Thursday, 12 December 2019

ER Stress and Protein Misfolding in Autism (and IP3R again) and perhaps what to do about it - Activation of Sigma-1 Chaperone Activity by Afobazole?

Today’s post may require even regular readers to refresh their memories and look up the meaning of some words.

There really is a lot in this post. I had to read it twice.
As is often the case, this post started at the end with the therapy (a trial of Afobazole) and then I just looked at why it might be effective.

Activate Chaparones

Today's post is all about sigma-1 receptors and the many clever things that happen when they are activated.

Even the above diagram showing the effect of Sigma-1R is incomplete!

In the mouse study below, the Russian researchers looked at the effect of Afobazole treatment just over a few days; I think other effects might have developed if they had looked at an extended time period. They focus on Sigma1-R receptors modulating NMDA-based neurotransmission, but there seem to many possible further effects within the Endoplasmic Reticulum that relate specifically to autism. These researchers have published other studies using Afobazole, including recently one on Parkinson's disease. 

The multifactorial nature of ASD precludes the use of its modern genetic models in the study of pharmacologic effects exerted on entire symptomatic complex of autism although they could relate functional correction of ASD with a certain gene. In experiments, the models of idiopathic ASD are based on inbred mice selected by behavioral phenotype. BALB/c mice demonstrate pronounced autism-relevant behavioral phenotype characterized by low level of social relations, high levels of anxiety and aggression, increased brain weight, undeveloped corpus callosum, and lower serotonin concentration in the brain [7,12]. The emotional stress reaction (ESR) in these animals is associated with weaker binding capacity of the benzodiazepine site in GABAA receptor [6]. Transformation of ESR into the cell stress augments reception in the domain responsible for binding the endo- and exogenous ligands of sigma 1 receptor chaperon protein (Sigma1R) [1] responsible for adaptive reactions [8]. In addition, Sigma1R stimulate BDNF and NGF synthesis, promote the growth and arborization of nerve terminals, and control functional activity of potassium, calcium, and chloride ion channels and a variety of neuroreceptors [5,8,13]. Thus, this chaperon protein can be an important player in physiological and pharmacological regulation of ASD features.

Afobazole is a non-benzodiazepine anxiolytic drug that acts via activation of Sigma1R and interaction with MT1 and MT3 melatonin receptors and a regulatory site of MAO-A [4]. Clinical observations showed that Afobazole optimizes psychophysiological parameters in emotionally unstable persons without impairing attention, psychomotor responsiveness, and decision-making alertness in the model of operator work. The drug is characterized by mild activation effect and reduces anxiety, thus promoting adaptation to novel environment [2]. This work was designed to examine the effects of Afobazole on cognitive rigidity in BALB/c mice.

Evidently, enhanced motor activity of Afobazole treated BALB/c mice reflected the anxiolytic effect of this drug, which stimulated exploratory behavior aimed at solving the novel task. Thus, Afobazole improved adaptation to changing environment

The present study revealed the potency of Afobazole to promote retraining and reversal learning of BALB/c mice, which manifested in increased rate of adaptation to novel environment and more effective solution of the modified task. Afobazole interacts with Sigma1R receptors and induces their activation [1]. It cannot be excluded that the anxiolytic effect of Afobazole is accompanied by up-regulation of Sigma1R chaperone functions, because this drug normalizes the stress-induced down-regulation of reception in benzodiazepine site of GABAA receptor [6]. A large cluster of Sigma1R receptor was revealed in the hippocampus that plays a key role in adaptive behavior related to building of spatial cognitive maps, learning, and memory. Sigma1R receptors modulate NMDA-based neurotransmission; they can enhance spontaneous release of glutamate in the hippocampus, potentiate glutamate-induced release of neurotrophic factor, and participate in synaptic plasticity [8]. However, Sigma1R receptors regulate cognitive processes under disturbed neurotransmitter balance only. All these data agree with our previous findings and with the current views on the mechanism of Afobazole action [1,4,5]. Thus, the mode of action and pharmacological effects of Afobazole are promising features, which justify the hopes to use it as an effective remedy to treat cognitive rigidity in ASD patients

More on sigma-1 and NMDA receptors:-

NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

Sigma-1 receptors (σ-1Rs) are endoplasmic reticulum resident chaperone proteins implicated in many physiological and pathological processes in the CNS. A striking feature of σ-1Rs is their ability to interact and modulate a large number of voltage- and ligand-gated ion channels at the plasma membrane. We have reported previously that agonists for σ-1Rs potentiate NMDA receptor (NMDAR) currents, although the mechanism by which this occurs is still unclear. In this study, we show that in vivo administration of the selective σ-1R agonists (+)-SKF 10,047 [2S-(2α,6α,11R*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride (N-allylnormetazocine) hydrochloride], PRE-084 (2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate hydrochloride), and (+)-pentazocine increases the expression of GluN2A and GluN2B subunits, as well as postsynaptic density protein 95 in the rat hippocampus. We also demonstrate that σ-1R activation leads to an increased interaction between GluN2 subunits and σ-1Rs and mediates trafficking of NMDARs to the cell surface. These results suggest that σ-1R may play an important role in NMDAR-mediated functions, such as learning and memory. It also opens new avenues for additional studies into a multitude of pathological conditions in which NMDARs are involved, including schizophrenia, dementia, and stroke.

