Epiphany

Friday, 21 April 2017

The Excitatory/Inhibitory Imbalance – GABAA stabilization via IP3R

This blog aims to synthesize the relevant parts of the research and make connections that point towards some potential therapeutic avenues. Most researchers work in splendid isolation and concentrate on one extremely narrow area of interest.

The GABA_A reset, not functional in some autism

On the one hand things are very simple, if the GABA_A receptors function correctly and are inhibitory and the glutamate receptors (particularly NMDA and mGluRx) function correctly, there is harmony and a perfect excitatory/inhibitory balance.

Unfortunately numerous different things can go wrong and you could write a book about each one.

As you dig deeper you see that the sub-unit make-up of GABA_A receptors is not only critical but changes. The plus side is that you can influence this.

Today we see that the receptors themselves are physically movable and sometimes get stuck in the “wrong place”. When the receptors cluster close together they produce a strong inhibitory effect, but continual activation of NMDA receptors by the neurotransmitter glutamate - as occurs naturally during learning and memory, or in epilepsy - leads to an excess of incoming calcium, which ultimately causes the receptors to become more spread out, reducing how much the neuron can be inhibited by GABA. There needs to be a mechanism to move the GABA_Areceptorsback into their original clusters.

Very clever Japanese researchers have figured out the mechanism and to my surprise it involves one of those hubs, where strange things in autism seem to connect to, this time IP₃R.

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

I guess the Japanese answer to my question above is simple. YES,

A very recent science-light article by Gargus on IP3:-

Mighty element plays major part in autism

Now to the Japanese.

I wonder if Gargus has read the Japanese research, because both the cause and cure for the GABA_A receptors dispersing and clustering is an increase in calcium and both mediated by glutamate.

The excitatory neurotransmitter glutamate binds to the mGluR receptor and activates IP₃ receptor-dependent calcium release and protein kinase C to promote clustering of GABA_A receptors at the postsynaptic membrane - the place on a neuron that receives incoming neurotransmitters from connecting neurons.

If Professor Gargus is correct, and IPR3 does not work properly in autism, the GABA_A receptors are likely not sitting there in nice neat clusters. As a result their inhibitory effect is reduced and neurons fire when they should not.

Gargus has found that in the types of autism he has investigated IP₃ receptor open as they should, but close too fast and so do not release enough calcium from the cell’s internal calcium store (the endoplasmic reticulum).

In particular the Japanese researchers found that:-

“Stabilization of GABA synapses by mGluR-dependent Ca²⁺ release from IP₃R via PKC”

If the IP₃ receptor does not stay open as long as it should, not enough Ca²⁺will be released and GABA synapses will not be stabilized. Then GABA_A receptors will be diffused rather than being in neat clusters.

The science-light version of the Japanese study:-

How excitatory/inhibitory balance is maintained in the brain

Just as a thermostat is used to maintain a balanced temperature in a home, different biological processes maintain the balance of almost everything in our bodies, from temperature and oxygen to hormone and blood sugar levels. In our brains, maintaining the balance -- or homeostasis -- between excitation and inhibition within neural circuits is important throughout our lives, and now, researchers at the RIKEN Brain Science Institute and Nagoya University in Japan, and École Normale Supérieure in France have discovered how disturbed inhibitory connections are restored. Published in Cell Reports, the work shows how inhibitory synapses are stabilized when the neurotransmitter glutamate triggers stored calcium to be released from the endoplasmic reticulum in neurons.

"Imbalances in excitation and inhibition in the brain has been linked to several disorders," explains lead author Hiroko Bannai. "In particular, forms of epilepsy and even autism appear to be related to dysfunction in inhibitory connections."

One of the key molecules that regulates excitation/inhibition balance in the brain is the inhibitory neurotransmitter GABA. When GABA binds to GABAA receptors on the outside of a neuron, it prevents that neuron from sending signals to other neurons. The strength of the inhibition can change depending on how these receptors are spaced in the neuron's membrane.

While GABAA receptors are normally clustered together, continual neural activation of NMDA receptors by the neurotransmitter glutamate -- as occurs naturally during learning and memory, or in epilepsy -- leads to an excess of incoming calcium, which ultimately causes the receptors to become more spread out, reducing how much the neuron can be inhibited by GABA.

To combat this effect, the receptors are somehow continually re-clustered, which maintains the proper excitatory/inhibitory balance in the brain. To understand how this is accomplished, the team focused on another signaling pathway that also begins with glutamate, and is known to be important for brain development and the control of neuronal growth.

In this pathway glutamate binds to the mGluR receptor and leads to the release of calcium from internal storage into the neuron's internal environment. Using quantum dot-single particle tracking, the team was able to show that after release, this calcium interacts with protein kinase C to promote clustering of GABAA receptors at the postsynaptic membrane--the place on a neuron that receives incoming neurotransmitters from connecting neurons.

