at birth is there for an evolutionary reason.  It is very easy to recreate it with synthetic oxytocin.

Another interesting point is in the conflict of interest statement:-

Laura Cancedda is on the Provisional Application: US 61/919,195, 2013. Modulators of Intracellular Chloride Concentration For Treating An Intellectual Disability

Regular readers will note that in this blog we have known for some time that modifying GABAA leads to improved cognitive function.  I even suggested to Ben-Ari that IQ should be measured in their autism trials for Bumetanide.  IQ is much less subjective than measures of autism.


My conclusion is that while genetic testing has its place, it is more productive to look at identifying and treating the downstream dysfunctions that are shared by many individual genetic dysfunctions.

By focusing on individual genes there is a big risk of just giving up, so if you have Pitt Hopkins Syndrome, like Peter the Wild Boy, it is a single gene cause of “autism” and there is no known therapy.  Well it seems that it shares downstream consequences with many other types of autism, so it is treatable after all.

I also think more people need to consider that cognitive dysfunction (Intellectual Disability/MR) may indeed be treatable, and not just via GABA; so good luck to Laura Cancedda.

Friday, 2 October 2015

Is dysregulated IP3R calcium signaling a nexus where genes altered in ASD converge to exert their deleterious effect?

Place de l'Étoile in Paris and the avenues radiating from it.  The Arc de Triomphe in the centre would be the IP3 receptor

There are a small number of researchers in the field of autism who really do seem to know what they are talking about;  one of those is Jay Gargus, from University of California at Irvine.  He is one of the few well versed on ion channel dysfunctions (channelopathies).  Today we look at his recent paper relating to the IP3R calcium channel in something called the endoplasmic reticulum (ER).

Gargus’ recent findings relate to calcium signaling, which we have seen previously in this blog to be dysfunctional in autism.  Blocking one type of calcium channel, with Verapamil, has had a remarkable effect in the children of some of those reading this blog; this has included resolving aggressive behavior, resolving GI problems and, most recently, greatly reducing seizures.  An interesting side effect of this drug is that it protects older people from Type 2 diabetes.

We will also encounter yet another kind of stress, ER stress (endoplasmic reticulum stress), which plays a role in many disorders including Type 2 diabetes and is suggested by some Japanese researchers to play a role in autism.  Interestingly some of my pet autism interventions are known to affect ER stress.

As usual in this blog, I will skip some of the complexities, but we do need to know some new words.  The explanation is mainly courtesy of the remarkable Wikipedia.


In cell biology, an organelle is a specialized subunit within a cell that has a specific function.  Individual organelles are usually separately enclosed within their own lipid bilayers.  These lipid bilayers are also extremely important and need to be perfectly intact.  It does appear that these lipid bilayers are a little different in autism.

Components of a typical animal cell:

  1.     Nucleolus
  2.     Nucleus
  3.     Ribosome (little dots)
  4.    Vesicle
  5.    Rough endoplasmic reticulum
  6.    Golgi apparatus (or "Golgi body")
  7.    Cytoskeleton
  8.   Smooth endoplasmic reticulum
  9.   Mitochondrion
  10.   Vacuole
  11.   Cytosol (fluid that contains organelles)
  12.    Lysosome
  13.    Centrosome
  14.    Cell membrane

Endoplasmic Reticulum (ER) and ER Stress

The endoplasmic reticulum (ER) is the cellular organelle in which protein folding, calcium homeostasis, and lipid biosynthesis occur. Stimuli such as oxidative stress, ischemic insult, disturbances in calcium homeostasis, and enhanced expression of normal and/or folding-defective proteins lead to the accumulation of unfolded proteins, a condition referred to as ER stress.

Inositol trisphosphate receptor (InsP3R) or IP3R

IP3R is a Ca2+ channel activated by inositol trisphosphate (InsP3). InsP3R is very diverse among organisms, and is necessary for the control of cellular and physiological processes including cell division, cell proliferation, apoptosis, fertilization, development, behavior, learning and memory. Inositol triphosphate receptor represents a dominant second messenger leading to the release of Ca2+ from intracellular store sites.

It has a broad tissue distribution but is especially abundant in the cerebellum. Most of the InsP3Rs are found in the cell integrated into the endoplasmic reticulum.

Genes and autism

It is a widely held view that autism is essentially a genetic condition with some environmental triggers.