Afobazole is primarily used to treat mild anxiety.  Indeed it appears that sigma-1 receptor activation ameliorates anxiety through NR2A-CREB-BDNF signalling.  NR2A is a sub-unit of NMDA receptors.

Sigma-1 receptor activation ameliorates anxiety-like behavior through NR2A-CREB-BDNF signaling pathway in a rat model submitted to single-prolonged stress.

It does seem that activating the sigma-1 receptor might be another of those nexuses in treatment, where different dysfunctions in autism might well respond to the same therapy.  Recall how many functions of the Endoplasmic Reticulum are impaired in autism, such as the all important calcium homeostasis. 

It also might account for some of the people with autism that respond to Memantine/Nameda and Donepezil. My old post on IP3R and the endoplasmic reticulum, looked at the interesting hypothesis proposed by Gargus.

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

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.

Prolonged ER stress typically results in cell death by apoptosis; an answer to “where did all the Purkinje cells go?”, in people with severe autism, perhaps.  

ER stress is known to affect "neurite outgrowth", which is all the bits like dendrites. Purkinje cells have the most dendrites.  Loss of Purkinje cells affects your motor skills and the Pukinje cell layer is found to be severely depleted in people with autism. Many people with autism, even some Aspies, have poor motor skills. 

Research shows that exercise suppresses Purkinje cell losss and that the ones remaining in autistic brains are likley dysfunctional. When synaptic pruning works correctly each Purkinje cell in an adult receives only one climbing fiber input, in ASD models there is an abundance of climbing fibers. It does seem that with enough practice you may overcome poor motor skills in autism.

Interestingly, in the research we see that both Atorvastatin and Rosuvastatin enhance neurite outgrowth. Atorvastatin has long been part of my PolyPill for severe autism.

For effective synaptic pruning you need microglia that are not activated, so shift them back to M0.  This is another part of my PolyPill.

Protein folding is the physical process by which a protein chain acquires its native 3-dimensional structure, a conformation that is usually biologically functional.

Molecular chaperones are a class of proteins that aid in the correct folding of other proteins, sigma-1 is one example.

A protein is considered to be misfolded if it cannot achieve its normal native state and function.

Incorrect protein folding is a common feature of neurodegenerative disease.

An emerging approach is to use pharmaceutical chaperones to fold mutated proteins to render them functional.

As will be seen in the research, ER stress is a feature of severe autism and indeed schizophrenia.

The result is that perfect genes do not produce perfect functional proteins.  They produce misfolded perfect proteins that cannot function.

Misfolded proteins can interact with one another and form structured aggregates and gain toxicity through intermolecular interactions, but that would lead to a degenerative brain disease (Alzheimer’s, Huntington’s, Parkinson’s etc).  So, the misfolding in autism, if present, it not catastrophic (except perhaps for those Purkinje cells); but a nice folded shirt does give a better result than a crumpled one. Better keep your proteins neatly folded. 

Is there ER Stress in Autism?

The short answer is yes, at least in the kind of autism that leads to young human brains  being donated to medical research.  

Autism research based on human brain tissue is biased towards severe autism (they can die in childhood), whereas many/most clinical trials are now biased towards mild autism (having participants who are fully verbal and cooperative makes life easier for researchers, but their young brains do not get donated to medical research).   

Altered Expression of Endoplasmic Reticulum Stress-Related Genes in the Middle Frontal Cortex of Subjects with Autism Spectrum Disorder

The endoplasmic reticulum (ER) is an important organelle responsible for the folding and sorting of proteins. Disturbances in ER homeostasis can trigger a cellular response known as the unfolded protein response, leading to accumulation of unfolded or misfolded proteins in the ER lumen called ER stress. A number of recent studies suggest that mutations in autism spectrum disorder (ASD)-susceptible synaptic genes induce ER stress. However, it is not known whether ER stress-related genes are altered in the brain of ASD subjects. In the present study, we investigated the mRNA expression of ER stress-related genes (ATF4, ATF6, PERK, XBP1, sXBP1, CHOP, and IRE1) in the postmortem middle frontal gyrus of ASD and control subjects. RT-PCR analysis showed significant increases in the mRNA levels of ATF4, ATF6, PERK, XBP1, CHOP, and IRE1 in the middle frontal gyrus of ASD subjects. In addition, we found a significant positive association of mRNA levels of ER stress genes with the diagnostic score for stereotyped behavior in ASD subjects. These results, for the first time, provide the evidence of the dysregulation of ER stress genes in the brain of subjects with ASD.

Increase in mRNA levels of endoplasmic reticulum stress genes in the middle frontal gyrus of autism spectrum disorder (ASD) subjects. mRNA levels of endoplasmic reticulum stress genes were determined by qRT-PCR in the middle frontal gyrus of ASD (n = 13) and control (n = 12) subjects. The Ct values were normalized to the mean of 18S and β-actin. a Activating transcription factor 4 (ATF4). b Activating transcription factor 6 (ATF6). c Protein kinase-like endoplasmic reticulum kinase (PERK). d X-box protein 1 (XBP1). e Spliced X-box protein 1 (sXBP1). f CCAAT-enhancer-binding protein homologous protein (CHOP). g Inositol-requiring enzyme 1 (IRE1). * p < 0.05, ** p < 0.01, and *** p < 0.0001 vs. controls.

