Showing posts with label neuroligin 2. Show all posts
Showing posts with label neuroligin 2. Show all posts

Monday, 23 January 2017


Add testosterone/estradiol to those dysfunctional hormones










androgen receptor = AR

estrogen receptor = ER

RORα (retinoic acid-related orphan receptor alpha.)


RORα does some clever things.

RORα is necessary for normal circadian rhythms






Many Genes affected by RORα




For those who like natural substances, some research from Japan.




This is true with very many products sold as supplements.



Purkinje Cells

Back in 2013 I wrote a post about Purkinje cells.

          Pep up those Purkinje cells











Estradiol – Neuroligin 2 to KCC2





How to address the rest of the Purkinje-KCC2 axis?

·        More RORα, or just a RORα agonist.

·        More aromatase


·        Estrogen receptor agonists

·        Exogenous estradiol



Friday, 23 December 2016

Neuroligins, Estradiol and Male Autism

Today’s post looks deeper into the biology of those people who respond to the drug bumetanide, which means a large sub-group of those with autism, likely those with Down Syndrome and likely some with schizophrenia.
It is a rather narrow area of science, but other than bumetanide treatment, there appears to be no research interest in further translating science into therapy.    So it looks like this blog is the only place to develop such ideas.
I did not expect this post would lead to a practical intervention, but perhaps it does. As you will discover, the goal would be to restore a hormone called estradiol to its natural higher level, perhaps by increasing an enzyme called aromatase, which appears to be commonly downregulated in autism.  This should increase expression of neuroligin 2, which should increase expression of the ion transporter KCC2; this will lower intracellular chloride and boost cognition.
It seems that those people using Atorvastatin may have already started this process, since this statin increases IGF-1 and insulin is one of the few things that increases the aromatise enzyme. 

This process is known as the testosterone-estradiol shunt.  In effect, by becoming slightly less male, you may be able to correct one of the key dysfunctions underlying autism. Options would include insulin, IGF-1, estradiol and a promoter of aromatase.

The testosterone – estradiol shunt

It would seem that this sub-group of autism is currently a little bit too male, which might be seen as early puberty and in other features. In this group the balance between testosterone and estradiol is affected not just in the brain, which is actually a good thing.  This should be measurable, if it is not visible.


NKCC1, KCC2 and AE3

As we have seen in earlier posts, some people with autism have too little of a transporter called KCC2 that takes chloride out of neurons and too much of NKCC1 that lets chloride in.  The result is an abnormally high level of chloride, which changes the way the GABA neurotransmitter functions.  This reduces cognitive function and increases the chance of seizures.

It is likely that a group may exist that has mis-expression of an ion exchanger called AE3. Potentially some have just an AE3 dysfunction and some may have AE3, KCC2 and NKCC1 mis-expression.  I will come back to this in a later post, but in case I forget, here is the link:

“NKCC1 seems to be responsible for approximately two thirds of the steady-state chloride accumulation, whereas AE3 is responsible for the remaining third”

Genetic dysfunction of AE3 is not surprisingly associated with seizures and should respond to treatment with Diamox/Acetazolamide.

Block NKCC1 with Bumetanide and/or increase KCC2 expression

I was recently updating the Bumetanide researchers about my son’s near four years of therapy with their drug and my ideas to take things further.

My plan is to apply other methods to reduce intracellular chloride levels.  I think that over time, blocking NKCC1 with bumetanide may trigger a feedback loop that leads to a further increase in NKCC1 expression.  So bumetanide continues to work, but the effect is reduced. One way to further reduce intracellular chloride levels is to increase expression of KCC2, the transport that takes chloride out of neurons.

The best way to do this would be to understand why KCC2 is down regulated in the first place. I have touched on this in earlier posts, where I introduced neuroligin 2.

Today’s post will look at neuroligins in autism and how they are connected to the female hormone Estradiol.  We will also look at how estrogen receptor expression may help explain why more males have autism. Taken together this suggests that an  estrogen receptor agonist might be an effective autism therapy in this sub-group.