What is strange is that many hundreds, and later I suspect thousands, of genes are known to be implicated.  Do these lead to thousands of unique dysfunctions that ultimately manifest themselves as what we, rather clumsily, describe as “autism”?  This appears to be unlikely, more likely is that a much smaller number of downstream dysfunctions are involved.  This is behind what is suggested later by Gargus.

What I have always found odd is that siblings with idiopathic autism do NOT generally share the same genetic variations.  Most autism is called idiopathic, which means of unknown cause.  This is why I have not done any genetic testing on my son.

If siblings have Fragile X, then of course they do have the same genetic defect; the brother will likely be much more severely affected than the sister.

It occurs to me that unless the idiopathic autistic siblings live under some high voltage power cables, next to a TV transmitter or a chemical factory, the genetic testing must be missing something.  We have seen that sequencing the exome, the current “ultimate genetic test”, in fact only looks at 5% of genome.  We have also seen that in the remaining 95% are the so called enhancers and silencers of the genes in the exome.  We have also seen that overexpression of a perfect gene (as in Down syndrome) can do as much damage as a faulty gene.

My advice is to look in the remaining 95% of the genome.

Gargus, IP3R and Autism

Having completed the introduction now we can move on to the Gargus paper.

He is suggesting that a dysfunction at a specific calcium channel in the ER may be the common dysfunction triggered by “autism genes”.

So far he has only tested his idea on some single gene autisms, fragile X and tuberous sclerosis.

Autism spectrum disorder (ASD) affects 2% of children, and is characterized by impaired social and communication skills together with repetitive, stereotypic behavior. The pathophysiology of ASD is complex due to genetic and environmental heterogeneity, complicating the development of therapies and making diagnosis challenging. Growing genetic evidence supports a role of disrupted Ca2+ signaling in ASD. Here, we report that patient-derived fibroblasts from three monogenic models of ASD—fragile X and tuberous sclerosis TSC1 and TSC2 syndromes—display depressed Ca2+ release through inositol trisphosphate receptors (IP3Rs). This was apparent in Ca2+ signals evoked by G protein-coupled receptors and by photoreleased IP3 at the levels of both global and local elementary Ca2+ events, suggesting fundamental defects in IP3R channel activity in ASD. Given the ubiquitous involvement of IP3R-mediated Ca2+ signaling in neuronal excitability, synaptic plasticity, gene expression and neurodevelopment, we propose dysregulated IP3R signaling as a nexus where genes altered in ASD converge to exert their deleterious effect. These findings highlight potential pharmaceutical targets, and identify Ca2+ screening in skin fibroblasts as a promising technique for early detection of individuals susceptible to ASD.

This part I found interesting:-

Because of the ubiquitous nature of IP3R signaling and its diverse roles in almost all cells of the body, deficits in IP3-mediated Ca2+ signaling may not be limited to neurological correlates of ASD, but may also explain other characteristic ASD-associated heterogeneous symptoms, such as those of the gastrointestinal tract and immune system.  Furthermore, since the ER serves as a sensor of a host of environmental stressors, this same mechanism may contribute to the known environmental component
to the ASD phenotype, and holds the potential to reveal relevant stressors.

Is it a coincidence that the Verapamil therapy I propose also benefits autism symptoms linked to the gastrointestinal tract and immune system (mast cells/allergy) and also now seizures (hyper excitability)?  I think not,

Here is the rather easier to read press release from the University:-

UCI researchers find biomarker for autism that may aid diagnostics

Irvine, Calif., Sept. 22, 2015 — By identifying a key signaling defect within a specific membrane structure in all cells, University of California, Irvine researchers believe, they have found both a possible reliable biomarker for diagnosing certain forms of autism and a potential therapeutic target.

Dr. J. Jay Gargus, Ian Parker and colleagues at the UCI Center for Autism Research & Translation examined skin biopsies of patients with three very different genetic types of the disorder (fragile X syndrome and tuberous sclerosis 1 and 2). They discovered that a cellular calcium signaling process involving the inositol trisphosphate receptor was very much altered.

This IP3R functional defect was located in the endoplasmic reticulum, which is among the specialized membrane compartments in cells called organelles, and may underpin cognitive impairments – and possibly digestive and immune problems – associated with autism.

“We believe this finding will be another arrow in the quiver for early and accurate diagnoses of autism spectrum disorders,” said Gargus, director of the Center for Autism Research & Translation and professor of pediatrics and physiology & biophysics. “Equally exciting, it also presents a target of a molecular class already well-established to be useful for drug discovery.”