These findings show that glutamate activates distinct receptors and patterns of calcium signaling for opposing control of inhibitory GABA synapses.

Notes Bannai, "it was surprising that the same neurotransmitter that triggers GABAA receptor dispersion from the synapse, also plays a completely opposite role in stabilizing GABAA receptors, and that the processes use different calcium signaling pathways. This shows how complex our bodies are, achieving multiple functions by maximizing a limited number of biological molecules.

Pre-activation of the cluster-forming pathway completely prevented the dispersion of GABAA receptors that normally results from massive excitatory input, as occurs in status epilepticus -- a condition in which epileptic seizures follow one another without recover of consciousness. Bannai explains, "further study of the molecular mechanisms underlying the process we have uncovered could help develop treatments or preventative medication for pathological excitation-inhibition imbalances in the brain.

"The next step in understanding how balance is maintained in the brain is to investigate what controls which pathway is activated by glutamate. Most types of cells use calcium signals to achieve biological functions. On a more basic level, we believe that decoding these signals will help us understand a fundamental biological question: why and how are calcium signals involved in such a variety of biological phenomena?"

The full Japanese study:-

Bidirectional Control of Synaptic GABA_AR Clustering by Glutamate and Calcium

· Bidirectional synaptic control system by glutamate and Ca²⁺ signaling

· Stabilization of GABA synapses by mGluR-dependent Ca²⁺ release from IP₃R via PKC

· Synaptic GABA_AR clusters stabilized through regulation of GABA_AR lateral diffusion

· Competition with an NMDAR-dependent Ca²⁺ pathway driving synaptic destabilization

GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABA_A receptor (GABA_AR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP₃ receptor-dependent calcium release and protein kinase C to promote GABA_AR clustering and GABAergic transmission. This GABA_AR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses.

In this study, we demonstrate that the mGluR/IICR/PKC pathway stabilizes GABAergic synapses by constraining lateral diffusion and increasing clustering of GABA_ARs, without affecting the total number of GABA_AR on the cell surface. This pathway defines a unique form of homeostatic regulation of GABAergic transmission under conditions of basal synaptic activity and during recovery from E/I imbalances. The study also highlights the ability of neurons to convert a single neurotransmitter (glutamate) into an asymmetric control system for synaptic efficacy using different calcium-signaling pathways.

The most striking conceptual finding in this study is that two distinct intracellular signaling pathways, NMDAR-driven Ca²⁺ influx and mGluR-driven Ca²⁺ release from the ER, effectively driven by the same neurotransmitter, glutamate, have an opposing impact on the stability and function of GABAergic synapses. Sustained Ca²⁺ influx through ionotropic glutamate receptor-dependent calcium signaling increases GABA_AR lateral diffusion, thereby causing the dispersal of synaptic GABA_AR, while tonic mGluR-mediated IICR restrains the diffusion of GABA_AR, thus increasing its synaptic density. How can Ca²⁺ influx and IICR exert opposing effects on GABA synaptic structure? Our research indicates that each Ca²⁺ source activates a different Ca²⁺-dependent phosphatase/kinase: NMDAR-dependent Ca²⁺ influx activates calcineurin, while ER Ca²⁺ release activates PKC.

Taken together, these results lead us to propose the following model for bidirectional competitive regulation of GABAergic synapses by glutamate signaling. Phasic Ca²⁺ influx through NMDARs following sustained neuronal excitation or injury leads to the activation of calcineurin, overcoming PKC activity and relieving GABA_AR diffusion constraints. In contrast, during the maintenance of GABAergic synaptic structures or the recovery from GABA_AR dispersal, the ambient tonic mGluR/IICR pathway constrains GABA_AR diffusion by PKC activity, overcoming basal calcineurin activity. One possible mechanism of dual regulation of GABA_AR by Ca²⁺ is that each Ca²⁺-dependent enzyme has a unique sensitivity to the frequency and number of external glutamate release events and can act to decode neuronal inputs (Fujii et al., 2013x , Li et al., 2012x , Stefan et al., 2008x ) in inhibitory synapses.

Tight control of E/I balance, the loss of which results in epilepsy and other brain and nervous system diseases/disorders, is dependent on GABAergic synaptic transmission (Mann and Paulsen, 2007x ). A recent study showed that the excitation-induced acceleration of GABA_AR diffusion and subsequent dispersal of GABA_ARs from synapses is the cause of generalized epilepsy febrile seizure plus (GEFS+) syndrome (Bouthour et al., 2012x ). Our results indicate that pre-activation of the mGluR/IICR pathway by DHPG could completely prevent the dispersion of synaptic GABA_ARs induced by massive excitatory input similar to status epilepticus. Thus, further study of the molecular mechanisms underlying the mGluR/IICR-dependent stabilization of GABAergic synapses via regulation of GABA_AR lateral diffusion and synaptic transmission could be helpful in the prevention or treatment of pathological E/I imbalances, for example, in the recovery of GABAergic synapses from epileptic states

DHPG = group I mGluR agonist dihydroxyphenylglycine.

