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Showing posts with label GABAa. Show all posts
Showing posts with label GABAa. Show all posts

Tuesday, 6 November 2018

When is an SSRI not an SSRI? Low dose SSRIs as Selective Brain Steroidogenic Stimulants (SBSSs) via Allopregnanolone modifying GABAa receptors and neonatal KCC2 expression


Today’s post might seem to have a very complicated tittle, but to regular readers it is really just another take on what we have seen time and time again.
Today we see how another steroid imbalance in autism – low levels of allopregnenolone in this case – affects the neurotransmitter GABA and indeed the chloride transporter KCC2.

Putting Prozac/Zoloft to a better use?

I did report previously on a trial in adults with autism where pregnenolone was used.


Recall that disturbed hormonal homeostasis is a key feature of autism. What matters is the level of each hormone inside the brain (i.e. centrally), not in your blood. The only way to get a reliable idea of what is going on would be to take a sample of spinal fluid.



Today we look at boosting allopregnenolone not with a steroid hormone, but with a 1/10th dose of Prozac (Fluoxetine) or indeed Zoloft (Sertraline). Prozac is a selective serotonin reuptake inhibitor (SSRI) when given at the usual dose of 20-80mg, but at 2.5mg it does not function as an SSRI.
At regular doses selective serotonin reuptake inhibitors (SSRI) drugs like Prozac are well known to cause problems, as do benzodiazepines like Clonazepam.
Thanks to Professor Catterall we saw in earlier posts how tiny doses of Clonazepam have an effect on one particular sub-unit of GABAA receptors. By fine tuning the response of this receptor we saw how a cognitive improvement can be achieved, in some people. The dose is so low there appear to be no long term side effects. At least one other professor of medicine, I am in contact with, has been treating his son with autism with low dose clonazepam for years.
Many adults and children with autism are prescribed Prozac for anxiety. Even Temple Grandin has said she takes Prozac.
At low, non-serotonergic doses, some drugs like Prozac show a different mode of action, they potently, positively, and allosterically modulate GABA action at GABAA receptors. These drugs achieve this by increasing the amount of the steroid hormone allopregnanolone.
Neurosteroid biosynthesis down‐regulation and changes in GABAA receptor subunit composition are a feature of several neurological conditions, including some autism.
Stimulating allopregnenalone biosynthesis will have multiple effects including on TSPO and endocannabinoid receptors.


Brain principal glutamatergic neurons synthesize 3α-hydroxy-5α-pregnan-20-one (Allo), a neurosteroid that potently, positively, and allosterically modulates GABA action at GABAA receptors. Cerebrospinal fluid (CSF) Allo levels are decreased in patients with posttraumatic stress disorder (PTSD) and major depression. This decrease is corrected by fluoxetine in doses that improve depressive symptoms. Emotional-like behavioral dysfunctions (aggression, fear, and anxiety) associated with a decrease of cortico-limbic Allo content can be induced in mice by social isolation. In socially isolated mice, fluoxetine and analogs stereospecifically normalize the decrease of Allo biosynthesis and improve behavioral dysfunctions by a mechanism independent from 5-HT reuptake inhibition. Thus, fluoxetine and related congeners facilitate GABAA receptor neurotransmission and effectively ameliorate emotional and anxiety disorders and depression by acting as selective brain steroidogenic stimulants (SBSSs).                               
When the results of these in vitro studies are compared to those of our in vivo studies, it becomes evident that in mice the doses of fluoxetine and norfluoxetine that cause a rapid increase in brain Allo levels do not exceed brain concentrations in the low nanomolar range, whereas the fluoxetine concentrations that directly activate 3a-HSD in vitro are in the micromolar range. Moreover, the high potency and stereospecificity of fluoxetine and norfluoxetine in decreasing aggressive behavior and normalizing brain Allo content during social isolation (see Table 1, and Figure 3) support the notion that these compounds facilitate the action of 5a-R type I or 3a-HSD by an unidentified indirect mechanism, which is most probably perturbed by protracted social isolation.

Thus, these drugs, which were originally termed ‘SSRI’ antidepressants, may be beneficial in psychiatric disorders because in doses that are inactive on 5-HT reuptake mechanisms, they increase the bioavailability of neuroactive GABAergic steroids. On the basis of these considerations, we now propose that the term ‘SSRIs’ should be changed to the more appropriate term ‘selective brain steroidogenic stimulants’ (SBSSs), which more accurately defines the pharmacological mechanisms expressed by fluoxetine and its congeners.

Conclusions

The pharmacology of the S stereoisomers of fluoxetine and norfluoxetine appears to be prototypic for molecules that possess specific neurosteroidogenic activity. The doses of S-fluoxetine and S-norfluoxetine required to normalize brain Allo content downregulation, pentobarbital action, aggressiveness, and anxiety in socially isolated mice are between 10-fold to 50-fold lower than those required to induce SSRI activity. However, the precise mechanisms of action by which S-fluoxetine and S-norfluoxetine increase neurosteroids remain to be investigated.

Derivatives of S-fluoxetine and S-norfluoxetine, acting with high potency and specificity on brain neurosteroid expression at doses devoid of significant action on brain 5-HT reuptake mechanisms, may represent a new class of pharmacological tools important for the management of anxiety, related mood disorders, dysphoria, fear, and impulsive aggression.

On the basis of these data, new drugs devoid of SSRI activity but that are potent neurosteroidogenic agents should be developed for the treatment of psychiatric disorders that result from the downregulation of neurosteroid expression, including major depression, and in the prevention of PTSD.

France often gets very negative comments about how it treats people with autism, but in the case studies below it looks like some innovative work is going on in some of their day hospitals, where boys and girls with severe autism are sent to pass their time. 

The system in England has recently been highlighted as being pretty appalling, where over 2,000 people with autism are currently detained in Assessment and Treatment Units (ATUs), privately run secure residential "hospitals", at great cost paid for by the State. Those inside might enter with the approval of their family to stay for 3 weeks for respite care, but end up being detained for 3 years, or even longer. The State assumes their guardianship and the individual and parents are powerless. The individuals are kept in prison-like conditions and not surprisingly get worse not better, the worse they get, the harder it is ever to be released. Hard to believe this is still happening.  If you live in England, best not to hand your child over to the State. Someone has even written a book about escaping from such a unit. This is no better than the old State Hospitals in the US, that finally were closed down in the 1970s, that warehoused mentally disabled people, until their premature death.