We found significant increases in ATF4, ATF6, PERK, XBP1, CHOP, and IRE1 mRNA levels in the middle frontal gyrus of ASD subjects. Among these molecules, CHOP is known to interact with the heterodimeric receptors GABAB1aR/GABAB2R and inhibits the formation of heterodimeric complexes resulting in the intracellular accumulation and reduced cell surface expression of receptors [34]. Interestingly, decreased levels of GABAB1R and GABAB2R have been found in the brain of ASD subjects [35]. What are the downstream mechanism mediating ER stress-induced changes in central nervous system function? One potential mechanism is inflammation. Accumulating evidence suggest that pathways activated by the ER stress response induce inflammation. When activated, all three sensors of the UPR, PERK, IRE1, and ATF6, participate in upregulating inflammatory processes. It is known that PERK and IRE1 activation can interfere with NFκB inhibitory signals, thereby promoting a proinflammatory response [36]. In addition, CHOP has been shown to induce the expression of proinflammatory cytokines such as IL-23 [37]. Moreover, ER stress activates NLRP3 inflammasomes via thioredoxin-interacting protein (TXNIP), leading to increases in proinflammatory cytokine levels [38,39]. In this regard, our earlier studies using the same tissue samples of the present study found increased levels of proinflammatory cytokines IL-1β and IFN-γ in the middle frontal gyrus of ASD subjects [30].
Also, chronic ER stress is known to induce cellular apoptosis through a number of pathways including CHOP, calcium signaling, and microRNAs [40]. Activation of PERK triggers a series of transcriptional responses mediated by ATF4 and CHOP, which in turn inhibit the expression of anti-apoptotic protein Bcl2 and induce pro-apoptotic proteins such as Bcl2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) [40]. The induction of pro-apoptotic signaling pathway results in the activation of BAX- and BAK-dependent apoptosis at the mitochondria and the activation of the caspase cascade [41]. Interestingly, decrease in Bcl2, but increase in p53 protein levels have been reported in the frontal cortex of ASD subjects [42].
We found that mRNA levels of ER stress genes are positively associated with the stereotyped behavior domain of the ADI-R. It has been shown that autism-associated mutations in NLGN3, which is known to induce ER stress, also increase stereotyped behavior in mice [43]. Similarly, mice lacking CNTNAP2 showed increased repetitive behaviors such as grooming and digging [44], further suggesting that abnormalities in ASD candidate genes implicated in ER stress induce stereotyped behavior in rodents. The present data was collected in a relatively small number of study subjects, which needs further investigation using larger samples before a conclusion can be drawn. Also, the change in gene expression as part of ER stress axis in ASD could be associated with other priming factors functional on different coordinates of this complex neurodevelopmental disorder. Additional studies are warranted to analyze the ER stress-inducing factors with direct relationship to the pathophysiological changes associated with ASD. To further establish a definitive role of ER stress in ASD pathophysiology, the following questions still need to be addressed: (1) Is ER stress in ASD of neurodevelopmental origin? (2) Are there factors other than mutant synaptic proteins that can trigger ER stress leading to ASD phenotype? (3) Is inflammation triggering ER stress or is ER stress triggering inflammation leading to ASD phenotype? (4) Does ER stress induce changes in neural connectivity between key brain regions implicated in ASD pathophysiology? Future studies addressing the above questions might lead to a better understanding of the pathophysiology and provide new avenues of treatment of this disorder.

Cellular stress and apoptosis contribute to the pathogenesis ofautism spectrum disorder


Lay Summary

Autism results in significant morbidity and mortality in children. The functional and molecular changes in the autistic brains are unclear. The present study utilized autistic brain tissues from the National Institute of Child Health and Human Development's Brain Tissue Bank for the analysis of cellular and molecular changes in autistic brains. Three key brain regions, the hippocampus, the cerebellum, and the frontal cortex, in six cases of autistic brains and six cases of non‐autistic brains from 6 to 16 years old deceased children, were analyzed. The current study investigated the possible roles of endoplasmic reticulum (ER) stress, oxidative stress, and apoptosis as molecular mechanisms underlying autism. The activation of three signals of ER stress (protein kinase R‐like endoplasmic reticulum kinase, activating transcription factor 6, inositol‐requiring enzyme 1 alpha) varies in different regions. The occurrence of ER stress leads to apoptosis in autistic brains. ER stress may result from oxidative stress because of elevated levels of the oxidative stress markers: 4‐Hydroxynonenal and nitrotyrosine‐modified proteins in autistic brains. These findings suggest that cellular stress and apoptosis may contribute to the autistic phenotype. Pharmaceuticals and/or dietary supplements, which can alleviate ER stress, oxidative stress and apoptosis, may be effective in ameliorating adverse phenotypes associated with autism.


Figure 1. Immunoblot analysis of endoplasmic reticulum (ER) stress signals in the autistic cerebellum. Immunoblot analysis of the cerebellum homogenate was performed using p-IRE1a, p-PERK, and total ATF6 antibodies.


In summary, we showed the elevation of ER stress signals, oxidative stress, and apoptosis in three regions of autistic brains. Based on these findings, we reason that increased cellular stress and apoptosis in the autistic brain may be associated with the pathogenesis of autism. Because autism is affected by multiple genetic and environmental factors that are case-specific and there are inherent limitations in the postmortem brain, the present observations will need further confirmation in future studies. Further research with larger sample sizes is needed to investigate the association of cellular stress and apoptotic events with the severity and clinical phenotypes of autism.