On a practical level you want to inhibit GABA_A dispersion and promote GABA_Astabilization. How you might do this would depend on exactly what was the underlying problem.

If the problem is IP₃R not releasing enough calcium, you might activate PKC in a different way or just increase the signal from Group 1 mGluR. If the problem is too much calcium influx through NMDA receptors due to excess glutamate, you could increase the re-uptake of glutamate, via GLT-1, using Riluzole. You could block the flow of Ca²⁺through NMDA receptors using an antagonist.

The Japanese used dihydroxyphenylglycine (DHPG) as their Group 1 mGluR agonist. DHPG is an agonist of mGluR1 and mGluR5. We have come across mGluR5 many times before in this blog. Mavoglurant is an experimental drug candidate for the treatment of fragile X syndrome. It is an antagonist of mGluR5.

We have seen many times before that there is both hypo and hyper function possible and indeed that fragile X is not necessarily a good model for autism.

The selective mGluR5 agonist CHPG protects against traumatic brain injury, which would indeed make sense. Although, that research suggests an entirely different mechanism.

The selective mGluR5 agonist CHPG protects against traumatic brain injury in vitro and in vivo via ERK and Akt pathway

The calcium released by IP₃ works in complex way together with DAG (diacylglycerol ) to activate PKC (protein kinase C).

Ideally you would have enough calcium released from IP3, but you could also increase DAG. It depends which part of the process is rate-limiting.

Diacylglycerol (DAG) has been investigated extensively as a fat substitute due to its ability to suppress the accumulation of body fat. Diglycerides, generally in a mix with monoglycerides are common food additives largely used as emulsifiers. In Europe, when used in food the mix is called E471.

Conclusion

On the one hand things are getting very complicated, but on the other we keep coming back to the same variables (IP3R, mGlur5, GABA_A etc.).

It is pretty clear that some very personalized therapy will be needed. Is it an mGlur5 agonist or antagonist? Or quite possibly neither, because in different parts of the brain it will have a good/bad effect.

It does look like Riluzole should work well in some people.

A safe IP3R agonist looks a possibility. As shown in the diagram earlier in this post,IP3 is usually made in situ, but agonists exist.

In effect autism could be the opposite of Huntington’s disease. In Huntington’s, type 1 IP₃ receptors are more sensitive to IP₃, which leads to the release of too much Ca²⁺ from the ER. The release of Ca²⁺ from the ER causes an increase in concentrations of Ca²⁺inside cells and in mitochondria.

According to Gargus we should have reduced concentrations of Ca²⁺inside cells in autism.

I suspect it is much more complicated in reality, because it is not just the absolute level of Ca²⁺but rather the flow of Ca²⁺; so it matters where it is coming from. I think we likely have impaired calcium channel activity of multiple types in autism and the actual level of intracellular calcium will not tell you much at all.

As the Japanese commented, it is surprising that glutamate is the neurotransmitter that controls the clustering, or not, of GABA_A receptors. This suggests that you cannot ignore glutamate and just “fix” GABA.

Thursday, 13 April 2017

Estradiol/Aromatase Deficiency in Autism, Schizophrenia and Bipolar

There was a rather complicated post in which I was linking some of the odd biological features of autism to something called RORα.

This was one of those posts that appeals to the scientist readers like Tyler.

The Purkinje-RORa-Estradiol-Neuroligin-KCC2 axis in Autism

Happy with his elevated estradiol level

Today’s post is more like the Psychiatrist's take on the same subject, so it is less complicated.

I was thinking that a logical way to treat boys, post puberty, and girls with autism would be to target RORα. In males this would be the treating aromatase deficiency. You would start by measuring by measuring the level of testosterone and estradiol in both boys and girls.

My assumption is that there will be a substantial group of males who will have high testosterone and low estradiol. In autism and its big brothers (and sisters) Schizophrenia and Bipolar, there are disturbed levels of these hormones. One logical therapy would be estradiol, which is much less problematic for girls than boys.

Boys with genetically caused aromatase deficiency lack the female hormone estradiol, but are not treated with transdermal estradiol until after puberty. Girls are treated with estradiol from a younger age.

Untreated males with aromatase deficiency have retarded bone age, but end up as very tall adults. They also have a problem with low bone density and so have weak bones.