Autism Spectrum Disorder (ASD) is defined by the copresence of two core symptoms: alteration in social communication and repetitive behaviors and/or restricted interests. In ASD children and adults, irritability, self-injurious behavior (SIB), and Attention Deficit and Hyperactivity Disorders- (ADHD-) like symptoms are regularly observed. In these situations, pharmacological treatments are sometimes used. Selective Serotonin Reuptake Inhibitors- (SSRI-) based treatments have been the subject of several publications: case reports and controlled studies, both of which demonstrate efficacy on the symptoms mentioned above, even if no consensus has been reached concerning their usage. In this article four clinical cases of children diagnosed with ASD and who also present ADHD-like symptoms and/or SIB and/or other heteroaggressive behaviors or irritability and impulsivity treated with low doses of fluoxetine are presented.
Case 1 
An 8-year-old girl (19 kg) had an ASD diagnosis according to the DSM-5 and ADI-R criteria based on information provided by parents. She also had significant mental retardation, with severe SIB (banging her head against objects and biting her hands), forcing her entourage to maintain a daily and permanent physical restraint. She spends most of her time in a day hospital. She received the following pharmacological treatment: risperidone 2 mg/d and cyamemazine 80 mg/d without modifications to her SIB and at the price of a major slowing down and a manifestation of a tendency toward blunting. The CGI severity of illness score was at five (markedly ill). We decreased and stopped risperidone and started valproic acid. After four weeks of valproic acid 400 mg/d in combination with cyamemazine (60 mg/day), SIBs did not improve. Then, we added fluoxetine 2.5 mg/d and increased it after one week to 5 mg/d and to 10 mg/d in the third week. After one week, the CGI improvement scale (CGI-I) was at two; after three weeks, it lowered to 1 (very much improved). We also observed a significant decrease in anxiety as well as the disappearance of SIB (disappearance of the behavior consisting of the banging and rubbing her head against objects). However, it should be noted that the entourage kept the bandages on her hands because she continued to bite them, even if she did it with less intensity than before. There were no side effects. After three months of fluoxetine, her clinical state remains stable.

Case 2 
A 12-year-old boy (70 kg), with DSM-5 criteria for an ASD and ADI-R confirming this diagnosis, exhibited extreme irritability, violence, and impulsiveness as well as SIB (he had thrown seven television sets out of the window). The CGI severity illness scoring was at six (severely ill). In the day hospital where he spent most of his time, it was difficult for staff to manage his impulsivity and unpredictability. His treatment included risperidone 4 mg/d as well as loxapine 80 mg/d. Despite this pharmacological treatment, episodes of aggression and SIBs continued. This treatment induced a significant weight gain (8 kg in 5 months). Treatment with fluoxetine 2.5mg/d was introduced and increased to5mg/d after one week and to 10 mg/d at the beginning of the third week. After one week, there was a CGI-I score of three, which decreased to two after two weeks of treatment and to one after three weeks. Such a positive clinical response allowed for a reduction in risperidone to 2mg/d and in loxapine to 60 mg/d. The treatment was tolerated well by the patient, and he began to lose weight (4 kg). After two months off luoxetine, his clinical state remains stable.

Case 3
 A 6-year-old male child (30 kg) with DSM-5 criteria and ADI-R for an ASD exhibited problems of SIB and repetitive behaviors (washing his hands for more than 30 minutes at least two to three times per day), severe irritability, frequent crying, social withdrawal, and inappropriate speech. Treatment with risperidone 2mg/d had improved irritability and partially the SIB, but it had also produced significant weight gain (four kg in three months). A decrease in the risperidone dosage seemed necessary. Treatment with fluoxetine2.5mg/d was begun, which quickly led to a reduction in inappropriate behavior (for example, impulsive crawling on the ground in the classroom). After one week, the CGI-I scoring was at two. The dosage was gradually increased to 5 mg/d the second week and to 7.5mg/d the third week. The repetitive behaviors gradually subsided. After three weeks the CGI-I score was at one, and it remained stable for nine weeks. The risperidone dosage could be decreased to 0,5 mg/day and the patient’s weight remained the same.
Case 4 
A 12-year-old boy (62kg) withDSM-5 and ADI-R criteria for a severe case of ASD, including severe ADHD-like symptoms, often required physical restraint and did not improve despite a long-term treatment of risperidone 3 mg/d as well as melaton in 4mg at bedtime. The CGI severity illness scoring was at 6 (severely ill). The behavioral pattern included irritability, marked agitation, crying, severe hyperactivity, and other behaviors typical of this disorder. He was also anxious, rendering the situation at his day hospital where he spent most of his time all the more difficult. A prescription of fluoxetine 2.5mg/d was initiated with an immediate and complete improvement of ADHD-like symptoms:CGI-I at one week of treatment was at a one, making this case the most remarkable of the four presented here. Treatment with fluoxetine was continued with a dosage increase up to 5 mg/d to allow for a decrease in the risperidone dose to 1 mg/d. CGI-I score remained stable at one for the duration of the nine weeks.

Our reader Mira, whose son has FXS, recently referred to Dr Hagerman’s trial of low dose Sertaline/Zoloft in Fragile X. GABAA malfunction appears to be a feature of Fragile X, but it is not necessarily the identical malfunction to those with idiopathic autism who respond to bumetanide.

Objective

Observational studies and anecdotal reports suggest sertraline, a selective serotonin reuptake inhibitor (SSRI), may improve language development in young children with fragile X syndrome (FXS). We evaluated the efficacy of six months of treatment with low-dose sertraline in a randomized, double-blind, placebo-controlled trial in 52 children with FXS ages 2–6 years.


Results

Eighty-one subjects were screened for eligibility and 57 were randomized to sertraline (27) or placebo (30). Two subjects from the sertraline arm and three from the placebo arm discontinued. Intent-to-treat analysis showed no difference from placebo on the primary outcomes: the Mullen Scales of Early Learning (MSEL) expressive language age equivalent and Clinical Global Impression-Improvement (CGI-I). However, analyses of secondary measures showed significant improvements, particularly in motor and visual perceptual abilities and social participation. Sertraline was well tolerated, with no difference in side effects between sertraline and placebo groups. No serious adverse events occurred.

Conclusion

This randomized controlled trial of six-months of sertraline treatment showed no primary benefit with respect to early expressive language development and global clinical improvement. However, in secondary, exploratory analyses there were significant improvements seen on motor and visual perceptual subtests, the Cognitive T score sum on the MSEL, and on one measure of Social Participation on the Sensory Processing Measure–Preschool. Further, post hoc analysis found significant improvement in early expressive language development as measured by the MSEL among children with ASD on sertraline. Treatment appears safe for this 6-month period in young children with FXS, but we do not know the long-term side effects of this treatment. These results warrant further studies of sertraline in young children with FXS using refined outcome measures, as well as longer term follow-up studies to address long-term side effects of low-dose sertraline in early childhood.


