Treating Rett syndrome, some autism and some dementia via TrkA, TrkB, BDNF, IGF-1, NGF and NDPIH. And logically why Bumetanide really should work in Rett

Source: Rett Syndrome: Crossing the Threshold to Clinical Translation

 

Today’s post is on the one hand very specific to Rett syndrome, but much is applicable to broader autism and other single gene autisms.

Today’s post did start out with the research showing Bumetanide effective in the mouse model of Rett syndrome. This ended up with figuring out why this should have been obvious based on what we already know about growth factors that are disturbed in autism and very much so in Rett.

We even know from a published human case studies that Bumetanide can benefit those with Fragile X and indeed Down syndrome, but the world takes little notice.

If Bumetanide benefits human Rett syndrome would anyone take any notice?  They really should.

To readers of this blog who have a child with Rett, the results really are important.  You can even potentially link the problem symptoms found in Rett to the biology and see how you can potentially treat multiple symptoms with the same drug.

One feature of Rett is breathing disturbances, which typically consist of alternating periods of hyperventilation and hypoventilation.

Our reader Daniel sent me a link to paper that suggest an old OTC cough medicine could be used to treat the breathing issues.

The antitussive cloperastine improves breathing abnormalities in a Rett Syndrome mouse model by blocking presynaptic GIRK channels and enhancing GABA release

Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene. One of the major RTT features is breathing dysfunction characterized by periodic hypo- and hyperventilation. The breathing disorders are associated with increased brainstem neuronal excitability, which can be alleviated with antagonistic agents.

Since neuronal hypoexcitability occurs in the forebrain of RTT models, it is necessary to find pharmacological agents with a relative preference to brainstem neurons. Here we show evidence for the improvement of breathing disorders of Mecp2-null mice with the brainstem-acting drug cloperastine (CPS) and its likely neuronal targets. CPS is an over-the-counter cough medicine that has an inhibitory effect on brainstem neuronal networks. In Mecp2-null mice, CPS (30 mg/kg, i.p.) decreased the occurrence of apneas/h and breath frequency variation. GIRK currents expressed in HEK cells were inhibited by CPS with IC50 1 μM. Whole-cell patch clamp recordings in locus coeruleus (LC) and dorsal tegmental nucleus (DTN) neurons revealed an overall inhibitory effect of CPS (10 μM) on neuronal firing activity. Such an effect was reversed by the GABAA receptor antagonist bicuculline (20 μM). Voltage clamp studies showed that CPS increased GABAergic sIPSCs in LC cells, which was blocked by the GABAB receptor antagonist phaclofen. Functional GABAergic connections of DTN neurons with LC cells were shown.

These results suggest that CPS improves breathing dysfunction in Mecp2-null mice by blocking GIRK channels in synaptic terminals and enhancing GABA release.

  

Cloperastine (CPS) is a central-acting antitussive working on brainstem neuronal networks The drug has several characteristics. 1) It affects the brainstem integration of multiple sensory inputs via multiple sites including K+ channels, histamine and sigma receptors. 2) Its overall effect is inhibitory, suppressing cough and reactive airway signals. 3) With a large safety margin, it has been approved as an over-the-counter medicine in several Asian and European countries.  

With the evidence that DTN cells receive GABAergic recurrent inhibition, we tested whether the inhibitory effect of CPS was caused by enhanced GABAergic transmission. Thus, we recorded the evoked firing activity of DTN cells before and during bath application of CPS in the presence of 20 μM bicuculline. Under this condition, CPS failed to decrease the excitability of DTN neurons (F(1,9) = 0.41, P > 0.05; two‐way repeated measures ANOVA) (n=9) (Fig. 8), indicating that the inhibitory effect relies on GABAA synaptic input 

 

It appeared to me that the breathing issues might be considered as another consequence of the excitatory/inhibitory (E/I) imbalance that is a core feature of much severe autism.

In the case of Rett the lack of BDNF will make any E/I imbalance worse and that by treating the E/I imbalance we will produce the inhibitory effect from GABAa receptors that is needed to ensure correct breathing.  Note that in bumetanide responsive autism there is no inhibitory effect from GABAa receptors, the effect is excitatory.