Clinical Features of Aromatase Deficiency

	Female	Male
Fetal life:	Masculinization of the mother during pregnancy	Masculinization of the mother during pregnancy
Genitalia at birth:	Severe clitoromegaly and posterior labioscrotal fusion	Normal male
Childhood:	Multi-cystic ovaries	Unremarkable
Puberty:	Absent growth spurt Absent breast development Primary amenorrhea Further enlargement of clitoris Enlarged cystic ovaries Normal development of pubic and axillary hair	Absent growth spurt Normal pubertal development
Adult:	Severe estrogen deficiency Virilization Enlarged cystic ovaries Continued linear bone growth	Tall, eunucoid proportions with continued linear growth into adulthood Osteoporosis Genu valgum

Source: AROMATASE DEFICIENCY

I have commented before that I think retarded bone age is a useful marker of some types of autism. You just need an X-ray of your hand and some interpretation that looks at the gaps between the small bones.

We have also seen in the comments section of this blog that numerous readers have low bone density problems in their families.

Extreme aromatase deficiency is very rare and is caused by mutations in the CYP19A1 gene.

We saw that RORα seems to be a hub for where things go wrong in autism (also schizophrenia and bipolar); I am not suggesting a problem with the CYP19A1 gene.

One approach in psychiatry research is to just try things, without bothering too much about the underlying science. This has been the case with Estradiol, where there have been several very positive studies in schizophrenia and bipolar.

Estradiol in Clinical Trials

Nobody is going get approval to use Estradiol in young boys, other than those who are promoting gender reassignment. These people want to chemically block puberty and then use high doses of estradiol to feminize the male body.

Estradiol is widely used in post-menopausal women, but also in clinical trials of middle aged women with schizophrenia or bipolar, where it appears to provide a clear improvement.

Estradiol for treatment-resistant schizophrenia: a large-scale randomized-controlled trial in women of child-bearing age.

Many women with schizophrenia remain symptomatic despite optimal use of current therapies. While previous studies suggest that adjunctive oestrogen therapy might be effective, large-scale clinical trials are required before clinical applications are possible. This study is the first large-scale randomized-controlled trial in women with treatment-resistant schizophrenia. This Definitive Oestrogen Patch Trial was an 8-week, three-arm, double-blind, randomized-controlled trial conducted between 2006 and 2011. The 183 female participants were aged between 18 and 45 (mean = 35 years), with schizophrenia or schizoaffective disorder and ongoing symptoms of psychosis (Positive and Negative Syndrome Scale, PANSS score>60) despite a stable dose of antipsychotic medication for at least 4 weeks. Mean duration of illness was more than 10 years. Participants received transdermal estradiol 200 μg, transdermal estradiol 100 μg or an identical placebo patch. For the 180 women who completed the study, the a priori outcome measure was the change in PANSS score measured at baseline and days 7, 14, 28 and 56. Cognition was assessed at baseline and day 56 using the Repeatable Battery of Neuropsychological Status. Data were analysed using latent growth curve modelling. Both estradiol groups had greater decreases in PANSS positive, general and total symptoms compared with the placebo this study shows estradiol is an effective and clinically significant adjunctive therapy for women with treatment-resistant schizophrenia, particularly for positive symptoms.

A Role for Estrogen in Schizophrenia: Clinical and Preclinical Findings

The role of estrogen in bipolar disorder, a review.

BACKGROUND:

It appears that the female reproductive events and hormonal treatments may impact the course of bipolar disorder in women. In particular, childbirth is known to be associated with onset of affective episodes in women with bipolar disorder. During the female reproductive events the sex hormones, e.g. estrogen, are fluctuating and particularly postpartum there is a steep fall in the levels of serum estrogen. The role of estrogen in women with bipolar disorder is, however, not fully understood.

AIM:

The main objective of this review is to evaluate the possible relation between serum estrogen levels and women with bipolar disorder including studies of the anti-manic effects of the selective estrogen receptor modulator tamoxifen.

METHOD:

A systematically literature search on PubMed was conducted: two studies regarding the connection between serum estrogen levels and women with bipolar disorder were identified. Furthermore, four studies were found concerning the antimanic effects of tamoxifen.

RESULTS:

Both studies in the estrogen studies showed very low levels of estrogen in women with postpartum psychosis and significant improvement of symptoms after treatment with estrogen. The four tamoxifen studies found that tamoxifen was effective in producing antimanic effects.

CONCLUSION:

These results indicate that estrogen fluctuations may be an important factor in the etiology of bipolar disorder and it is obvious that more research on this topic is needed to clarify the role of estrogen in women with bipolar disorder.