Neurosteroid biosynthesis down‐regulation and changes in GABAA receptor subunit composition: a biomarker axis in stress‐induced cognitive and emotional impairment

By rapidly modulating neuronal excitability, neurosteroids regulate physiological processes, such as responses to stress and development. Excessive stress affects their biosynthesis and causes an imbalance in cognition and emotions. The progesterone derivative, allopregnanolone (Allo) enhances extrasynaptic and postsynaptic inhibition by directly binding at GABAA receptors, and thus, positively and allosterically modulates the function of GABA. Allo levels are decreased in stress-induced psychiatric disorders, including depression and post-traumatic stress disorder (PTSD), and elevating Allo levels may be a valid therapeutic approach to counteract behavioural dysfunction. While benzodiazepines are inefficient, selective serotonin reuptake inhibitors (SSRIs) represent the first choice treatment for depression and PTSD. Their mechanisms to improve behaviour in preclinical studies include neurosteroidogenic effects at low non-serotonergic doses. Unfortunately, half of PTSD and depressed patients are resistant to current prescribed 'high' dosage of these drugs that engage serotonergic mechanisms. Unveiling novel biomarkers to develop more efficient treatment strategies is in high demand. Stress-induced down-regulation of neurosteroid biosynthesis and changes in GABAA receptor subunit expression offer a putative biomarker axis to develop new PTSD treatments. The advantage of stimulating Allo biosynthesis relies on the variety of neurosteroidogenic receptors to be targeted, including TSPO and endocannabinoid receptors. Furthermore, stress favours a GABAA receptor subunit composition with higher sensitivity for Allo. The use of synthetic analogues of Allo is a valuable alternative. Pregnenolone or drugs that stimulate its levels increase Allo but also sulphated steroids, including pregnanolone sulphate which, by inhibiting NMDA tonic neurotransmission, provides neuroprotection and cognitive benefits. In this review, we describe current knowledge on the effects of stress on neurosteroid biosynthesis and GABAA receptor neurotransmission and summarize available pharmacological strategies that by enhancing neurosteroidogenesis are relevant for the treatment of SSRI-resistant patients. Linked Articles This article is part of a themed section on Pharmacology of Cognition: a Panacea for Neuropsychiatric Disease? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.19/issuetoc.

Too little allopregnanalone can induce autism.


Results
Autism spectrum disorder (ASD) is a neurodevelopmental disorder with core symptoms of social impairments and restrictive repetitive behaviors. Recent evidence has implicated a dysfunction in the GABAergic system in the pathophysiology of ASD. We investigated the role of endogenous allopregnanolone (ALLO), a neurosteroidal positive allosteric modulator of GABAA receptors, in the regulation of ASD-like behavior in male mice using SKF105111 (SKF), an inhibitor of type I and type II 5α-reductase, a rate-limiting enzyme of ALLO biosynthesis. SKF impaired sociability-related performance, as analyzed by three different tests; i.e., the 3-chamber test and social interaction in the open field and resident-intruder tests, without affecting olfactory function elucidated by the buried food test. SKF also induced repetitive grooming behavior without affecting anxiety-like behavior. SKF had no effect on short-term spatial working memory or long-term fear memory, but enhanced latent learning ability in male mice. SKF-induced ASD-like behavior in male mice was abolished by the systemic administration of ALLO (1mg/kg, i.p.) and methylphenidate (MPH: 2.5mg/kg, i.p.), a dopamine transporter inhibitor. The effects of SKF on brain ALLO contents in male mice were reversed by ALLO, but not MPH. On the other hand, SKF failed to induce ASD-like behavior or a decline in brain ALLO contents in female mice. These results suggest that ALLO regulates episodes of ASD-like behavior by positively modulating the function of GABAA receptors linked to the dopaminergic system. Moreover, a sex-dependently induced decrease in brain ALLO contents may provide an animal model to study the main features of ASD.



Results
Some steroids, whose levels are raised in autism (allopregnanolone, androsterone, pregnenolone, dehydroepiandrosterone and their sulfate conjugates) are neuroactive and modulate GABA, glutamate, and opioid neurotransmission, affecting brain development and functioning. These steroids may contribute to autism pathobiology and symptoms such as elevated anxiety, sleep disturbances, sensory deficits, and stereotypies among others.

Tuning the Brain
I did write a post a while back to show the effect of tuning GABAa receptors.




The effect of allopregnanolone of KCC2 expression and hence the level of chloride within neurons.

Neonatal allopregnanolone or finasteride administration modifies hippocampal K(+) Cl(-) co-transporter expression during early development in male rats.

Abstract

The maintenance of levels of endogenous neurosteroids (NS) across early postnatal development of the brain, particularly to the hippocampus, is crucial for their maturation. Allopregnanolone (Allop) is a NS that exerts its effect mainly through the modulation of the GABAA receptor (GABAAR). During early development, GABA, acting through GABAAR, that predominantly produces depolarization shifts to hyperpolarization in mature neurons, around the second postnatal week in rats. Several factors contribute to this change including the progressive increase of the neuron-specific K(+)/Cl(-) co-transporter 2 (KCC2) (a chloride exporter) levels. Thus, we aimed to analyze whether a different profile of NS levels during development is critical and can alter this natural progression of KCC2 stages. We administrated sustained Allop (20mg/kg) or Finasteride (5α-reductase inhibitor, 50mg/kg) from the 5th postnatal day (PD5) to PD9 and assessed changes in the hippocampal expression of KCC2 at transcript and protein levels as well as its active phosphorylated state in male rats. Taken together data indicated that manipulation of NS levels during early development influence KCC2 levels and point out the importance of neonatal NS levels for the hippocampal development.                                                                                                                           
Conclusion

Add very low dose Prozac to the long list of possible SIB therapies, more practical than electroconvulsive therapy (ECT), that is for sure!

This post was long waiting in my “to-complete” pile. I thought it would be a short one, but it kept growing.  It does draw together several interesting issues and shows there is a pattern developing in all these blog posts.
The majority of psychiatric drugs have such severe drawbacks that the great majority of children are better off without them.  However, there are many existing drugs that have little known neurological effects that can be highly beneficial and are known to be safe to use long term.
Psychiatric drugs that can be repurposed at lower dosages for different purposes may indeed be free of the major drawbacks encountered at higher doses.
It looks like humans with Fragile X Syndrome (FXS) are leading the way with low dose SSRI therapy to modulate GABA.  It would seem highly plausible that other idiopathic autism might also benefit and the French case studies in this post are examples of those who did benefit.
I think this is another example of fine-tuning the brain to optimize its functioning. It probably will not produce miracles, but the science shows that allopregnenalone can be tuned to vary mood in humans.  Low levels of allopregnenalone can produce autistic-like behaviours in mouse models.
The effect of allopregnenalone on KCC2 expression may only be present in tiny babies, if it continues into childhood that would be another reason to consider it as a target for modulation.  If that were the case, then Finasteride the cheap generic drug for prostate enlargement, should be investigated.
As is always the case in autism, both extremes are likely to exist; some people will likely benefit from low dose SSRIs but it will make some others worse (anxiety, SIB etc). If you start with elevated allopregnenalone, you would want less, not more.
Repurposing existing drugs has huge unrealized potential.
The OTC antihistamine Clemastine, which I highlighted in an earlier post as being a Positive Allosteric Modulator (PAM) of P2X7, and so helps remyelination, is yet another example of repurposing a safe drug.  Reportedly, it has this effect even below the regular dosage for allergy; at the high dosage usage in MS trials it will send you to sleep and risk some other side effects. As MS is not a singular condition, it seems that some people respond much more so than others. It also seems to have a benefit is some psychiatric disorders; not bad for a cheap OTC antihistamine.