Study results appear online in Translational Psychiatry, a Nature publication.

Autism spectrum disorder is a range of complex neurodevelopmental disorders affecting 2 percent of U.S. children. The social and economic burden of ASD is enormous, currently estimated at more than $66 billion per year in the U.S. alone. Drug development has proven problematic due to the limited understanding of the underlying causes of ASD, as demonstrated by the recent failure of several much anticipated drug trials.

There are also no current, reliable diagnostic biomarkers for ASD. Genetic research has identified hundreds of genes that are involved, which impedes diagnosis and, ultimately, drug development. There simply may be too many targets, each with too small an effect.

Many of these genes associated with ASD, however, have been found to be part of the same signaling pathway, and multiple defects in this pathway may converge to produce a large functional change.

The UCI scientists detected such a convergence in the IP3R calcium channel in an organelle called the endoplasmic reticulum. Organelles are membrane structures within cells with specialized cellular functions. According to Gargus, diseases of the organelles, such as the ER, are an emerging field in medicine, with several well-recognized neurological ailments linked to two other ones, the mitochondria and lysosomes.

The IP3R controls the release of calcium from the ER. In the brain, calcium is used to communicate information within and between neurons, and it activates a host of other cell functions, including ones regulating learning and memory, neuronal excitability and neurotransmitter release – areas known to be dysfunctional in ASD.
“We propose that the proper function of this channel and its signaling pathway is critical for normal performance of neurons and that this signaling pathway represents a key ‘hub’ in the pathogenesis of ASD,” said Parker, a fellow of London’s Royal Society and UCI professor of neurobiology & behavior, who studies cellular calcium signaling.

To see if IP3R function is altered across the autism spectrum, clinical researchers at The Center for Autism & Neurodevelopmental Disorders – which is affiliated with the Center for Autism Research & Translation – are currently expanding the study and have begun to examine children with and without typical ASD for the same signaling abnormalities. These patients undergo complete behavioral diagnostic testing, and sophisticated EEG, sleep and biochemical studies are performed. This includes the sequencing of their entire genome. Also, skin cell samples are cultured and made available to lab-based researchers for functional assays.

In the area of drug discovery, scientists at the Center for Autism Research & Translation continue to probe the IP3R channel, specifically how it regulates the level of neuron excitability. The brains of people who have autism show signs of hyperexcitability, which is also seen in epilepsy, a disorder increasingly found to be associated with ASD. Cells from individuals who have autism exhibit depressed levels of calcium signaling, and this might explain why these patients experience this hyperexcitability. By restoring the release of calcium from the IP3R, the researchers believe, they can apply a “brake” on this activity.

ER Stress

As we saw above, the endoplasmic reticulum (ER) is the cellular organelle in which protein folding, calcium homeostasis, and lipid biosynthesis occur. Stimuli such as oxidative stress, ischemic insult, disturbances in calcium homeostasis, and enhanced expression of normal and/or folding-defective proteins lead to the accumulation of unfolded proteins, a condition referred to as ER stress.
We know that we usually have oxidative stress in autism and we know that calsium homeostasis is disturbed, so it is not surprising if we found ER stress in autism.

The following paper is not open access but it does suggest that ER stress leads to impaired synaptic function and specifically GABAB dysfunction.  If you respond well to Baclofen, you likely have a GABAB dysfunction.  Based on anecdotal evidence I would suggest that people with Asperger’s and anxiety might well have ER stress, since they are the ones that respond well to baclofen.

The molecular pathogenesis of ASD (autism spectrum disorder), one of the heritable neurodevelopmental disorders, is not well understood, although over 15 autistic-susceptible gene loci have been extensively studied. A major issue is whether the proteins that these candidate genes encode are involved in general function and signal transduction. Several mutations in genes encoding synaptic adhesion molecules such as neuroligin, neurexin, CNTNAP (contactin-associated protein) and CADM1 (cell-adhesion molecule 1) found in ASD suggest that impaired synaptic function is the underlying pathogenesis. However, knockout mouse models of these mutations do not show all of the autism-related symptoms, suggesting that gain-of-function in addition to loss-of-function arising from these mutations may be associated with ASD pathogenesis. Another finding is that family members with a given mutation frequently do not manifest autistic symptoms, which possibly may be because of gender effects, dominance theory and environmental factors, including hormones and stress. Thus epigenetic factors complicate our understanding of the relationship between these mutated genes and ASD pathogenesis. We focus in the present review on findings that ER (endoplasmic reticulum) stress arising from these mutations causes a trafficking disorder of synaptic receptors, such as GABA (γ-aminobutyric acid) B-receptors, and leads to their impaired synaptic function and signal transduction. In the present review we propose a hypothesis that ASD pathogenesis is linked not only to loss-of-function but also to gain-of-function, with an ER stress response to unfolded proteins under the influence of epigenetic factors.