Men also need to have a certain level of estradiol, but as they age, and particularly if they get overweight, they often end up with too much. So the usual problem is too much estradiol.

In males, estrogen is produced in fat (adipose) tissue by the action of the enzyme aromatase on testosterone. So it would not be surprising if males with five times more adipose tissue produced more estrogen/estradiol. This would might explain mild feminization of the body and the lack of more aggressive male behaviors.

Many males with schizophrenia and a substantial number with autism are on medication that causes weight gain. While there are is research on how to reduce this weight gain, if the weight gain causes more estradiol, there actually is some potential benefit.

Estrogen Plays Key Role in Male Brain Development

Male Brains Need Estrogen for Good Memory

"We found that testosterone alone can improve an aspect of memory known as spatial memory -- the kind of memory needed to drive, get dressed, use a knife and fork -- what you need to learn to navigate three-dimensional space," Asthana says. "But men with both testosterone and estrogen had better verbal memory."

Asthana thinks that new estrogen-like drugs that lack sex-hormone effects such as breast enlargement might be useful to preserve memory in aging men. He says he is planning to test this theory in human trials

An estrogen-like drug, raloxifene, was trialed unsuccessfully in women with Alzheimer’s, but not in men.

Estradiol is a research therapy for males with prostate cancer.

Estradiol in Autism

We saw in the science heavy earlier post that that children with autism do not have sufficient estrogen receptor beta expression to mediate the protective benefits of estrogen.

Estrogen receptor beta agonists, which are already known to improve brain plasticity and memory in animals, have been proposed to help reverse autism's behavioral deficits.

High testosterone, low aromatase and correspondingly low estradiol are features of autism and will compound the effect of reduced estrogen receptor beta expression.

Conclusion

There are far less issues with the use of estradiol in females with autism. Given there have already been trials in Schizophrenia and Bipolar on females using estradiol, it is about time a psychiatrist made a trial in autism.

I think that via the effect on RORα, there will be numerous positive effects. The risks and side effects will be exactly the same as in the previous Schizophrenia and Bipolar trials.

Having seen what, if any, positive effects the females with autism experience, it would be time to consider adult males. Is there a behavioral benefit in small enough doses of estradiol that do not cause feminization?

It would also be useful to measure the level of estradiol in overweight males to get some benchmarks of what is “normal" today in males.

Later on, using bone-age and indeed estradiol levels it might be possible to identify a sub-group of autism who might be likely to benefit from this therapy. There may even be familial markers, like problems associated with low bone density, which might predispose the person with autism to have low levels of estradiol.

The other issue is the lack of estrogen beta-type receptors in people with autism.

Monday, 10 April 2017

Mouse Models of Autism

Researchers use animals in place of humans, for research purposes; in the case of autism it is usually the unfortunate mouse, but sometimes rats.

The Jackson Laboratory in the US is the source for more than 8,000 strains of genetically defined mice used for research purposes.

SFARIgene has a fascinating on-line database that lists all the mouse/rat models of autism and the research linked to them. Most importantly it also lists all the “rescue lines”, the research showing therapies that improved the mouse’s autism.

For example, you can look up the model of human Fragile-X, which is called Fmr1, and then see the long list of drugs that helped that particular type of mouse.

There are already well over 200 different mouse/rat genetic models of autism and 1,000 rescue lines.

So while medicine has no approved drugs to treat human autism, autistic mice appear to be better placed.

There remains the question of how close humans are to mice. They are more closely related than you might think, but there are still big differences.

There are also induced models of autism, where the scientists have not tinkered with a specific single gene; these might closer relate to most human autism. You will find a model of advanced paternal age, a model of diesel exhaust particles, and all kinds of other things.

One very widely used model is called the Maternal Immune Activation (MIA) model. In the research you may find it called Polyinosinic:polycytidylic acid, or just poly(I:C).

In the MIA model the pregnant mouse is injected will an immune stimulant (Polyinosinic:polycytidylic acid) that triggers a big immune response, which affects the development of her pup. The pup is born with features that resemble human autism.

There is a similar model where the mother is given an infection rather than induced inflammation.

Depending on the gestational age at which MIA or infection is administered, the offspring can be studied in the context not only of autism, but also schizophrenia. This should not be surprising if you have read the post discussing the overlapping polygenic nature of autism and schizophrenia.

You can even induce temporary autism using proprionic acid. Proprioic acid is produced naturally in your intestines when the food you eat reacts with the bacteria that live there. Proprionic acid is a SCFA (short chained fatty acid), you need to have some SCFAs, but as it often the case, too much may not be good for you. In the case of a mouse, when injected with a large dose of poprionic acid, its behaviour changes to that of autism. This is entirely reversible over time, or faster still, by administering the antioxidant NAC (N-acetyl cysteine).