Chaperone Sigma1R mediates the neuroprotective action of afobazole in the 6-OHDA model of Parkinson’s disease


Parkinson’s disease (PD) is a progressive neurodegenerative disease with limited treatment options. Therefore, the identification of therapeutic targets is urgently needed. Previous studies have shown that the ligand activation of the sigma-1 chaperone (Sigma1R) promotes neuroprotection. The multitarget drug afobazole (5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride) was shown to interact with Sigma1Rs and prevent decreases in striatal dopamine in the 6-hydroxydopamine (6-OHDA)-induced parkinsonism model. The aim of the present study was to elucidate the role of Sigma1Rs in afobazole pharmacological activity. Using ICR mice we found that administration of afobazole (2.5 mg/kg, i.p.) or selective agonist of Sigma1R PRE-084 (1.0 mg/kg, i.p.) over 14 days normalizes motor disfunction and prevents decreases in dopamine in the 6-OHDA-lesioned striatum. Afobazole administration also prevents the loss of TH + neurons in the substantia nigra. The pre-administration of selective Sigma1R antagonist BD-1047 (3.0 mg/kg, i.p.) abolishes the activity of either afobazole or PRE-084, as determined using the rotarod test and the analysis of striatal dopamine content. The current study demonstrates the contribution of Sigma1Rs in the neuroprotective effect of afobazole in the 6-OHDA model of Parkinson’s disease and defines the therapeutic perspective of Sigma1R agonists in the clinic.                                                                                                                                                

Sigma-1 (σ1) Receptor in Memory and Neurodegenerative Diseases

The sigma-1 (σ1) receptor has been associated with regulation of intracellular Ca2+ homeostasis, several cellular signaling pathways, and inter-organelle communication, in part through its chaperone activity. In vivo, agonists of the σ1 receptor enhance brain plasticity, with particularly well-described impact on learning and memory. Under pathological conditions, σ1 receptor agonists can induce cytoprotective responses. These protective responses comprise various complementary pathways that appear to be differentially engaged according to pathological mechanism. Recent studies have highlighted the efficacy of drugs that act through the σ1 receptor to mitigate symptoms associated with neurodegenerative disorders with distinct mechanisms of pathogenesis. Here, we will review genetic and pharmacological evidence of σ1 receptor engagement in learning and memory disorders, cognitive impairment, and neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, and Huntington’s disease.

Crosstalk between endoplasmic reticulum stress and oxidative stress in schizophrenia: The dawn of new therapeutic approaches


The complete understanding of the pathways and the point of convergence of ER and oxidative stress in schizophrenia is still quite fragmentary.

Neuronal migration along with altered secretion of neurotrophins modulates neuronal circuits and synaptic function during schizophrenia.

Chemical chaperones including Sigma-1 receptor agonists may prevent stress-induced protein misfolding associated with schizophrenia.

ER-stress inhibitors, sigma-1 receptor agonists and gene therapies holds a strong therapeutic potential against schizophrenia.
Disruption of oxidant/anti-oxidant ratio as well as endoplasmic reticulum (ER) stress are thought to be involved in the pathophysiology of schizophrenia. These stresses can lead to impairments in brain functions progressively leading to neuronal inflammation followed by neuronal cell death. Moreover, the cellular stresses are interlinked leading us to the conclusion that protein misfolding, oxidative stress and apoptosis are intricately intertwined events requiring further research into their mechanistic and physiological pathways. These pathways can be targeted by using different therapeutic interventions like anti-oxidants, sigma-1 receptor agonists and gene therapy to treat the neurodegenerative course of schizophrenia. We have also put empahsis on use of synthetic and natural ER stress inhibitors like 4-phenylbutyrate or salubrinal for the treatment of this disorder. This would provide an opportunity to create new therapeutic benchmarks in the field of neuropsychiatric disorders like schizophrenia, dissociative identity disorder and obsessive compulsive disorder.

Targeting ligand-operated chaperone sigma-1 receptors in the treatment of neuropsychiatric disorders

Current conventional therapeutic drugs for the treatment of psychiatric or neurodegenerative disorders have certain limitations of use. Psychotherapeutic drugs such as typical and atypical antipsychotics, tricyclic antidepressants, and selective monoamine reuptake inhibitors, aim to normalize the hyper- or hypo-neurotransmission of monoaminergic systems. Despite their great contribution to the outcomes of psychiatric patients, these agents often exert severe side effects and require chronic treatments to promote amelioration of symptoms. Furthermore, drugs available for the treatment of neurodegenerative disorders are severely limited.

Areas covered

This review discusses recent evidence that has shed light on sigma-1 receptor ligands, which may serve as a new class of antidepressants or neuroprotective agents. Sigma-1 receptors are novel ligand-operated molecular chaperones regulating a variety of signal transduction, ER stress, cellular redox, cellular survival, and synaptogenesis. Selective sigma-1 receptor ligands exert rapid antidepressant-like, anxiolytic, antinociceptive and robust neuroprotective actions in preclinical studies. The review also looks at recent studies which suggest that reactive oxygen species might play a crucial role as signal integrators at the downstream of Sig-1Rs

Expert opinion

The significant advances in sigma receptor research in the last decade have begun to elucidate the intracellular signal cascades upstream and downstream of sigma-1 receptors. The novel ligand-operated properties of the sigma-1 receptor chaperone may enable a variety of interventions by which stress-related cellular systems are pharmacologically controlled.