Friday, 1 June 2018

Autism, Power Outages and the Starving Brain?



There are certain Critical Periods in the development of the human brain and these are the most vulnerable times to any genetic or environmental insult.  Critical Periods (CPs) will be the subject of post appearing shortly.


Another power outage waiting to happen

 Have you wondered why autism secondary to mitochondrial disease (regressive autism) almost always seem to occur before five years of age, and usually much earlier?  Why does it not happen later? Why is it's onset often preceded by a viral infection?
I think you can consider much of this in terms of the brain running out of energy. Humans have evolved to require a huge amount of energy to power their developing brains, a massive 40% of the body’s energy is required by the brain in early childhood.  If your overload a power grid it will end in a blackout.
We know many people with autism have a tendency towards mitochondrial dysfunction, they lack some key enzyme complexes. This means that the process of OXPHOS (Oxidative phosphorylation), by which the body converts glucose to usable energy (ATP), is partially disabled. 

We saw in earlier posts how the supply of glucose and oxygen to the brain can be impaired in autism because there is unstable blood flow.


It is just like in your house, all your electrical appliances might mean you need a 25KW supply, because you do not use them all at the same time. Just to be on the safe side you might have a 40KW limit. What if the power company will only give you a 20 KW connection? If you turn on the clothes drier, the oven, the air conditioning and some other things all of a sudden you blow the main fuse and perhaps damage the hard drive of your old computer.
So, in the power-hungry brain of a three-year-old, you add a viral infection and all of a sudden you exceed the available power supply from the mitochondria, that have soldered on for 3years with impaired supply of complex 1 and imperfect cerebral blood flow. By the sixth year of life, the peak power requirement from the brain would have fallen to within the safe limit of the mitochondria and its impaired supply of complex 1.  Instead of blowing the fuse, which is easy to reset, you have blown some neuronal circuitry, which is not so easy to repair.    

Too Many Synapses?
We know that it is the synapses in the brain that are the big energy users and we also know that in most autism there are too many synapses. So, in that group of autism there is an even bigger potential energy demand.



Note that in Alzheimer’s type dementia (AD in the above chart) you see a severe loss of synapses/spines as atrophy takes place. This occurs at the same time as a loss of insulin sensitivity occurs (type 3 diabetes). Perhaps the AD brain is also starved of energy, it does seem to respond to ketosis (ketones replacing glucose as the fuel) and it responds to Agmatine (increasing blood flow via eNOS).
We also know that adolescent synaptic pruning is dysfunctional in autism and we even know why. Interestingly by modifying GABAA function with bumetanide we may indeed allow the brain to eliminate more synapses (a good thing), so possibly an unexpected benefit from Ben Ari’s original idea.

"Working with a mouse model we have shown that, at puberty, there is an increase in inhibitory GABA receptors, which are targets for brain chemicals that quiet down nerve cells. We now report that these GABA receptors trigger synaptic pruning at puberty in the mouse hippocampus, a brain area involved in learning and memory." The report, published by eLife, "Synaptic pruning in the female hippocampus is triggered at puberty by extrasynaptic GABAA receptors on dendritic spines."            
These findings may suggest new treatments targeting GABA receptors for "normalizing" synaptic pruning in diseases such as autism and schizophrenia, where synaptic pruning is abnormal. Research has suggested that children with autism may have an over-abundance of synapses in some parts of the brain.

Synaptic pruning in the female hippocampus is triggered at puberty by extrasynaptic GABAA receptors on dendritic spines

Adolescent synaptic pruning is thought to enable optimal cognition because it is disrupted in certain neuropathologies, yet the initiator of this process is unknown. One factor not yet considered is the α4βδ GABAA receptor (GABAR), an extrasynaptic inhibitory receptor which first emerges on dendritic spines at puberty in female mice. Here we show that α4βδ GABARs trigger adolescent pruning. Spine density of CA1 hippocampal pyramidal cells decreased by half post-pubertally in female wild-type but not α4 KO mice. This effect was associated with decreased expression of kalirin-7 (Kal7), a spine protein which controls actin cytoskeleton remodeling. Kal7 decreased at puberty as a result of reduced NMDAR activation due to α4βδ-mediated inhibition. In the absence of this inhibition, Kal7 expression was unchanged at puberty. In the unpruned condition, spatial re-learning was impaired. These data suggest that pubertal pruning requires α4βδ GABARs. In their absence, pruning is prevented and cognition is not optimal.


Strange Patterns of Growth
Longitudinal studies are when researchers collect the same data over long period of years. Most autism research is just based on a single snapshot in time.
One observation of mine is that some people with strictly defined autism (SDA) are born at the 90+ percentile for height, but then fall back to something like the 20 percentile. Body growth has dramatically slowed. Was this because energy has been diverted to the overgrowing brain? 
A five-year old’s brain is an energy monster. It uses twice as much glucose (the energy that fuels the brain) as that of a full-grown adult, a new study led by Northwestern University anthropologists has found.
It was previously believed that the brain’s resource burden on the body was largest at birth, when the size of the brain relative to the body is greatest. The researchers found instead that the brain maxes out its glucose use at age 5. At age 4 the brain consumes glucose at a rate comparable to 66 percent of the body’s resting metabolic rate (or more than 40 percent of the body’s total energy expenditure). 

“The mid-childhood peak in brain costs has to do with the fact that synapses, connections in the brain, max out at this age, when we learn so many of the things we need to know to be successful humans,” Kuzawa said.

“At its peak in childhood, the brain burns through two-thirds of the calories the entire body uses at rest, much more than other primate species,” said William Leonard, co-author of the study. “To compensate for these heavy energy demands of our big brains, children grow more slowly and are less physically active during this age range. Our findings strongly suggest that humans evolved to grow slowly during this time in order to free up fuel for our expensive, busy childhood brains.” 

Full paper: -


The high energetic costs of human brain development have been hypothesized to explain distinctive human traits, including exceptionally slow and protracted preadult growth. Although widely assumed to constrain life-history evolution, the metabolic requirements of the growing human brain are unknown. We combined previously collected PET and MRI data to calculate the human brain’s glucose use from birth to adulthood, which we compare with body growth rate. We evaluate the strength of brain–body metabolic trade-offs using the ratios of brain glucose uptake to the body’s resting metabolic rate (RMR) and daily energy requirements (DER) expressed in glucose-gram equivalents (glucosermr% and glucoseder%). We find that glucosermr% and glucoseder% do not peak at birth (52.5% and 59.8% of RMR, or 35.4% and 38.7% of DER, for males and females, respectively), when relative brain size is largest, but rather in childhood (66.3% and 65.0% of RMR and 43.3% and 43.8% of DER). Body-weight growth (dw/dt) and both glucosermr% and glucoseder% are strongly, inversely related: soon after birth, increases in brain glucose demand are accompanied by proportionate decreases in dw/dt. Ages of peak brain glucose demand and lowest dw/dt co-occur and subsequent developmental declines in brain metabolism are matched by proportionate increases in dw/dt until puberty. The finding that human brain glucose demands peak during childhood, and evidence that brain metabolism and body growth rate covary inversely across development, support the hypothesis that the high costs of human brain development require compensatory slowing of body growth rate. 