I did wonder if arrhythmia (irregular heartbeat) is present in Rett, since the breathing problems in Rett are also seen as being caused by a dysfunction in the autonomic nervous system. Arrhythmia is actually a big problem for girls with Rett syndrome.  Regular readers of this blog might then ask about Propranolol, does that help?  It turns out to have been tried and it is not so helpful.  What is effective is another drug we have come across for autism, the sodium channel blocker Phenytoin.  Phenytoin is antiepileptic drug (AED) and it works by blocking voltage gated sodium channels.

Low dose phenytoin was proposed as an autism therapy and a case study was published from Australia. In a separate case study, phenytoin was used to treat self-injury that was triggered by frontal lobe seizures.

When you treat arrhythmia in Rett girls with Phenytoin does it have an impact on their breathing problems?

If you treat the girls with Phenytoin do they still go on to develop epilepsy?

What about if you add treatment with Bumetanide to reduce symptoms of autism? 

Lots of questions looking for answers.

 

What is Rett Syndrome?

Rett syndrome was first identified in the 1950s by Dr Andreas Rett as a disorder that develops in young girls.  Only as recently as 1999 was it determined that the syndrome is caused by a mutation in the MECP2 gene on the X chromosome.  The X chromosome is very important because girls have two copies, but boys have just one.  Rett was an Austrian like many other early researchers in autism like Kanner and Asperger. Even Freud was educated in Vienna. Eugen Bleuler lived pretty close by in Switzerland and he coined the terms schizophrenia, schizoid and autism. 

Rett syndrome is a rare genetic disorder that affects brain development, resulting in severe mental and physical disability.

It is estimated to affect about 1 in 12,000 girls born each year.

Rett is a rare condition, but among these rare conditions it is quite common and so there is a lot of research going on to find treatments.  The obvious one is gene therapy to get the brain to make the missing MeCP2 protein.

Rett syndrome is thankfully rare in absolute terms, but it is one of the best known development conditions that is associated with autism symptoms.

While Rett syndrome may not officially be an ASD in the DSM-5, the link to autism remains. Many children are diagnosed as autistic before the MECP2 mutation is identified and then the diagnosis is revised to RTT/Rett. 

Fragile X  syndrome (FXS), on the other hand, is the most common inherited cause of intellectual disability (ID), as well as the most frequent single gene type of autism.

In the meantime, the logical strategy is to treat the downstream consequences of the mutated gene. Much is known about these downstream effects and there overlaps with some broader autism and indeed dementia.

One area known to be disturbed in Rett, some other autisms and dementia is growth factors inside the brain. The best known growth factors are IGF-1 (Insulin-like Growth Factor 1), BDNF (brain-derived neurotrophic factor) and my favorite NGF (Nerve growth factor).

Without wanting to get too complicated we need to note that BDNF acts via a receptor called TrkB.  You can either increase BDNF or just find something else to activate TrkB, as pointed out to me by Daniel.

For readers whose children respond to Bumetanide they are benefiting from correcting elevated levels of chloride in neurons. Too much had been entering by the transporter NKCC1 and too little exiting via KCC2.

One of the effects of having too little BDNF and hence not enough activation of TrkB is that chloride becomes elevated in neurons.  If you do not activate TrkB you do not get enough KCC2, which is what allows chloride to exit neurons.

To what extent would TrkB activation be an alternative/complement to bumetanide in broader autism?

To what extent would TrkB activation be success in treating some types of chronic pain (where KCC2 is known to be down regulated)?

Low levels of BDNF are a feature of Rett and much dementia.

So you would want to:

·        Increase BDNF

·        Activate TRKB with something else

·        Block NKCC2 to compensate for the lack of KCC2

Note that BDNF is not reduced in all types of autism, just in a sub-group.