Researchers create a mouse model that matches as closely as possible the human condition they are trying to treat. Then they can investigate various drugs that might be of therapeutic benefit. In some cases a large number of drugs from a library of compounds are tried on the off chance of stumbling upon one that is effective.

An alternative approach is when a researcher has a theory that a specific drug should be effective, he then tests it in several different mouse models of autism. If the drug is effective in several mouse models that would suggest it might be beneficial in some humans. This is how Ben-Ari advanced his bumetanide research and Catterall his low dose Clonazepam research; the difference is that Ben-Ari has moved on to humans, as regular readers know.

Those of you who look at the SFARgene database will see how hundreds of so very different things, both genetic or environmental, lead to the same autism.

Friday, 7 April 2017

Treating Mitochondrial Disease/Dysfunction in Autism

In my book I will be covering the science behind hopefully almost all autism, which then naturally leads to translating it into therapy. In the ideal world you would just skip straight to the therapy and the final section of the book will be just that. Clearly it would make sense to read the science first, so that you know what are the dysfunctions that you might need to treat.

Hopefully there will also be some case studies from people who have applied a science-based approach to identify and implement effective therapies.

Roger would clearly make a very good example of a reversible in-born metabolic-caused type of autism.

I will be posting on my blog some drafts from the Part III - Translating Science to Treat Autism. This is of course just one person's collection of other people's ideas and some of his one. The reader and his/her medical medical team ultimately decide what to implement and must monitor its ongoing implementation.

* * *

Mitochondrial disease is managed rather than cured. It seems to be present in autism in widely varying degrees of severity. Extreme cases result in very severe regressive autism with MR/ID.

It is either diagnosed based on detailed analysis of numerous blood tests, or more recently via a sample taken from inside the cheek. These tests cannot be perfect, because mitochondrial disease can be organ-specific.

Someone with body-wide mitochondrial disease will have poor exercise endurance and this will be very noticeable compared to siblings and peers.

Dr Kelley, from Johns Hopkins, has published his therapy for autism secondary to mitochondrial disease (AMD):-

1. Augment residual mitochondrial enzyme complex I activity

2. Enhance natural systems for protection of mitochondria from reactive oxygen species

3. Avoid conditions known to impair mitochondrial function or increase energy demands, such as prolonged fasting, inflammation, and the use of drugs that inhibit complex I.

Combining the first and second parts of the treatment plan, the following is a typical prescription for treating AMD:

L-Carnitine 50 mg/kg/d Alpha Lipoic acid 10 mg/kg/d

Coenzyme Q10 10 mg/kg/d Pantothenate 10 mg/kg/d

Vitamin C 30 mg/kg/d Nicotinamide 7.5 mg/kg/d (optional)

Vitamin E 25 IU/kg/d Thiamine 15 mg/kg/d (optional)

There are actually five stages in the OXPHOS process in mitochondria and there are five enzyme complexes. Dr Kelley's plan above is for the most common dysfunction, complex 1.

Different clinicians have different treatments.

Also appearing elsewhere are :-

Calcium folinate (2 x 25 mg), but not because of peroxynitrite

Biotin 5-10 mg/day

NAC

Methylcobalamin B12

Creatine

On the basis that peroxynitrite, from nitrosative stress, damages the mitochondria, you might consider:

· Calcium folinate (leucoverin) in very high doses like 25mg twice a day.

· Xanthine oxidase inhibitors, typically used to lower uric acid to treat gout. A good example is Allopurinol. It will both lower uric acid and peroxynitrite. Uric acid is itself a potent scavenger of peroxynitrite; this may look odd given the previous sentence. If someone has low uric acid and wants to reduce peroxynitrite then uric acid itself should be therapeutic. The purine metabolism may play a key role in some types of autism, as proposed by Professor Robert Naviaux.

· Rosmarinic acid, a natural scavenger of peroxynitrite.

There are many anomalies in autism and one is uric acid. Some people have low levels and some have high levels. Uric acid is itself a scavenger of peroxynitrite. People with high levels of uric acid do get gout, but almost never MS (multiple sclerosis) and it has been suggested that scavenging peroxynitrite is neuroprotective.

Special, electrically charged, antioxidants have been developed to target the mitochondria. MitoE is a charged version of vitamin E and MitoQ is a charged version of coenzyme Q10.