To quantify the metabolic costs of the human brain, in this study we used a unique, previously collected age series of PET measures of brain glucose uptake spanning birth to adulthood (32), along with existing MRI volumetric data (36), to calculate the brain’s total glucose use from birth to adulthood, which we compare with body growth rate. We estimate total brain glucose uptake by age (inclusive of all oxidative and nonoxidative functions), which we compare with two measures of whole-body energy expenditure: RMR, reflecting maintenance functions only, and daily energy requirements (DER), reflecting the combination of maintenance, activity, and growth. We hypothesized that ages of peak substrate competition (i.e., competition for glucose) between brain and body would be aligned developmentally with the age of slowest childhood body growth, and more generally that growth rate and brain glucose use would covary inversely during development, as is predicted by the concept of a trade-off between brain metabolism and body growth in human life-history evolution. 

Daily glucose use by the brain peaks at 5.2 y of age at 167.0 g/d and 146.1 g/d in males and females, respectively. These values represent 1.88- and 1.82-times the daily glucose use of the brain in adulthood (Fig. 1 A and B and SI Appendix, Fig. S2), despite the fact that body size is more than three-times as large in the adult.




Glucose use of the human brain by age. (A) Grams per day in males. (B) Grams per day in females; dashed horizontal line is adult value (A and B). (C) Glucosermr% (solid line) and glucoseder% (dashed line) in males. (D) Glucosermr% (solid line) and glucoseder% (dashed line) in females.

The most relevant data is the line highlighted in yellow below, showing brain consumption of glucose peaks at 40% (of total body consumption) around 5 years old and drops to 20% in adulthood.

Our findings agree with past estimates indicating that the brain dominates the body’s metabolism during early life (31). However, our PET-based calculations reveal that the magnitude of brain glucose uptake, both in absolute terms and relative to the body’s metabolic budget, does not peak at birth but rather in childhood, when the glucose used by the brain comprises the equivalent of 66% of the body’s RMR, and roughly 43% of total expenditure. These findings are in broad agreement with past clinical work showing that the body’s mass-specific glucose production rates are highest in childhood, and tightly linked with the brain’s metabolic needs (40). Whereas past attempts to quantify the contribution of the brain to the body’s metabolic expenditure suggested that the brain accounted for a continuously decreasing fraction of RMR as the brain-to-body weight ratio declined with age (25, 31), we find a more complex pattern of substrate trade-off. Both glucosermr% and glucoseder% decline in the first half-year as a fast but decelerating pace of body growth established in utero initially outpaces postnatal increases in brain metabolism. Beginning around 6 mo, increases in relative glucose use are matched by proportionate decreases in weight growth, whereas ages of declining brain glucose uptake in late childhood and early adolescence are accompanied by proportionate increases in weight growth. The relationships that we document between age changes in brain glucose demands and body-weight growth rate are particularly striking in males, who maintain these inverse linear trends despite experiencing threefold changes in brain glucose demand and body growth rate between 6 mo and 13 y of age. In females, an earlier onset of pubertal weight gain leads to earlier deviations from similar linear inverse relationships.
                                     

What the researchers then did was to see how the growth rate of the brain is correlated to the growth rate of the body. In effect that what they found was that the growth of the body has to slow down to allow the energy hungry brain to develop.  One the brain has passed its peak energy requirement at about 5 years old, body growth can then gradually accelerate. 
The brain is the red line, the body is blue. The chart on the left is males and the one on the right is females. 
So, we might suspect that in 2 to 4-year olds who seem not to be growing as fast as we might expect, the reason is that their brain is over-growing, a key feature of classic autism.

Glucoseder% and body-weight growth rate. Glucoseder% and weight velocities plotted as SD scores to allow unitless comparison. (A) Glucoseder% (red dots) and dw/dt (blue dots) by age in males. (B) Glucoseder% (red dots) and dw/dt (blue dots) by age in females


Brain Overgrowth in Autism
As has been previous commented on in this blog, Eric Courchesne has pretty much figured out what goes wrong in the growth trajectory of the autistic brain; that was almost 15 years ago.

Brain development in autism: early overgrowth followed by premature arrest of growth.


Author information


Abstract


Due to the relatively late age of clinical diagnosis of autism, the early brain pathology of children with autism has remained largely unstudied. The increased use of retrospective measures such as head circumference, along with a surge of MRI studies of toddlers with autism, have opened a whole new area of research and discovery. Recent studies have now shown that abnormal brain overgrowth occurs during the first 2 years of life in children with autism. By 2-4 years of age, the most deviant overgrowth is in cerebral, cerebellar, and limbic structures that underlie higher-order cognitive, social, emotional, and language functions. Excessive growth is followed by abnormally slow or arrested growth. Deviant brain growth in autism occurs at the very time when the formation of cerebral circuitry is at its most exuberant and vulnerable stage, and it may signal disruption of this process of circuit formation. The resulting aberrant connectivity and dysfunction may lead to the development of autistic behaviors. To discover the causes, neural substrates, early-warning signs and effective treatments of autism, future research should focus on elucidating the neurobiological defects that underlie brain growth abnormalities in autism that appear during these critical first years of life.


Research from 2017: -





Conclusion
A record of children’s height and weight and even head circumference is usually collected by their doctor. In an earlier post I did ask why they bother if nobody is checking this data. If a child falls from the 90th percentile in height to the 20th, something clearly is going on.
When I discussed this with a pediatric endocrinologist a few years ago, we then measured bone-age and IGF-1. If you have low IGF-1 and retarded bone age you might opt for some kind of growth hormone therapy.
In what is broadly defined as autism, I think we have some distinctly different things possibly happening: -

Group AMD
Energy conversion in the brain is less efficient than it should be due to a combination of impaired vascular function and impaired mitochondrial enzyme complex production. No symptoms are apparent and developmental milestones are achieved.  As the brain creates more synapses it energy requirement grows until the day when the body has some external insult like a viral infection, and the required power is not available, triggering a “power outage” which appears as the regression into autism. In biological terms there has been death of neurons and demyelination.

Group Sliding Down the Percentiles 
This group looks like a sub-set of classic autism. The brain grows too rapidly in the first two years after birth and this causes the expected slowing of body growth to occur much earlier than in typical children. This manifests itself in the child tumbling down the percentiles for height and weight.
The brain then stops growing prematurely, reducing energy consumption and allowing body growth to accelerate and the child slowly rises back up the height/weight percentiles.