I was surprised how much is known about ER stress, there is even a scientific journal devoted to it.

As is often the case, the literature is again full papers like the one below suggesting something, ER stress in this case, is a good drug target, but then do not suggest any drugs.

Cardiovascular disease constitutes a major and increasing health burden in developed countries. Although treatments have progressed, the development of novel treatments for patients with cardiovascular diseases remains a major research goal. The endoplasmic reticulum (ER) is the cellular organelle in which protein folding, calcium homeostasis, and lipid biosynthesis occur. Stimuli such as oxidative stress, ischemic insult, disturbances in calcium homeostasis, and enhanced expression of normal and/or folding-defective proteins lead to the accumulation of unfolded proteins, a condition referred to as ER stress. ER stress triggers the unfolded protein response (UPR) to maintain ER homeostasis. The UPR involves a group of signal transduction pathways that ameliorate the accumulation of unfolded protein by increasing ER-resident chaperones, inhibiting protein translation and accelerating the degradation of unfolded proteins. The UPR is initially an adaptive response but, if unresolved, can lead to apoptotic cell death. Thus, the ER is now recognized as an important organelle in deciding cell life and death. There is compelling evidence that the adaptive and proapoptotic pathways of UPR play fundamental roles in the development and progression of cardiovascular diseases, including heart failure, ischemic heart diseases, and atherosclerosis. Thus, therapeutic interventions that target molecules of the UPR component and reduce ER stress will be promising strategies to treat cardiovascular diseases. In this review, we summarize the recent progress in understanding UPR signaling in cardiovascular disease and its related therapeutic potential. Future studies may clarify the most promising molecules to be investigated as targets for cardiovascular diseases.

However all is not lost, a little digging uncovers several existing substances that affect ER Stress.

Atorvastatin, long part of my autism Polypill, is quite prominent.  Atorvastatin is lipophilic statin, which means it can better cross the blood brain barrier.  By chance it is the statin with the least side effects.

Statins inhibit HMG-CoA reductase, target mevalonic acid synthesis, and limit cholesterol biosynthesis. HMG-CoA reductase is expressed in the membrane of the endoplasmic reticulum (ER). Statins are prescribed to prevent cardiovascular events.
In cultured neonatal mouse cardiac myocytes the lipophilic statin atorvastatin and the hydrophilic statin pravastatin both up-regulated PDI, indicating unfolded protein response (UPR) meant to relieve ER stress. Only atorvastatin increased ER stress, growth arrest, and induced apoptosis via induction of CHOP, Puma, active Caspase-3 and PARP. Dose-dependent release of LDH was only observed in atorvastatin treated cells (1–10 μM). Hearts of mice treated with atorvastatin (5mg/kg/day for 7 months) showed protein aggresomes and autophagosomes when compared to vehicle treated controls. While atorvastatin changed mitochondrial ultrastructure, no differences in cardiac function, exercise ability or creatine kinase levels were found.
We show differential activation of ER stress by atorvastatin and pravastatin in cardiac myocytes. Our results provide a novel mechanism through which specific statins therapeutically modulate the balance of UPR/ER stress responses thereby possibly influencing cardiac remodeling.

Cerebral ischemia triggers secondary ischemia/reperfusion injury and endoplasmic reticulum stress initiates cell apoptosis. However, the regulatory mechanism of the signaling pathway remains unclear. We hypothesize that the regulatory mechanisms are mediated by the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α in the endoplasmic reticulum stress signaling pathway. To verify this hypothesis, we occluded the middle cerebral artery in rats to establish focal cerebral ischemia/reperfusion model. Results showed that the expression levels of protein kinase-like endoplasmic reticulum kinase and caspase-3, as well as the phosphorylation of eukaryotic initiation factor 2α, were increased after ischemia/reperfusion. Administration of atorvastatin decreased the expression of protein kinase-like endoplasmic reticulum kinase, caspase-3 and phosphorylated eukaryotic initiation factor 2α, reduced the infarct volume and improved ultrastructure in the rat brain. After salubrinal, the specific inhibitor of phosphorylated eukaryotic initiation factor 2α was given into the rats intragastrically, the expression levels of caspase-3 and phosphorylated eukaryotic initiation factor 2α in the were decreased, a reduction of the infarct volume and less ultrastructural damage were observed than the untreated, ischemic brain. However, salubrinal had no impact on the expression of protein kinase-like endoplasmic reticulum kinase. Experimental findings indicate that atorvastatin inhibits endoplasmic reticulum stress and exerts neuroprotective effects. The underlying mechanisms of attenuating ischemia/reperfusion injury are associated with the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/caspase-3 pathway.