I note that there already is solid evidence in the research:-

Restoration of motor learning in a mouse model of Rett syndrome following long-term treatment with a novel small-molecule activator of TrkB

Reduced expression of brain-derived neurotrophic factor (BDNF) and impaired activation of the BDNF receptor, tropomyosin receptor kinase B (TrkB; also known as Ntrk2), are thought to contribute significantly to the pathophysiology of Rett syndrome (RTT), a severe neurodevelopmental disorder caused by loss-of-function mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Previous studies from this and other laboratories have shown that enhancing BDNF expression and/or TrkB activation in Mecp2-deficient mouse models of RTT can ameliorate or reverse abnormal neurological phenotypes that mimic human RTT symptoms. The present study reports on the preclinical efficacy of a novel, small-molecule, non-peptide TrkB partial agonist, PTX-BD4-3, in heterozygous female Mecp2 mutant mice, a well-established RTT model that recapitulates the genetic mosaicism of the human disease. PTX-BD4-3 exhibited specificity for TrkB in cell-based assays of neurotrophin receptor activation and neuronal cell survival and in in vitro receptor binding assays. PTX-BD4-3 also activated TrkB following systemic administration to wild-type and Mecp2 mutant mice and was rapidly cleared from the brain and plasma with a half-life of ∼2 h. Chronic intermittent treatment of Mecp2 mutants with a low dose of PTX-BD4-3 (5 mg/kg, intraperitoneally, once every 3 days for 8 weeks) reversed deficits in two core RTT symptom domains – respiration and motor control – and symptom rescue was maintained for at least 24 h after the last dose. Together, these data indicate that significant clinically relevant benefit can be achieved in a mouse model of RTT with a chronic intermittent, low-dose treatment paradigm targeting the neurotrophin receptor TrkB. 

Early alterations in a mouse model of Rett syndrome: the GABA developmental shift is abolished at birth

Genetic mutations of the Methyl-CpG-binding protein-2 (MECP2) gene underlie Rett syndrome (RTT). Developmental processes are often considered to be irrelevant in RTT pathogenesis but neuronal activity at birth has not been recorded. We report that the GABA developmental shift at birth is abolished in CA3 pyramidal neurons of Mecp2^−/y mice and the glutamatergic/GABAergic postsynaptic currents (PSCs) ratio is increased. Two weeks later, GABA exerts strong excitatory actions, the glutamatergic/GABAergic PSCs ratio is enhanced, hyper-synchronized activity is present and metabotropic long-term depression (LTD) is impacted. One day before delivery, maternal administration of the NKCC1 chloride importer antagonist bumetanide restored these parameters but not respiratory or weight deficits, nor the onset of mortality. Results suggest that birth is a critical period in RTT with important alterations that can be attenuated by bumetanide raising the possibility of early treatment of the disorder.

    

The GABA Polarity Shift and Bumetanide Treatment: Making Sense Requires Unbiased and Undogmatic Analysis

 

GABA depolarizes and often excites immature neurons in all animal species and brain structures investigated due to a developmentally regulated reduction in intracellular chloride concentration ([Cl⁻]_i) levels. The control of [Cl⁻]_i levels is mediated by the chloride cotransporters NKCC1 and KCC2, the former usually importing chloride and the latter exporting it. The GABA polarity shift has been extensively validated in several experimental conditions using often the NKCC1 chloride importer antagonist bumetanide. In spite of an intrinsic heterogeneity, this shift is abolished in many experimental conditions associated with developmental disorders including autism, Rett syndrome, fragile X syndrome, or maternal immune activation. Using bumetanide, an EMA- and FDA-approved agent, many clinical trials have shown promising results with the expected side effects. Kaila et al. have repeatedly challenged these experimental and clinical observations. Here, we reply to the recent reviews by Kaila et al. stressing that the GABA polarity shift is solidly accepted by the scientific community as a major discovery to understand brain development and that bumetanide has shown promising effects in clinical trials.

 

Back in 2013 a case study was published showing Bumetanide worked for a boy with Fragile X syndrome. A decade later and still nobody has looked to see if it works in all Fragile X. 

Treating Fragile X syndrome with the diuretic bumetanide: a case report

https://pubmed.ncbi.nlm.nih.gov/23647528/

We report that daily administration of the diuretic NKCC1 chloride co-transporter, bumetanide, reduces the severity of autism in a 10-year-old Fragile X boy using CARS, ADOS, ABC, RDEG and RRB before and after treatment. In keeping with extensive clinical use of this diuretic, the only side effect was a small hypokalaemia. A double-blind clinical trial is warranted to test the efficacy of bumetanide in FRX.