Perhaps all those excessive synapses that were not pruned correctly are wasting glucose and so delay the growth of the rest of the body?   
In the sliding down the growth percentiles group, does this overgrowing brain ever exceed maximum available power? Maybe it just grows too fast and so mal-develops, as suggested by Courchesne, or maybe it grows too fast and cannot fuel correct development?  What happens if you increase maximum available power in this group, in the way some athletes use to enhance their performance/cheat?
All I know for sure is that in Monty, aged 14 with autism, increasing eNOS (endothelial nitric oxide synthase) using agmatine seems to make him achieve much more, with the same daily glucose consumption. I wonder what would happen if Agmatine was given to very young children as soon as it was noted that they were tumbling down the height percentiles?  This is perhaps what the pediatric endocrinologists should be thinking about, rather than just whether or not to administer growth hormones/IGF-1.
If you could identify Group AMD before the “power outage” you might be able to boost maximum power production or reduce body growth slightly and hence avoid the brain ever being starved of energy. That way you would not have most regressive autism.







Sunday, 25 September 2016

Excitotoxicity triggered by GABAa dysfunction




  
This blog, as you will have noticed, does rather meander through science of autism.  As a result there are some gaps and unanswered questions.

The blog talks a lot about the neurotransmitter GABA and the excitatory/inhibitory imbalance.  We have ended up with some therapies based on this that do seem to help many people.

The opposing (excitatory) neurotransmitter is glutamate which affects the NMDA, AMP and mGlu receptors.

It appears that in autism there is an unusually high level of glutamate, but another issue looks likely to be at specific receptors, for example mGluR5



This does get very complicated and lacks any immediate therapies. 

One very interesting insight was that you can repurpose the existing cheap generic GABAB drug Baclofen to treat NMDAR-hypofunction. 

This seems to work really well at low doses with many people with Asperger’s.  People with more severe autism do not seem to respond to low doses, however some do to higher doses.  The more potent version R Baclofen is a research drug.

GABAb-mediated rescue of altered excitatory–inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction



Reduced N-methyl-D-aspartate-receptor (NMDAR) signaling has been associated with schizophrenia, autism and intellectual disability. NMDAR-hypofunction is thought to contribute to social, cognitive and gamma (30–80 Hz) oscillatory abnormalities, phenotypes common to these disorders.

Constitutive NMDAR-hypofunction caused a loss of E/I balance, with an increase in intrinsic pyramidal cell excitability and a selective disruption of parvalbumin-expressing interneurons. Disrupted E/I coupling was associated with deficits in auditory-evoked gamma signal-to-noise ratio (SNR). Gamma-band abnormalities predicted deficits in spatial working memory and social preference, linking cellular changes in E/I signaling to target behaviors. The GABAB-receptor agonist baclofen improved E/I balance, gamma-SNR and broadly reversed behavioral deficits.



Excitotoxicity

We have touched on this subject on a few occasions but today, excitotoxicity is the focus of this post.
  
Excitotoxicity looks likely to be present in much autism and helps to connect all the various dysfunctions that we can read about in the literature.

It is a little scary because you cannot know to what extent this process is reversible.  It looks like in milder cases it should be treatable, whereas in extreme cases damage will be irreversible.

Excitotoxicity is the pathological process by which nerve cells are damaged or killed by excessive stimulation by neurotransmitters, particularly glutamate. This occurs when receptors for the excitatory neurotransmitter glutamate (glutamate receptors) such as the NMDA receptor and AMPA receptor are overactivated by glutamatergic storm. 

Unfortunately you can trigger glutamate excitotoxity via a dysfunction in GABAA receptors.

For example if you severely inhibit GABAA receptors you kill brain cells, but it was the reaction in glutamate signaling that did the damage.  GABA is supposed to be inhibitory; in some autism it is not and then Glutamate gets out of balance.  This does lead to excess firing of neurons, which seems to degrade cognition, but it will tend towards glutamate excitotoxity.

When you see the cascade of events triggered by glutamate excitotoxity you will see how this really helps to explain biological finding in autism, even mitochondrial dysfunctions.

You can then trace this all back to the faulty GABA switch caused by too little KCC2 and too much NKCC1.

Then you can look at other neurological conditions that feature glutamate excitotoxity, like traumatic brain injury and neuropathic pain, and you see that the research shows low expression of KCC2.

This then suggests that much of autism would have been prevented if you could increase KCC2.  You would not just fix the E/I imbalance but you would avoid all the damage done by excitotoxity.

Just how early you would have to correct KCC2 expression is not clear.  For sure it is a case of better late than never, but how much damage caused by excitotoxicity is reversible?


Good News

The good news is that because KCC2 underexpression is a feature of many conditions there is plenty of research money being spent looking for answers.  When they find a solution for increasing KCC2 to treat neuropathic pain, or spinal cord injury (SCI), the drug can be simply re-purposed for autism.

The French government is funding research into increasing KCC2 to treat SCI.  They are starting with serotin  5-HT2A receptor agonists.  Regular readers without any memory loss may recall that back in the 1960 Lovaas was giving LSD to people with autism at UCLA.  LSD is a potent 5-HT2A receptor agonist.  The French are also looking at BDNF to upregulate KCC2 and then they plan to have a blind test where they try all the chemicals they have in their library.  The French are of course doing their trials in test tubes.

When I looked at this subject a while back, I looked for existing therapies that are known to be safe and should be effective.

Treating KCC2 Down-Regulation in Autism, Rett/Down Syndromes, Epilepsy and Neuronal Trauma ?




My conclusion then was that intranasal insulin was the best choice.



Excitoxicity in Autism




Autism is a debilitating neurodevelopment disorder characterized by stereotyped interests and behaviours, and abnormalities in verbal and non-verbal communication. It is a multifactorial disorder resulting from interactions between genetic, environmental and immunological factors. Excitotoxicity and oxidative stress are potential mechanisms, which are likely to serve as a converging point to these risk factors. Substantial evidence suggests that excitotoxicity, oxidative stress and impaired mitochondrial function are the leading cause of neuronal dysfunction in autistic patients. Glutamate is the primary excitatory neurotransmitter produced in the CNS, and overactivity of glutamate and its receptors leads to excitotoxicity. The over excitatory action of glutamate, and the glutamatergic receptors NMDA and AMPA, leads to activation of enzymes that damage cellular structure, membrane permeability and electrochemical gradients. The role of excitotoxicity and the mechanism behind its action in autistic subjects is delineated in this review










The influx of intracellular calcium triggers the induction of inducible nitric oxide (iNOS) and phosphorylation of protein kinase C. Increased iNOS enhances nitric oxide (NO•) production in excess, whereas protein kinase C activates phospholipase A2 which in turn results in the generation of pro-inflammatory molecules The subsequent generation of free radicals can inhibit oxidative phosphorylation and damage mitochondrial enzymes involved in the electron transport chain, which mitigate energy production .