The nuclear receptor peroxisome proliferator-activated receptor γ (PPAR-γ) is an important target in diabetes therapy, but its direct role, if any, in the restoration of islet function has remained controversial. To identify potential molecular mechanisms of PPAR-γ in the islet, we treated diabetic or glucose-intolerant mice with the PPAR-γ agonist pioglitazone or with a control. Treated mice exhibited significantly improved glycemic control, corresponding to increased serum insulin and enhanced glucose-stimulated insulin release and Ca2+ responses from isolated islets in vitro. This improved islet function was at least partially attributed to significant upregulation of the islet genes Irs1, SERCA, Ins1/2, and Glut2 in treated animals. The restoration of the Ins1/2 and Glut2 genes corresponded to a two- to threefold increase in the euchromatin marker histone H3 dimethyl-Lys4 at their respective promoters and was coincident with increased nuclear occupancy of the islet methyltransferase Set7/9. Analysis of diabetic islets in vitro suggested that these effects resulting from the presence of the PPAR-γ agonist may be secondary to improvements in endoplasmic reticulum stress. Consistent with this possibility, incubation of thapsigargin-treated INS-1 β cells with the PPAR-γ agonist resulted in the reduction of endoplasmic reticulum stress and restoration of Pdx1 protein levels and Set7/9 nuclear occupancy. We conclude that PPAR-γ agonists exert a direct effect in diabetic islets to reduce endoplasmic reticulum stress and enhance Pdx1 levels, leading to favorable alterations of the islet gene chromatin architecture.

PPAR-γ agonist pioglitazone is known to have a positive effect in some autism, but it does have side effects.

Other PPAR-γ agonists include Ibuprofen and Tangeretin (sold as Sytrinol).

ER stress plays a key role in diabetes and some obesity.


So as to Gargus’ question and the tittle of this post:

Is dysregulated IP3R calcium signaling a nexus where genes altered in ASD converge to exert their deleterious effect?

The researchers are now looking at children with and without idiopathic autism to see if dysregulated IP3R calcium is indeed a reliable marker.

Given so many things can lead to behavior diagnosed as autism, I think they will just identify an IP3R cluster.  Hopefully it is a big one.  Then they can find a therapy to  release calcium from IP3R.

Where does ER stress fit into this picture?  Gargus briefly mentions stressors and unfolded protein responses:-

In addition to its role in Ca2+ homeostasis, the ER serves as a key integrator of environmental stressors with metabolism and gene expression, as it mediates a host of broad ranging cell stress responses such as the heat shock and unfolded protein responses

I think he is missing something here. 

The endoplasmic reticulum (ER) is the cellular organelle in which lipid biosynthesis occurs as well as protein folding and calcium homeostasis.

I suspect all three may be dysfunctional.  We have ample evidence of lipid abnormalities in autism and even lipid bilayer abnormalities. The Japanese research referred to above suggests protein folding dysfunction.  Note that what reduces ER stress (statins and tangeretin) also reduces cholesterol.

The good news is that plenty of therapeutic avenues already exist.

The other good news is that after 261 posts of this blog, so many pieces of the autism puzzle seem to be fitting together, not perfectly, but well enough to figure out how to treat multiple aspects of classic autism.

I did stumble across a recent quote by Ricardo Dolmetsch, formerly of Stanford and currently Global Head of Neuroscience at drug maker Novartis.  He also has a son with classic autism.  He was quoted again saying there are currently no drug treatments for core autism.  He knows a thousand times more about biology than me, but he is totally wrong to keep saying that there is nothing you can do beyond behavioral education and, if that fails, institutionalization.  I did write to him a while back and I do feel rather sorry for him, since it was his research on Timothy Syndrome that indirectly led to my Verapamil “discovery”.

Some people are just too clever (him, not me).