The difficulty with hormones is that, due to evolution, each one performs numerous different functions in different parts of the body and they react with each other.  So a little extra estradiol/estrogen might indeed increase neuroligin 2 expression and hence increase KCC2 expression in the brain, but it would have other effects elsewhere.  In female hormone replacement therapy care is usually taken to direct estradiol/estrogen to where it is needed, rather than sending it everywhere.

I suspect that in this subgroup of autism the lack of estradiol is body-wide, not just in the brain.  If not you would either need an estrogen receptor agonist that is cleverly developed to be brain specific, or take the much easier route of delivering an existing agonist direct to the brain, which may also be possible.

In the paper below NL2 and neuroligin-2 mean the same thing. 


GABAA receptors are ligand-gated Cl- channels, and the intracellular Cl- concentration governs whether GABA function is excitatory or inhibitory. During early brain development, GABA undergoes functional switch from excitation to inhibition: GABA depolarizes immature neurons but hyperpolarizes mature neurons due to a developmental decrease of intracellular Cl- concentration. This GABA functional switch is mainly mediated by the up-regulation of KCC2, a potassium-chloride cotransporter that pumps Cl- outside neurons. However, the upstream factor that regulates KCC2 expression is unclear.


We report here that KCC2 is unexpectedly regulated by neuroligin-2 (NL2), a cell adhesion molecule specifically localized at GABAergic synapses. The expression of NL2 precedes that of KCC2 in early postnatal development. Upon knockdown of NL2, the expression level of KCC2 is significantly decreased, and GABA functional switch is significantly delayed during early development. Overexpression of shRNA-proof NL2 rescues both KCC2 reduction and delayed GABA functional switch induced by NL2 shRNAs. Moreover, NL2 appears to be required to maintain GABA inhibitory function even in mature neurons, because knockdown NL2 reverses GABA action to excitatory. Gramicidin-perforated patch clamp recordings confirm that NL2 directly regulates the GABA equilibrium potential. We further demonstrate that knockdown of NL2 decreases dendritic spines through down-regulating KCC2.


Our data suggest that in addition to its conventional role as a cell adhesion molecule to regulate GABAergic synaptogenesis, NL2 also regulates KCC2 to modulate GABA functional switch and even glutamatergic synapses. Therefore, NL2 may serve as a master regulator in balancing excitation and inhibition in the brain.

Neuroligins and Neurexins

The following paper has an excellent explanation of neuroligins, neurexins and their role in autism.  It does get complicated.

Neurexins (Nrxns) and neuroligins (Nlgns) are arguably the best characterized synaptic cell-adhesion molecules, and the only ones for which a specifically synaptic function was established8,9. In the present review, we will describe the role of Nrxns and Nlgns as synaptic cell-adhesion molecules that act in an heretofore unanticipated fashion. We will show that they are required for synapse function, not synapse formation; that they affect trans-synaptic activation of synaptic transmission, but are not essential for synaptic cohesion of the pre- and postsynaptic specializations; and that their dysfunction impairs the properties of synapses and disrupts neural networks without completely abolishing synaptic transmission as1012. As cell-adhesion molecules, Nrxns and Nlgns probably function by binding to each other and by interacting with intracellular proteins, most prominently PDZ-domain proteins, but the precise mechanisms involved and their relation to synaptic transmission remain unclear. The importance of Nrxns and Nlgns for synaptic function is evident from the dramatic deficits in synaptic transmission in mice lacking Nrxns or Nlgns.

As we will describe, the role of Nrxns and Nlgns in synaptic function almost predestines them for a role in cognitive diseases, such as schizophrenia and autism spectrum disorders (ASDs), that have been resistant to our understanding. One reason for the difficulties in understanding cognitive diseaseas is that they may arise from subtle changes in a subset of synapses in a neural circuit, as opposed to a general impairment of all synapses in all circuits. As a result, the same molecular alteration may produce different circuit changes and neurological symptoms that are then classified as distinct cognitive diseases. Indeed, recent studies have identified mutations in the genes encoding Nrxns and Nlgns as a cause for ASDs, Tourette syndrome, mental retardation, and schizophrenia, sometimes in patients with the same mutation in the same family1327. Viewed as a whole, current results thus identify Nrxns and Nlgns as trans-synaptic cell-adhesion molecules that mediate essential signaling between pre- and postsynaptic specializations, signaling that performs a central role in the brain’s ability to process information and that is a key target in the pathogenesis of cognitive diseases.