Based on the research, you might also seek to activate PGC-1α, the master regulator of mitochondrial biogenesis. This can potentially be achieved via:-

· Exercise (gradual endurance training)

· Activate PPARγ and perhaps PPARα (e.g. Bezafibrate and Rosiglitazone)

· Activate AMPK (Metformin)

· Activate Sirt-1 (resveratrol and other polyphenolic ‎compounds)

Carnitine-like analogs may also help in theory. The standard L-Carnitine, widely used as a supplement, is very poorly absorbed even at high doses. An analog is a modified version of a molecule that keeps the therapeutic beneficial effect, but overcomes a drawback, bioavailability in the case of carnitine. There is some basis in the literature to believe that the Latvian drug Mildronate might be useful to treat complex 1 mitochondrial dysfunction.

more detail at https://epiphanyasd.blogspot.com/2017/02/mitochondrial-disease-and-autsim.html

Monday, 3 April 2017

Different Types of Excitatory/Inhibitory Imbalance in Autism, Fragile-X & Schizophrenia

There is much written in the complex scientific literature about the Excitatory/Inhibitory (E/I) imbalance between neurotransmitters in autism.

Many clinical trials have already been carried out, particularly in Fragile-X. These trials were generally ruled as failures, in spite of a significant minority who responded quite well in some of these trials.

As we saw in the recent post on the stage II trial of bumetanide in severe autism, there is so much “background noise” in the results from these trials and it is easy to ignore a small group who are responders. I think if you have less than 40%, or so, of positive responders they likely will get lost in the data.

You inevitably get a significant minority who appear to respond to the placebo, because people with autism usually have good and bad days and testing is very subjective.

There are numerous positive anecdotes from people who participated in these “failed” trials. If you have a child who only ever speaks single words, but while on the trial drug starts speaking full sentences and then reverts to single words after the trial, you do have to take note. I doubt this is a coincidence.

Here are some of the trialed drugs, just in Fragile-X, that were supposed to target the E/I imbalance:-

Metabotropic glutamate receptor 5 (mGluR5) antagonist

· Mavoglurant

· Lithium

mGluR5 negative allosteric modulator

· Fenobam

N-methyl-D-aspartic acid (NMDA) antagonist

· Memantine

Glutamate re-uptake promoter

· Riluzole

Suggested to have effects on NMDA & mGluR5 & GABA_A

· Acamprosate

GABA_B agonist

· Arbaclofen

Positive allosteric modulator (PAM) of GABA_A receptor

· Ganaxolone

Best not to be too clever

Some things you might use to modify the E/I imbalance can appear to have the opposite effect, as was highlighted in the comments in the post below:-

NMDAR hypo-function causing E/I imbalance in Autism and Schizophrenia – Baclofen, Sodium benzoate and Cinnamon (again)

So whilst it is always a good idea to try and figure things out, you may end up getting things the wrong way around, mixing up hypo and hyper.

The MIT people who work on Fragile-X are really clever and they have not figured it all out.

Fragile-X and Idiopathic Autism

Fragile-X gets a great deal of attention, because its biological basis is understood. It results in a failure to express the fragile X mental retardation protein (FMRP), which is required for normal neural development.

We saw in the recent post about eIF4E, that this could lead to an E/I imbalance and then autism.

eIF4E inhibitors for Autism – Why not Ribavirin?

Our reader AJ started looking at elF4E and moved on to EIF4E- binding protein number 1.

In the green and orange boxes below you can find elF4E and elF4E-BP2.

This has likely sent some readers to sleep, but for those whose child has Fragile-X, I suggest they read on, because it is exactly here that the lack of fragile X mental retardation protein (FMRP) causes a big problem. The interaction between FMRP on the binding proteins of elF4E, cause the problem with neuroligins (NLGNs), which causes the E/I imbalance. Look at the red oval shape labeled FMRP and green egg-shaped NLGNs.

In which case, while AJ might naturally think Ribavirin is a bit risky for idiopathic autism, it might indeed be very effective in some Fragile-X. You would hope some researcher would investigate this.

Can you have more than one type of E/I imbalance?

Readers whose child responds well to bumetanide probably wonder if they have solved their E/I imbalance.

I think they have most likely improved just one dysfunction that fits under the umbrella term E/I imbalance. There are likely other dysfunctions that if treated could further improve cognition and behavior.

On the side of GABA, it looks like turning up the volume on α3 sub-unit and turning down the volume on α5 may help. We await the (expensive) Down syndrome drug Basmisanil for the latter, given that the cheap 80 year old drug Cardiazol is no longer widely available. Turning up the volume on α3 sub-unit can be achieved extremely cheaply, and safely, using a tiny dose of Clonazepam.

It does appear that targeting glutamate is going to be rewarding for at least some of those who respond to bumetanide.

One agonist of NMDA receptors is aspartic acid. Our reader Tyler is a fan of L-Aspartic Acid, that is sold as a supplement that may boost athletic performance.

Others include D-Cycloserine, already used in autism trials; also D-Serine and L-Serine.