 

What do Rett syndrome and Fragile X have in common? 

In a healthy mature neuron the level of chloride needs to be low for it to function correctly (the neurotransmitter GABA to be inhibitory).

 

Rett and Fragile X are part of a large group of conditions that feature elevated levels of chloride in neurons.

 

Elevated chloride in neurons is treatable.

 

Is Bumetanide a cure for Rett syndrome, or Fragile X?

No it is not, but it is a step in that direction because it reverses a key defect present in at least some Rett and some Fragile X.

In the mouse model of Rett, bumetanide corrected some, but not all the problems caused by the loss of function of the MECP2 gene.

 

Moving on to IGF-1

IGF-1 is a growth hormone with multiple functions throughout aging. Production of IGF-1 is stimulated by GH (growth hormone).

The lowest levels occur in infancy and old age and highest levels occur around the growth spurt before puberty.

Girls with Turner syndrome, lack their second X chromosome and this causes a lack of growth hormones and female hormones. They end up with short stature and with features of autism. Treatment is possible with GH or indeed IGF-1.

In dementia one strategy is to increase IGF-1.  This same strategy is also being applied to single gene autisms like Rett and Pitt Hopkins.

Trofinetide and NNZ-2591 are improved synthetic analogues of peptides that occur naturally in the brain and are related to IGF-1. Trofinetide is being developed to treat Rett and Fragile X syndromes, NNZ-2591 is being developed to treat Angelman, Phelan-McDermid, Pitt Hopkins and Prader-Willi syndromes.

 

NGF (nerve growth factor)

Nerve growth factor does what it says (boosting nerve growth), plus much more. NGF plays a key role in the immune system, it is produced in mast cells, and it plays a role in how pain in perceived.

NGF acts via NGF receptors, not surprisingly, but also via TrkA receptors. We saw earlier in this post that BDNF acts via TrkB receptors.

Once NGF binds to the TrkA receptor it triggers a cascade of signalling via  the Ras/MAPK pathway and the PI3K/Akt pathway.  Both pathways relate to autism and Ras itself can play a role in intellectual disability. 

These are also cancer pathways and indeed NGF seems to play a role.  Beta cells in the pancreas produce insulin and these beta cells have TrkA receptors. In type 1 diabetes these beta cells die.  Beta cells need NGF to activate their TrkA receptors to survive.

Clearly for multiple reasons you need plenty of NGF.

Lack of NGF would be one cause of dementia and that is why Rita Levi-Montalcini choose to self-treat with NGF eye drops for 30 years. Rita won a Nobel prize for discovering NGF.

In Rett syndrome we know that the level of NGF is very low in the brain.

Logical therapies for Rett would seem to include:

·        NGF itself, perhaps taken as eye drops, but tricky to administer

·        A TrkA agonist, that would mimic the effect of NGF

·        The traditional medicinal mushroom  Lion’s Mane (Hericium erinaceus) 

We should note that effect of NGF acting via TrkA is mainly in the peripheral nervous system, not the brain.

It has long been known that Lions’ Mane (Hericium erinaceus) increases NGF but it was not clear why.  This has very recently been answered.

The active chemical has been identified to be N-de phenylethyl isohericerin (NDPIH).

The opens the door to synthesizing NDPIH as drug to treat a wide range of conditions from Alzheimer’s to Rett. 

Mushrooms Magnify Memory by Boosting Nerve Growth  

Active compounds in the edible Lion’s Mane mushroom can help promote neurogenesis and enhance memory, a new study reports. Preclinical trials report the compound had a significant impact on neural growth and improved memory formation. Researchers say the compound could have clinical applications in treating and preventing neurodegenerative disorders such as Alzheimer’s disease.

Professor Frederic Meunier from the Queensland Brain Institute said the team had identified new active compounds from the mushroom, Hericium erinaceus.

“Extracts from these so-called ‘lion’s mane’ mushrooms have been used in traditional medicine in Asian countries for centuries, but we wanted to scientifically determine their potential effect on brain cells,” Professor Meunier said.

