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.

Organelle

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.

 

Shared functional defect in IP₃R-mediated calcium signaling in diverse monogenic autism syndromes

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

Study also points to potential new drug discovery advances

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 GABA_B dysfunction.  If you respond well to Baclofen, you likely have a GABA_B 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.

Genetic factors and epigenetic factors for autism: Endoplasmic reticulum stress and impaired synaptic function

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.

Endoplasmic Reticulum Stress As a Therapeutic Target in Cardiovascular Disease

Abstract

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.

Lipophilic And Hydrophilic Statins Differentially Modulate Endoplasmic Reticulum Stress Response In Cardiac Myocytes

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.

Neuroprotective effects of atorvastatin against cerebral ischemia/reperfusion injury through the inhibition of endoplasmic reticulum stress

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.

Peroxisome Proliferator-Activated Receptor γ Activation Restores Islet Function in Diabetic Mice through Reduction of Endoplasmic Reticulum Stress and Maintenance of Euchromatin Structure^▿

ABSTRACT

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.

The role for endoplasmic reticulum stress in diabetes mellitus

Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes

Conclusion

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).