Sigma-1 receptor ligands
Clinically used drugs:
·         Afobazole (5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride): Anxiolytic drug
·         Carbetapentane: Cough suppressant
·         Dextromethorphan (DM): Antitussive drug; DM-quinidine (Q) therapy is effective in reducing pseudobulbar affect in ALS and multiple sclerosis
·         DonepezilSigma-1 agonist; acetylcholine esterase inhibitor used in Alzheimer’s disease
·         Fluvoxamine: Clinically used SSRI; Sig-1R agonist
·         Sertraline: Clinically used SSRI with a putative Sig-1R antagonist property
·         Haloperidol: Clinically used antipsychotic; potent, but non selective sigma antagonist
·         Haloperidol-metabolite II (reduced HP, 4-(4-chlorophenyl)-alpha-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol): In contrast to haloperidol, having higher selectivity to Sig-1Rs
·         MemantineA novel Alzheimer’s disease medication blocking NMDA glutamate receptors
·         Zonisamide: Anti-Parkinson drug approved in Japan

Involvement of endoplasmic reticulum stress and neurite outgrowth in the model mice of autism spectrum disorder

Implication of Endoplasmic Reticulum Stress in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is categorized as a neurodevelopmental disorder according to the Diagnostic and Statistical Manual of Disorders, Fifth Edition and is defined as a congenital impairment of the central nervous system. ASD may be caused by a chromosomal abnormality or gene mutation. However, these etiologies are insufficient to account for the pathogenesis of ASD. Therefore, we propose that the etiology and pathogenesis of ASD are related to the stress of the endoplasmic reticulum (ER). ER stress, induced by valproic acid, increased in ASD mouse model, characterized by an unfolded protein response that is activated by this stress. The inhibition of neurite outgrowth and expression of synaptic factors are observed in ASD. Similarly, ER stress suppresses the neurite outgrowth and expression of synaptic factors. Additionally, hyperplasia of the brain is observed in patients with ASD. ER stress also enhances neuronal differentiation. Synaptic factors, such as cell adhesion molecule and shank, play important roles in the formation of neural circuits. Thus, ER stress is associated with the abnormalities of neuronal differentiation, neurite outgrowth, and synaptic protein expression. ER stress elevates the expression of the ubiquitin-protein ligase HRD1 for the degradation of unfolded proteins. HRD1 expression significantly increased in the middle frontal cortex in the postmortem of patients with ASD. Moreover, HRD1 silencing improved the abnormalities induced by ER stress. Because other ubiquitin ligases are related with neurite outgrowth, ER stress may be related to the pathogenesis of neuronal developmental diseases via abnormalities of neuronal differentiation or maturation.

Sigma-1 receptor: The novel intracellular target of neuropsychotherapeutic drugs

The sigma-1 receptor localized at the ER modulates via its chaperone activity inter-organelle communications. Sigma-1 receptors thus regulate a variety of cellular events, such as neuronal differentiation, cellular survival, and bioenergetics. By numerous animal studies, these actions of the sigma-1 receptors have been linked to the pathophysiology of certain human diseases such as depression, ischemia, drug abuse, pain, and cancer. Considering the current pharmacotherapy of neuropsychiatric diseases that largely depends on drugs developed based on the monoamine theory, the sigma-1 receptor is expected to serve as a molecule, which provides a novel target of “post-monoamine” drugs, thus bringing a new approach for treatment of patients suffering from neuropsychiatric diseases.


Fig. 1. Molecular functions of the sigma-1 receptor. The sigma-1 receptor possesses two transmembrane domains and mainly localize at the ER membrane. Sigma-1 receptors are clustered at the mitochondria-associated ER membrane (MAM) and ER membranes juxtaposing postsynaptic density of specific types of neurons. The ER lumenal domain of the sigma-1 receptor exerts chaperone activities by which ER membrane proteins are stabilized. The figure depicts the recently reported actions of the sigma-1 receptors including: 1) Sigma-1 receptors associating with BiP stabilizes IP3 receptors type-3 (IP3R) at the MAM, leading to regulation of Ca2+ influx into mitochondria and following ATP production; 2) Sigma-1 receptors stabilize the ER stress sensor IRE1 at the MAM in an ROS-dependent manner, leading to prolongation of the IRE1-XBP1 cell survival signal; 3) Sigma-1 receptors suppress generation of reactive oxygen species (ROS) and following activation of the NFkB signaling (How the sigma-1 receptor regulates ROS generation is unknown); 4) Sterols such as 25-hydroxycholesterol promote the association of sigma-1 receptors with Insig-1 [Collaborating with Insig-1, sigma-1 receptors regulate ER-associated degradation (ERAD) of HMG-CoA reductase and galactosylceramide synthase at the ER]; 5) Sigma-1 receptors regulate the trafficking of potassium channel subunits from the ER to the plasma membrane or processing/secretion of brain-derived trophic factor (BDNF). Sigma-1 receptors likely associate with potassium channel subunits or pro-BDNF at the ER. In spinal neurons, sigma-1 receptors, which colocalize with a K channel subunit are clustered at the ER membrane apposing postsynaptic densities (PSD). How the sigma-1 receptor regulates processing/secretion of BDNF is unknown.  (in the earlier part of this post the mechanism that increases BDNF is explained, if you activate sigma-1R you inevitably will increase BDNF)


In our simplified view of autism, aimed at actually treating it, we should have a list of stresses and what to do about them:-

·        Oxidative stress
·        Nitrosative stress
·        Endoplasmic Reticulum (ER) stress

Reducing oxidative stress has multiple biological and behavioral effects; the overall effect is generally positive.