“Pre-clinical testing found the lion’s mane mushroom had a significant impact on the growth of brain cells and improving memory.

“Laboratory tests measured the neurotrophic effects of compounds isolated from Hericium erinaceus on cultured brain cells, and surprisingly we found that the active compounds promote neuron projections, extending and connecting to other neurons.

“Using super-resolution microscopy, we found the mushroom extract and its active components largely increase the size of growth cones, which are particularly important for brain cells to sense their environment and establish new connections with other neurons in the brain.” 

 

Hericerin derivatives activates a pan‐neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spatial memory

The traditional medicinal mushroom Hericium erinaceus is known for enhancing peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. Here, we purified and identified biologically new active compounds from H. erinaceus, based on their ability to promote neurite outgrowth in hippocampal neurons. N-de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom, together with its hydrophobic derivative hericene A, were highly potent in promoting extensive axon outgrowth and neurite branching in cultured hippocampal neurons even in the absence of serum, demonstrating potent neurotrophic activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA-12 only partly prevented the NDPIH-induced neurotrophic activity, suggesting a potential link with BDNF signaling. However, we found that NDPIH activated ERK1/2 signaling in the absence of TrkB in HEK-293T cells, an effect that was not sensitive to ANA-12 in the presence of TrkB. Our results demonstrate that NDPIH acts via a complementary neurotrophic pathway independent of TrkB with converging downstream ERK1/2 activation. Mice fed with H. erinaceus crude extract and hericene A also exhibited increased neurotrophin expression and downstream signaling, resulting in significantly enhanced hippocampal memory. Hericene A therefore acts through a novel pan-neurotrophic signaling pathway, leading to improved cognitive performance.

 

Since the discovery of the first neurotrophin, NGF, more than 70 years ago, countless studies have demonstrated their ability to promote neurite regeneration, prevent or reverse neuronal degeneration and enhance synaptic plasticity. Neurotrophins have attracted the attention of the scientific community in the view to implement therapeutic strategies for the treatment of a number of neurological disorders. Unfortunately, their actual therapeutic applications have been limited and the potential use of their beneficial effects remain to be exploited. Neurotrophins, for example, have poor oral bioavailability, and very low stability in serum, with half-lives in the order of minutes  as well as minimal BBB permeability and restricted diffusion within brain parenchyma. In addition, their receptor signaling networks can confer undesired off-target effects such as pain, spasticity and even neurodegeneration. As a consequence, alternative strategies to increase neurotrophin levels, improve their pharmacokinetic limitations or target specific receptors have been developed. Identification of bioactive compounds derived from natural products with neurotrophic activities also provide new hope in the development of sustainable therapeutical interventions. Hericerin derivative are therefore attractive compounds for their ability to promote a pan-neurotrophic effect with converging ERK1/2 downstream signaling pathway and for their ability to promote the expression of neurotrophins. Further work will be needed to find the direct target of Hericerin capable of mediating such a potent pan-neurotrophic activity and establish whether this novel pathway can be harnessed to improve memory performance and for slowing down the cognitive decline associated with ageing and neurodegenerative diseases.

 

What this means is that there are 2 good reasons why Lion’s Mane should be helpful in Rett syndrome, both increasing BDNF and NGF.

  

Conclusion

Interestingly, one of the above papers is co-authored by a researcher from the European Brain Research Institute, founded by Rita Levi-Montalcini, the Nobel laureate who discovered NGF (Nerve growth factor). My top pick to test next in Rett syndrome would be NGF. Administration would have to follow Rita’s own example and be in the form of eye drops or follow the Lion’s Mane option, that has recently been further validated.

Rett syndrome is very well documented and many researchers are engaged in studying it.

As with broader autism, the problem is translating all the research into practical therapy today.

Clearly polytherapy will be required.

More than one type of neuronal hyperexcitability seems to be in play.

It looks like one E/I imbalance is the bumetanide responsive kind, that can be treated and will reduce autism symptoms and improve learning skills.  Then we have the hypoventilation/apnea for which Cloperastine looks a fair bet.  For the arrhythmia we have Phenytoin.  If there are still seizures after all that therapy it looks like sodium valproate is the standard treatment for Rett.