Reactive intermediates such as peroxynitrates and other peroxidation products hamper the normal function of mitochondrial enzymes by impairing oxidative phosphorylation and inhibiting complex II of the electron transport chain. Moreover, lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) can interact with synaptic protein and impair transport of glucose and glutamate, thereby decreasing energy production and increasing excitotoxic sensitivity

Overstimulation of the glutamate receptors, NMDA and AMPA, leads to the release of other excitotoxins resulting in the accumulation of glutamate. Indeed, excess glutamate concentrations results in an increase in calcium levels in the cytosol. This effect is attributed to the fact that excessive glutamate allows calcium channel to open for longer periods of time, leading to increased influx of calcium into cells. Calcium triggers inducible nitric oxide and protein kinase C that produce free radicals, ROS and arachidonic aid. Generation of these oxidants results in mitochondrial dysfunction and accumulation of pro-inflammatory molecules and finally cell death. Free radicals interact with the mitochondrial and cellular membrane to form lipid peroxidation. 4-HNE is a major destructive product of this process. Lipid peroxidation prevents the dephosphorylation of excessively phosphorylated tau protein, significantly interfering with microtubule function. It has also been shown to inhibit glutathione reductase needed to convert oxidised glutathione to its functional reduced form

The mechanism responsible for excitotoxicity and neuronal cell death is diverse. Experimental studies have shown that the apoptotic and/or necrotic cell death may be due to the severity of NMDA damage or can be dependent on receptor subunit composition of neurons (Bonfoco et al. 1995; Portera-Cailliau et al. 1997). Pathological events related to this mode of action can be loss of cellular homoeostasis with acute mitochondrial dysfunction leading to hindrance in ATP production. Moreover, glutamatergic insults can cause cell death by the action of one or more molecular pathways which involves the action of signaling molecules such as cysteine proteases, mitochondrial endonucleases, peroxynitrite, PARP-1 and GAPDH in the excitotoxic neurodegeneration pathway.

Intracellular calcium levels also rely on voltage-dependent calcium channels and Na exchangers . The Na?/Ca2? exchanger is a bi-directional membrane ion transporter, which during membrane depolarisation or the opening of the gated sodium channels, transports sodium out of the cell and calcium into the cell. AMPA-type glutamate receptors are highly permeable to calcium and its over expression can lead to excitotoxicity. The Ca2? permeability capability of AMPA-type glutamate receptors relies on the presence or the absence of the GluR2 subunit in the receptor complex. Reduced GluR2 expression permits the construction of AMPA receptors with high Ca2? permeability and contributes to neuronal defect and excitotoxicity. Another mechanism is the release of calcium from internal stores such as the endoplasmic reticulum and mitochondria. It results in mitochondrial dysfunction, reduction in ATP synthesis and ROS generation.

Voltage gated channels found in dendrites and cell bodies of neurons modulate neuronal excitability and calcium-regulated signaling cascades (Dolmetsch et al. 2001; Catterall et al. 2005). Point mutations in the gene encoding the L-type voltage-gated channels Ca v1.2 (CACNA1C) and Ca v1.4. (CACNA1F) prevent voltage-dependent inactivation of these genes. This causes the channel to open for longer time, leading to excessive influx of calcium.

Conclusion

Autism is a multifactorial disorder characterized by neurobehavioral and neurological dysfunction. Excitotoxicity is the major neurobiological mechanism that modulates diverse risk factors associated with autism. It is triggered by potential mutation in ion channels and signalling pathways, viral and bacterial pathogens, toxic metals and free radical generation. Over expression of glutamate receptors and increased glutamate levels leads to increased calcium influx and oxidative stress and progressive cellular degeneration and cell death. Genetic defect, such as mutation in voltage gated or ligand channels that regulate neuronal excitability leads to defect in synaptic transmission and excitotoxic condition in autism. Mutation in BKCa and Ca v1.2 channels also results in excess calcium influx Sodium, potassium and chloride channels also play important roles in maintaining homoeostasis of neuronal cells, and decreased channel activity leads to destabilization of membrane potential and excitotoxicity. Moreover, over expression of BDNF results in hyperexcitability. Excessive BDNF and NMDA receptor activity increases the neurotransmitter release and excitotoxic vulnerability. Given that autism is a multifaceted disorder with multiple risk factors, more precise studies are needed to explore the signalling pathways that influence emergence of excitotoxicity in ASDs.


Some relevant reading for those interested:-


GABAergic/glutamatergic imbalance relative to excessive neuroinflammation in autism spectrum disorders


Abstract

Background

Autism spectrum disorder (ASD) is characterized by three core behavioral domains: social deficits, impaired communication, and repetitive behaviors. Glutamatergic/GABAergic imbalance has been found in various preclinical models of ASD. Additionally, autoimmunity immune dysfunction, and neuroinflammation are also considered as etiological mechanisms of this disorder. This study aimed to elucidate the relationship between glutamatergic/ GABAergic imbalance and neuroinflammation as two recently-discovered autism-related etiological mechanisms.

Methods

Twenty autistic patients aged 3 to 15 years and 19 age- and gender-matched healthy controls were included in this study. The plasma levels of glutamate, GABA and glutamate/GABA ratio as markers of excitotoxicity together with TNF-α, IL-6, IFN-γ and IFI16 as markers of neuroinflammation were determined in both groups.

Results

Autistic patients exhibited glutamate excitotoxicity based on a much higher glutamate concentration in the autistic patients than in the control subjects. Unexpectedly higher GABA and lower glutamate/GABA levels were recorded in autistic patients compared to control subjects. TNF-α and IL-6 were significantly lower, whereas IFN-γ and IFI16 were remarkably higher in the autistic patients than in the control subjects.

Conclusion

Multiple regression analysis revealed associations between reduced GABA level, neuroinflammation and glutamate excitotoxicity. This study indicates that autism is a developmental synaptic disorder showing imbalance in GABAergic and glutamatergic synapses as a consequence of neuroinflammation.
Keywords: Autism, Glutamate excitotoxicity, Gamma aminobutyric acid (GABA), Glutamate/GABA, Tumor necrosis factor-α, Interleukin-6, Interferon-gamma, Interferon-gamma-inducible protein 16


Postmortem brain abnormalities of the glutamate neurotransmitter system in autism.



CONCLUSIONS:

Subjects with autism may have specific abnormalities in the AMPA-type glutamate receptors and glutamate transporters in the cerebellum. These abnormalities may be directly involved in the pathogenesis of the disorder.



Pathophysiologyof traumatic brain injury


General pathophysiology of traumatic brain injury
The first stages of cerebral injury after TBI are characterized by direct tissue damage and impaired regulation of CBF and metabolism. This ‘ischaemia-like’ pattern leads to accumulation of lactic acid due to anaerobic glycolysis, increased membrane permeability, and consecutive oedema formation. Since the anaerobic metabolism is inadequate to maintain cellular energy states, the ATP-stores deplete and failure of energy-dependent membrane ion pumps occurs. The second stage of the pathophysiological cascade is characterized by terminal membrane depolarization along with excessive release of excitatory neurotransmitters (i.e. glutamate, aspartate), activation of N-methyl-d-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolpropionate, and voltage-dependent Ca2+- and Na+-channels. The consecutive Ca2+- and Na+-influx leads to self-digesting (catabolic) intracellular processes. Ca2+ activates lipid peroxidases, proteases, and phospholipases which in turn increase the intracellular concentration of free fatty acids and free radicals. Additionally, activation of caspases (ICE-like proteins), translocases, and endonucleases initiates progressive structural changes of biological membranes and the nucleosomal DNA (DNA fragmentation and inhibition of DNA repair). Together, these events lead to membrane degradation of vascular and cellular structures and ultimately necrotic or programmed cell death (apoptosis).