Reducing endoplasmic reticulum (ER) stress, if present, does look a good idea.  It will have numerous effects; it should even reduce oxidative stress. The sigma-1 chaperone looks like it will have many effects that, on balance, should be positive, but undoubtedly may upset something and produce an overall negative effect in some people – it is inevitable.  I hope the effect on NMDA receptors does not cause a problem where an E/I (excitatory/inhibitory) imbalance is already being treated.

A highly selective sigma-1R agonist, one that does not affect any other receptors, does not exist.

Many psychiatric drugs like antidepressants do affect sigma-1R, but they are not suitable for long term use because of side effects, tolerance, addiction etc.

Afobazole is interesting because clinical trials have shown it to be well tolerated, non-addictive and reasonably effective for the treatment of anxiety.  Afobazole also affects the melatonin receptor MT1, it is not directly sedating but might affect some types of sleep abnormality. 

Afobazole is only researched in Russia, but findings are shared internationally, for example at this conference

The drug was developed, and is currently researched, by the “Research Zakusov Institute of Pharmacology” in Moscow. They recently also published a paper on the use of Afobazole in Parkinson’s disease.  In the Parkinson’s paper ( the researchers argue the role of the drug is in targeting ER stress, protein misfolding, IP3R etc.  The very things I am suggesting may be relevant to autism in today’s post.

Another interesting drug from the former USSR, though actually from Latvia (now in the European Union) is Mildronate.  I did suggest a long time ago, based on the research studies, that this might be effective to treat people with a lack of the Mitochondrial Complex 1.

I think mitochondrial disease is likely over diagnosed by MAPS-type doctors, but it is a genuine cause/contributing factor to some people’s autism.

So many people are using Mildronate to boost sporting performance and some for academic performance, it is now widely available from the same vendors /platforms selling Afobazole. (eBay, Amazon etc)

The underlying message is that when considering repurposing safe old drugs to treat neurological conditions, consider all of them, including Japanese, Russian and indeed Latvian.

Many interesting novel substances are mentioned in this blog, like Basmisanil  a highly selective negative allosteric modulator of α5 subunit-containing GABAA receptors for the treatment of cognitive impairment specifically associated with Down syndrome.  The problem with such novel substances is that they will be ultra expensive and often they fail in their clinical trials and are never commercialized.  Roche cancelled Basmisanil because it failed in the Down Syndrome trial.  Tuning down the response from GABAa receptors containing the α5 subunit may very well be an effective way to improve cognitive function in some people, but the failure of this trial likely means no new substances will be developed.

While it is okay to write about new drugs in development, the real interest is in applying what can be used today. All four of the following need to be satisfied:

1.     Safe (no/minimal side effects, no tolerance, no addiction, interactions)
2.     Affordable
3.     Available
4.     Effective

Some drugs, not commonly used in Western countries, likely do tick the first 3 points, whether effective in autism depends on the individual sub-type.  Many do look interesting - from Ibudilast (Japan), to Mildronate (Latvia) for Complex 1 mitochondrial disease and perhaps Afobazole (Russia) for some schizophrenia/autism.

Afobazole is a cheap over-the-counter anxiety treatment in Russia.  It is apparently “effective” in the BALB/c mouse model, that may be relevant to autism. BALB/c mice show low sociability, relatively high levels of anxiety and aggressive behaviors, large brain size, underdevelopment of the corpus callosum, and low levels of brain serotonin.

Is Afobazole the answer to ER stress in autism?  If not, then what might be?  The schizophrenia research suggests 4-phenylbutyrate, salubrinal, cordycepin, taurosodeoxycholic acid.  Cordycepin comes from a mushroom that I recall one of our Aspie readers favours.   

This post could go on forever; I think I have made my point, but a little more:-

The lipophilic 4-phenylbutyric acid derivative prevents aggregation and retention of misfolded proteins 

Chemical chaperones prevent protein aggregation. However, the use of chemical chaperones as drugs against diseases due to protein aggregation is limited by the very high active concentrations (mM range) required for mediating their effect. One of the most common chemical chaperones is 4-phenylbutyric acid (4-PBA). Despite its non-favorable pharmacokinetic properties, 4-PBA was approved as a drug to treat ornithine cycle diseases. Here we report that 2-isopropyl-4-phenylbutanoic acid (compound 5) was (2-10 fold) more effective than 4- PBA in several in vitro models of protein aggregation. Importantly, compound 5 reduced the secretion rate of autism-linked Arg451Cys Neuroligin3 (R451C NLGN3).

Protein misfolding, detectable in blood samples, predicts Alzheimer's Disease up to 14 years before onset, perhaps in time to start effective therapy? perhaps targeting sigma-1R, or perhaps with betanin, that pigment in beetroot, that seems to disrupt plaque formation.

Protein misfolding as a risk marker for Alzheimer's disease

In symptom-free individuals, the detection of misfolded amyloid-beta protein in the blood indicated a considerably higher risk of Alzheimer's disease -- up to 14 years before a clinical diagnosis was made. Amyloid-beta folding proved to be superior to other risk markers evaluated.