Sodium valproate is also an HDAC inhibitor and so has possibly beneficial epigenetic effects as a bonus.

I have always liked the idea of the Lion’s Mane mushrooms as a means to increase NGF (Nerve growth factor).  In today’s post we saw that it is the NDPIH from the mushrooms that acts to increase both BDNF and NGF.  You would struggle to buy NDPIH but you can buy these mushrooms. I did once buy the supplement version of these mushrooms and it was contaminated, so I think the best bet is the actual chemical or the actual mushroom.  One reader did write in once who is a big consumer of these mushrooms.

 

Lion's Mane Mushroom

Source: Igelstachelbart Nov 06

 

A Trk-B agonist that can penetrate the blood brain barrier would look a good idea.  There are some sold by the nootropic people.

7,8-dihydroxyflavone is such an agonist that showed a benefit in the mouse model.

 

7,8-dihydroxyflavone exhibits therapeutic efficacy in a mouse model of Rett syndrome

Following weaning, 7,8-DHF was administered in drinking water throughout life. Treated mutant mice lived significantly longer compared with untreated mutant littermates (80 ± 4 and 66 ± 2 days, respectively). 7,8-DHF delayed body weight loss, increased neuronal nuclei size and enhanced voluntary locomotor (running wheel) distance in Mecp2 mutant mice. In addition, administration of 7,8-DHF partially improved breathing pattern irregularities and returned tidal volumes to near wild-type levels. Thus although the specific mechanisms are not completely known, 7,8-DHF appears to reduce disease symptoms in Mecp2 mutant mice and may have potential as a therapeutic treatment for RTT patients.

Rett syndrome also features mitochondrial dysfunction and a variant of metabolic syndrome.  We have quite a resource available from broader autism, not much of it seems to have been applied in Rett.

You can see that in Rett less oxygen is available due to breathing issues and yet more oxygen is required due to “faulty” mitochondria. 

“Intensified mitochondrial O2 consumption, increased mitochondrial ROS generation and disturbed redox balance in mitochondria and cytosol may represent a causal chain, which provokes dysregulated proteins, oxidative tissue damage, and contributes to neuronal network dysfunction in RTT.”

https://www.frontiersin.org/articles/10.3389/fphys.2019.00479/full#:~:text=Rett%20syndrome%20(RTT)%2C%20an,inner%20membrane%20is%20leaking%20protons.

 

We have seen in this blog that 2 old drugs exist to increase oxygen levels in blood.  The Western world has Diamox (Acetazolamide) and the former soviet world has Mildronate/Meldonium. Mildronate also was suggested to have some wider potential benefit to mitochondria.

Rett is proposed as a neurological disorder with metabolic components, so based on what we have seen in this blog, you would think along the lines of Metformin, Pioglitazone and a lipophilic statin (Atorvastatin, Simvastatin or Lovastatin). 

The Anti-Diabetic Drug Metformin Rescues Aberrant Mitochondrial Activity and Restrains Oxidative Stress in a Female Mouse Model of Rett Syndrome

Statins improve symptoms of Rett syndrome in mice

The ultimate Rett cure will be one of the new gene therapies given to a baby before any significant progression of the disorder has occurred.

For everyone else, it looks like there is scope to develop a pretty potent individualized polytherapy, just by applying the very substantial knowledge that already exists in the research.

Good luck to Daniel and all the others seeking answers.

 

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. 

Afobazole Alleviates Cognitive Rigidity in Experimental Model of Autism Spectrum Disorders.

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 GABA_B1aR/GABA_B2R and inhibits the formation of heterodimeric complexes resulting in the intracellular accumulation and reduced cell surface expression of receptors [34]. Interestingly, decreased levels of GABA_B1R and GABA_B2R 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

Abstract

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

Highlights

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

Sigma-1 receptor ligands used in recent preclinical studies (2007~2010)

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

·         Donepezil: Sigma-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

·         Memantine: A 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)

Conclusion

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 (https://www.nature.com/articles/s41598-019-53413-w) 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 GABA_A 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.