Excitotoxicity and oxidative stress
TBI is primarily and secondarily associated with a massive release of excitatory amino acid neurotransmitters, particularly glutamate.854 This excess in extracellular glutamate availability affects neurons and astrocytes and results in over-stimulation of ionotropic and metabotropic glutamate receptors with consecutive Ca2+, Na+, and K+-fluxes.2273 Although these events trigger catabolic processes including blood–brain barrier breakdown, the cellular attempt to compensate for ionic gradients increases Na+/K+-ATPase activity and in turn metabolic demand, creating a vicious circle of flow–metabolism uncoupling to the cell.1650
Oxidative stress relates to the generation of reactive oxygen species (oxygen free radicals and associated entities including superoxides, hydrogen peroxide, nitric oxide, and peroxinitrite) in response to TBI. The excessive production of reactive oxygen species due to excitotoxicity and exhaustion of the endogenous antioxidant system (e.g. superoxide dismutase, glutathione peroxidase, and catalase) induces peroxidation of cellular and vascular structures, protein oxidation, cleavage of DNA, and inhibition of the mitochondrial electron transport chain.31160 Although these mechanisms are adequate to contribute to immediate cell death, inflammatory processes and early or late apoptotic programmes are induced by oxidative stress.11



Knocking down of the KCC2 in rat hippocampal neurons increases intracellular chloride concentration and compromises neuronal survival



Non-technical summary

‘To be, or not to be’– thousands of neurons are facing this Shakespearean question in the brains of patients suffering from epilepsy or the consequences of a brain traumatism or stroke. The destiny of neurons in damaged brain depends on tiny equilibrium between pro-survival and pro-death signalling. Numerous studies have shown that the activity of the neuronal potassium chloride co-transporter KCC2 strongly decreases during a pathology. However, it remained unclear whether the change of the KCC2 function protects neurons or contributes to neuronal death. Here, using cultures of hippocampal neurons, we show that experimental silencing of endogenous KCC2 using an RNA interference approach or a dominant negative mutant reduces neuronal resistance to toxic insults. In contrast, the artificial gain of KCC2 function in the same neurons protects them from death. This finding highlights KCC2 as a molecule that plays a critical role in the destiny of neurons under toxic conditions and opens new avenues for the development of neuroprotective therapy.


New understanding of brainchemistry could prevent brain damage after injury





Sciences de la vie, de la santé et des écosystèmes : Neurosciences (Blanc SVSE 4) 2010
Projet 
KCC2-SCI

The potassium-chloride transporter KCC2 : a new target for the treatment of neurological diseases




A decrease in synaptic inhibition –disinhibition- appears to be an important substrate in several neuronal disorders, such as spinal cord injury (SCI), neuropathic pain... Glycine and GABA are the major inhibitory transmitters in the spinal cord. An important emerging mechanism by which the strength of inhibitory synaptic transmission can be controlled is via modification of the intracellular concentration of chloride ions ([Cl-]i) to which receptors to GABA/glycine are permeable. Briefly, a low [Cl-]i is a pre-requisite for inhibition to occur and is maintained in healthy neurons by cation-chloride co-transporters (KCC2) in the plasma membrane, which extrude Cl-. We showed recently (Nature Medicine, accepted for publication) that these transporters are down-regulated after SCI, thereby switching the action of GABA and glycine from inhibition to excitation; this can account for both SCI-induced spasticity and chronic pain. KCC2 transporters therefore appear as a new target to restore inhibition within neuronal networks in pathological conditions. The present project aims at reducing spasticity and chronic pain after SCI by up-regulating KCC2. 
An important part will consist in identifying new compounds that increase the cell surface expression and/or the functionality of KCC2. Two strategies are considered. 1) Serotonin and BDNF will be tested on the basis of preliminary experiments and/or previous reports in other areas of the central nervous system indicating that these two compounds may affect the expression of KCC2. 2)Testing a large amount of compounds available in a library (“blind test”) to sort out KCC2-modulating molecules. This task can only be done in vitro on an assay that enables to easily visualize and quantify cell surface expression of KCC2, in response to these molecules (HEK293 cells). The few compounds isolated at the end of this task will then be tested on cultures of motoneurons (both mouse motoneurons and human motoneurons derived from induced pluripotent cells) and characterized further (potential toxicity, ability to cross the Brain Blood Barrier and effect on internalization and endocytosis of KCC2). 
The selected candidate compounds will enter into the in vivo validation phase aimed at increasing the expression of KCC2 following spinal cord injury (SCI; both contusion and complete spinal cord transection). The selected hits will be applied by intrathecal injections in SCI rats and their effects on KCC2 expression in the plasma membrane of motoneurons will be tested by means of western blots and immunohistochemistry. Their efficacy in increasing the cell-surface expression of KCC2 will also be tested electrophysiologically in vitro (i.e. their ability to hyperpolarize ECl). Functionally, their efficacy in reducing both SCI-induced spasticity and chronic pain will be assessed. 
Genetic tools will be used to increase the expression of KCC2 in some spinal neurons. This task will be done in collaboration with teams in the USA. Lentiviral vectors aimed at increasing KCC2 in the host cells, after parenchymal injection, have been developed in San Diego. A transgenic mouse model with a conditional tamoxifen-induced overexpression of KCC2 has been developed in Pittsburgh. The rationale for this part of the project is to use these genetic tools in the chronic phase of SCI to reduce spasticity and chronic pain. 
The last part of the project will focus on more fundamental issues regarding the relationship between the SCI-induced downregulation of KCC2 and the development of spasticity and chronic pain. 
The significance of the expected results goes far beyond the scope of SCI, since altered chloride homeostasis resulting from mutation or dysfunction of cation-chloride cotransporters has been implicated in various neurological disorders such as, for instance, ischemic seizures neonatal seizures and temporal lobe epilepsy. 


KCC2 escape from neuropathic pain






Activationof 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2.



 In healthy adults, activation of γ-aminobutyric acid (GABA)(A) and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl(-)](i)), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E(IPSP), in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT(2A) receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT(2A)R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).



Conclusion

Very little is certain in autism, in great part because only about 200 brains have ever been examined post mortem.  There are many theories, but very many more sub-types of autism.

GABAA dysfunction due to the faulty GABA switch never increasing KCC2 expression in the first weeks of life, triggering glutamate excitotoxicity and all that follows would go a long way to explaining my son’s type of autism. It might well explain 30+% of all autism.

Clearly other causes of excess glutamate would lead to a similar result.