  1. Yay, finally! I've been hoping for this post to happen for a year now when I first discovered 'my' gene was involved in the machinery. I'll certainly read up on the sigma-1 receptor now, I didn't know about the connection and it does seem to fit my equation very well.
    Thank you for this great post, and I hope others will have some use of it too. I might pop in later with a comment if I see I have some complementary information in my notes.


    1. Here is my take on the subject:

      The endoplasmic reticulum (ER) synthesizes proteins.
      Almost any kind of disturbance seen in autism can lead to ER stress (redox regulation, calcium regulation, glucose deprivation, infections...)
      During ER stress the folding of new proteins slows down and unfolded/misfolded proteins accumulate.

      This is when the Unfolded Protein Response (UPRer) sets in:
      -Protein translation is halted. This includes the production of very central synaptic proteins.
      -Production of chaperones - the little guys that fold proteins - is increased. Some of them are also heat shock proteins, like HSP70 and HSP90 with important roles in immune regulation.
      -Misfolded proteins are degraded.

      If the UPR fails to achieve the above within a time limit, it will instead aim towards apoptosis/programmed cell death.

      There are three pathways/sensors of ER stress; PERK, IRE1 and ATF6.
      It seems as the PERK path is the one responsible for ether halting or reversing the halt of protein translation. It is also resonsible for either inducing apoptosis via ATF4/ATF2/CHOP or decreasing apoptosis via NRF2. The PERK pathway (which rather is a loop) is perhaps the most interesting one and would need its own review because the devil is in the details here. Depending on the underlying disease and/or disease stage you will want to modulate it differently.
      All three paths are involved in chaperone/HSP production and IRE1 and ATF6 are involved in the degradation of faulty proteins.

      When it comes to interventions ER stress can be alleviated with things like NAC or pterostilbene.
      Initiation of the PERK pathway can be inhibited with things like TUDCA or Arctigenin.
      The UPR can be enhanced with Guanabenz, or decreased with ISRIB.
      Chaperones and heat shock proteins can be increased with for example Curcumin, Bacopa and Withaferin-A.
      Fisetin induces both ATF4 and NRF2, which repress oxidative stress under normal and stress conditions respectively. But it does not induce PERK, and actually inhibits apoptosis by CHOP.

      While ER stress can be bad for brains (it decreases the production of synaptic proteins, and kills neurons if stuck for too long) and can cause things such as diabetes, fatty liver, IBD and perhaps autoimmunity it is good if you want to kill of certain cancer cells.
      Some substances at a low dose will relieve parts of the ER stress while inducing it in higher doses.

      Note that there also is a separate mitochondrial unfolded protein response.

      (I didn't give any references, either google them yourself or ask me for them)

    2. Ling, not included in my post is a new 'sigma-1R drug being trialed for a very wide range of conditions from Rett syndrome, Fragile X to Alzheimers.

      Preliminary Data From Anavex-2-73 Phase 2 Trial Suggest Safety, Efficacy

      Through the activation of S1R, Anavex-2-73 will decrease protein misfolding. That, in turn, will result in less cellular stress and better functioning of the mitochondria, known as the “powerhouses” of cells, which could improve the survival of neurons (brain cells). This is a unique strategy for treating neurological diseases like Rett syndrome.

      producer's web site :'

      Afobazole might be Anavex for people who do not have USD 50,000 a year handy and who do not want to wait another 5+ years.

    3. Talking about interventions for ER stress, I'm doing redrawal trials of a bunch of interventions, one at a time. Bacopa is the current one, and this is more or less ruining our holidays now.

      Two weeks in (or rather off) and we're experiencing:
      -Restless sleep, waking up every second night and moving around a lot while asleep
      -Irritability every day, a lot of it
      -Aggressive actions like hitting, pushing and hair pulling
      -Fever (lasting one day, no other symptoms but fatigue, not even a runny nose, nobody else in the family is sick, nobody to catch viruses from...)
      -Handflapping is back (it went away last time I upped the dose, so this confirms the relationship).
      -Facial rash from skin contact with certain known foods but now it doesn't go away within an hour as usual but stays for days and requires attendance.

      The effects from (adding) Bacopa slowly builds up over 3 months or so which makes them hard to spot. I didn't expect to see such a drastic worsening when removing it.
      Fisetin is very similar and seems to stack with Bacopa at my current dose, but it has a much faster effect. Starting it you notice effect within 3 days, and removing it takes 2 days. When using both Fisetin and Bacopa I can't see any effect from sulphoraphane at all, though it also acts in this realm.

      I'm not sure the family can live another week like this, but I need to remove the effect Bacopa has on serotonin/MAO to be able to test C8/keto again without hitting the 'happy mania' state.


  2. Thanks for this post. I have been taking TUDCA every day for more than a year---largely to protect my liver (I am on a very long antibiotic protocol). I had also read anecdotes on a Parkinson's forum that suggested TUDCA (taken every day) was helpful in mitigating the rigidity/shaking problems. I'll have to spend some time going through this article as it is quite dense.

  3. Hi Peter, big thanks for this great post, I will make a trial of Afobazole, as my son shows extreme anxiety and OCD. Next year he will start high school with a new assistant. A lot of changes are coming. I will get it after Christmas.

    1. Valentina, let us all know how your trial goes.

      It does look like it should be helpful in some autism. Anxiety is very often an issue and so there may be multiple potential benefits.

    2. Ok Peter, I will let all know, do you think that 10 mg twice a day will be enough?

    3. Valentina, the adult dose is 30mg a day, so 10mg twice a day would seem a good basis for a trial. Some American adults using it as a cognitive enhancer are using higher doses.