D-Serine is synthesized in the brain from L-serine, its enantiomer, it serves as a neuromodulator by co-activating NMDA receptors, making them able to open if they then also bind glutamate. D-serine is a potent agonist at the glycine site of NMDA receptors. For the receptor to open, glutamate and either glycine or D-serine must bind to it; in addition a pore blocker must not be bound (e.g. Mg2+ or Pb2+).

D-Serine is being studied as a potential treatment for schizophrenia and L-serine is in FDA-approved human clinical trials as a possible treatment for ALS/Motor neuron disease.

You may be thinking, my kid has autism, what has this got to do with ALS/Motor neuron disease (from the ice bucket challenge)? Well one of the Fragile-X trial drugs at the beginning of this post is Riluzole, a drug developed for specially for ALS. Although it does not help that much in ALS, it does something potentially very useful for some autism, ADHD and schizophrenia; it clears away excess glutamate.

Fragile-X is likely quite different to many other types of autism

I suspect that within Fragile-X there are many variations in the downstream biological dysfunctions and so that even within this definable group, there may be no universal therapies. So for some people an mGluR5 antagonist may be appropriate, but not for others.

Even within this discrete group, we come back to the need for personalized medicine.

I do not think Fragile-X is a good model for broader autism.

Glutamate Therapies

There are not so many glutamate therapies, so while the guys at MIT might disapprove, it would not be hard to apply some thoughtful trial and error.

You have:

mGluR5

· mGluR5 agonists (only research compounds)

· mGluR5 positive allosteric modulators (only research compounds)

· mGluR5 antagonists (Mavoglurant, Lithium)

· mGluR5 negative allosteric modulators (Fenobam, Pu-erh tea decreases mGluR5 expression )

Today you can only really treat too much mGluR5 activity. It there is too little activity, the required drugs are not yet available. I wonder how many people with Fragile-X are drinking Pu-erh tea, it is widely available.

NMDA agonists

D-Cycloserine an antibiotic with similar structure to D-Alanine (D-Cycloserine was trialed in autism and schizophrenia)

ɑ-amino acids:

· Aspartic acid (trialed and used by Tyler, suggested for schizophrenia)

· D-Serine (trialed in schizophrenia)

L-Serine

D-Alanine (trialed in schizophrenia)

L-Alanine

NMDA antagonists

· Memantine (widely used off-label in autism, but failed in clinical trials)

· Ketamine (trialed intra-nasal in autism)

Glutamate re-uptake promoters via GLT-1

· Riluzole

· Bromocriptine

· Beta-lactam antibiotics

Friday, 31 March 2017

The Glutamate Side of Things

Some readers have suggested that since we have discovered so many ways to treat the GABA_A dysfunctions common in autism, it is time to look at the glutamate side of things. Glutamate is the main excitatory neurotransmitter and has to be in balance with the opposing influence of GABA.

The chart below is really a summary of what has already been covered in this blog. To newcomers it will look complicated, to regular readers it is just bringing together everything we have already covered, even those tauopathies appear. Tau protein tangles appear in Alzheimer’s and some autism.

Glutamate excitoxicity is what happens when things go really wrong, for example in a severe autistic regression. I doubt you could be in a permanent state like this.

I am beginning to wonder is my son’s summer time raging, though triggered by allergy, develops to a so-called glutamatergic storm. It fades to nothing by using a Cav1.2 channel blocker, which does indeed stop those allergy mast cells de-granulating, but it stops the calcium influx in the above chart. Existing dysfunction in Cav1.2 and Cav1.4 puts you at risk of excitotoxicity.

The oxidative damage to mitochondria causes lipid peroxidation and in particular the 4-HNE produced will cause tau protein, from a recent post and Alzheimer’s, to produce tau tangles, a damaging feature of so-called tauopathies.

The nitrosative stress in particular damages the production of the Complex 1 enzyme leading to mitochondrial disease/dysfunction. The damaging peroxynitrates can be quenched using high doses of calcium folinate. Oxidative stress and the reduced level of GSH can be treated with antioxidants like NAC and ALA.

Reduced reuptake of glutamate, known to be caused by elevated TNF-α and immune dysfunction, is treatable via upregulating the GLT-1 transporter (beta-lactam antibiotics, riluzole and bromocriptine).

Elevated BDNF is a biomarker of autism and unfortunately this increases the chances of glutamate excitotoxicity.

An inactivated GABA switch that leaves neurons immature, will result in GABA acting excitatory rather than inhibitory, this itself can trigger of glutamate excitotoxicity. Use bumetanide.

Some types of autism feature NMDA hyper-function, this is treatable. A deviation of NMDA function in either direction (hypo or hyper) leads to autism, but you need to know which way it is, to treat it.

It is also possible to have over/under expression of NMDA receptors.