      It is reported to take a week to start to reduce anxiety with the effect peaking after 4 weeks. The anxiety effect continues for 1-2 weeks after stopping taking it.

  4. Hi Peter,

    Just a note to say that this is a fantastic post!

    Thank you for the great work you do!


  5. Here is yet another, recent, article on the subject. It describes the effects of carnosic acid (found in rosemary and salvia) on both NRF2 and ATF4 and shows that simultaneous activation of these induces Nerve Growth Factor.

    Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes.

    It also briefly mentions other active substances here such as ISRIB, fisetin and royal jelly.
    I'm pretty sure someone in the community previously posted about a synergistic blend of carnosic acid+something+something, but I can't find it.


  6. Hi Peter,
    I'm going to start with afobazole for our 8y old sun, mild MR/ID with autistic traits and of course OCD, anxiety episodes. I wonder if it is wise to statt with 10mg dose for 25kg kid and another question do you think it is better to take it at the start of the day or before eve?

    1. Los Palos, my son is twice a big as yours. He takes 10mg after breakfast and 10mg at about 4pm. The adult dose is 30mg a day.

      If you give 10mg a day, you might want to split the dose into 5mg twice a day.

  7. Perhaps look into TUDCA for ER stress/dysfunction. I've come across a few good PubMed articles relating the two.

    1. Dragon, TUDCA (Tauroursodeoxycholic acid) in the form of animal bile has long been used in Chinese medicine.

      TUDCA prevents cell death. This is controlled by BAX/BCL2, discussed earlier in this blog. BCL2 family genes/proteins play a key role in cancer, but also in some autism.

      Bcl-2 / Bax

      The gene/protein Bcl-2 is relevant to both cancer and autism and has been covered previously in this blog.

      A total of 25 genes make up the Bcl-2 family of proteins. Bcl-2 itself is anti-apoptotic while family member Bax is pro-apoptotic.

      Apoptosis is programmed cell death. The Bax/Bcl-2 ratio determines the apoptotic potential of a cell. Increasing the Bax/Bcl-2 ratio can be highly desirable if you have cancer, since what you want is cell death.

      Bcl-2 is dysregulated in autism. Studies have shown that the expression of Bcl-2 is significantly decreased in the brain of autistic subjects. This means a reduction in a protein that blocks apoptotic cell death, i.e. this favours growth and too much growth is a bad thing.

      The big head type of autism (macrocephaly) is associated with hyperactive pro-growth signalling pathways, so reduced expression of Bcl-2 is not a surprise.

    2. Low dose Naltrexone will also help prevent cell death.

    3. Low dose Naltrexone has been shown in the research to reduce ER stress. In people with inflammatory bowel disease, like Crohn's disease or ulcerative colitis, it reduces mucosal ER stress levels and this is part of the reason it is so therapeutic.

      ER stress does not necessarily lead to cell death and cell death is only sometimes a bad thing. Cell death is part of life, cancer is caused by cells refusing to die.

      Low dose Naltrexone would likely benefit many people with autism and not just the ones with GI inflammation.

    4. Anecdotaly, so far LDN has drastically improved my IBS. Weird as the colonoscopy I had last year didn't indicate "inflammation" in the colon. I don't know whether there was inflammation in the small bowel, or whether the colorectal surgeons only regard "inflammation" as being disease pathology such as Crohn's, UC etc. I don't think LDN works immediately like something such as Verapamil.. reading on the forums it seems to take a while for the effects to be seen as it shifts the immune response.. at least 1-2 weeks, maybe months.

  8. Peter, what was your experience with Afobazole?

    1. Lisa, I found Afobazole reduced anxiety, but seemed to have a mild negative effect on cognitive function.

      For us, cognitive function is a priority, since Monty attends mainstream school without an IEP (Individual educational plan), so he needs to be at the top of his potential, otherwise he will be lost.

    2. Thank you Peter for responding, you've helped me a lot, cognitive function is also important for my son, I realized he had great benefits with low dose clonazepam when I stopped giving it to him, he was very frustrated about not being able to say what he wanted.

      I also wanted to ask you about Verapamil, could it have lowered my son's testosterone levels if he was taking 1.25mg/kg? He has been masturbating a lot and I don't think it's normal, I don't know if it's related, I read it may be due to low Estradiol, he started when I stopped giving him Verapamil. Days have passed and nothing has improved, he wakes up in the night or talks while asleep and has still dark circles under his eyes and is a bit more aggressive, his GI issues are there too, I think Verapamil was helping him in more ways than I thought, but still I am unsure about the risks and its interactions with other medications that I may add to his treatment.

    3. Lisa, Verapamil is known to sometimes raise levels of the hormone prolactin, which in a woman can lower the levels of the hormones estrogen and progesterone and affect fertility, in men the effect is to lower testosterone levels. These are rare events.

      There is a wide range in what is considered "normal" for testosterone in children.

      If you are worried you can always go and measure prolactin and testosterone.

      Our doctor reader Agnieszka is very interested to hear from readers using Verapamil.

      Verapamil use in Autism – Request for Case Reports from Parents

      I suggest you contact her, as your son does seem to benefit from Verapamil.

    4. Peter, thank you for your advice, I will contact her.

      It's awesome that your son is doing so well in school. Congratulations :), have a nice day!


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