Neuroligins and neurexins in autism

ASDs are common and enigmatic diseases. ASDs comprise classical idiopathic autism, Asperger’s syndrome, Rett syndrome, and pervasive developmental disorder not otherwise specified73,74. Moreover, several other genetic disorders, such as Down syndrome, Fragile-X Mental Retardation, and tuberous sclerosis, are frequently associated with autism. Such syndromic forms of autism and Rett syndrome are usually more severe due to the nature of the underlying diseases. The key features of ASDs are difficulties in social interactions and communication, language impairments, a restricted pattern of interests, and/or stereotypic and repetitive behaviors. Mental retardation (~70% of cases) and epilepsy (~30% of cases) are frequently observed; in fact, the observation of epilepsy in patients with ASDs has fueled speculation that autism may be caused by an imbalance of excitatory vs. inhibitory synaptic transmission. In rare instances, idiopathic autism is associated with specialized abilities, for example in music, mathematics, or memory. The relation of ASDs to other cognitive diseases such as schizophrenia and Tourette’s syndrome is unclear. As we will see below with the phenotypes caused by mutations in Nlgns and Nrxns, the boundaries between the various disorders may not be as real as the clinical manifestations suggest.

A key feature of ASDs is that they typically develop before 2–3 years of age73,74. ASDs thus affect brain development relatively late, during the time of human synapse formation and maturation. Consistent with this time course, few anatomical changes are associated with ASDs75. An increase in brain size was repeatedly reported76, but is not generally agreed upon75. Thus, similar to other cognitive diseases, ASDs are not a disorder of brain structure but of brain function. Among cognitive diseases, ASDs are the most heritable (~ 80%), suggesting that they are largely determined by genes and not the environment. ASDs exhibit a male:female ratio of approximately 4:1, indicating that ASDs involve the X-chromosome directly, or that the penetrance of pathogenic genes is facilitated in males73,74.

Mutations in many genes have been associated with familial ASDs. A consistent observation emerging from recent studies is the discovery of mutations in the genes encoding Nrxn1, Nlgn3, and Nlgn4. Specifically, seven point mutations, two distinct translocation events, and four different large-scale deletions in the Nrxn1 gene were detected in autistic patients1318. Ten different mutations in the Nlgn4 gene were observed (2 frameshifts, 5 missense mutations, and 3 internal deletions), and a single mutation in the Nlgn3 gene (the R451C substitution)2124. Besides these mutations, five different larger deletions of X-chromosomal DNA that includes the Nlgn4 locus (referred to as copy-number variations) were detected in autism patients18,2527.

In addition to the Nrxn/Nlgn complex, mutations in the gene encoding Shank3 – an intracellular scaffolding protein that binds indirectly to Nlgns via PSD-95 and GKAP (Fig. 1)66 – may also be a relatively frequent occurrence in ASDs. An astounding 18 point mutations were detected in the Shank3 gene in autistic patients, in addition to several cases containing CNVs that cover the gene18,7782. Indeed, the so-called terminal 22q deletion syndrome is a relatively frequent occurrence that exhibits autistic features, which have been correlated with the absence of the Shank3 gene normally localized to this chromosome section. Shank3 is particularly interesting because it not only indirectly interacts with Nlgns, but also directly binds to CIRL/Latrophilins which in turn constitute α-latrotoxin receptors similar to Nrxns, suggesting a potential functional connection between Shank3 and Nrxns83.

Overall, the description of the various mutations in the Nrxn/Nlgn/Shank3 complex appears to provide overwhelming evidence for a role of this complex in ASDs, given the fact that in total, these mutations account for a significant proportion of autism patients. It should be noted, however, that two issues give rise to skepticism to the role of this complex in ASDs.

First, at least for some of the mutations in this complex, non-symptomatic carriers were detected in the same families in which the patients with the mutations were found. Whereas the Nlgn3 and Nlgn4 mutations appear to be almost always penetrant in males, and even female carriers with these mutations often have a phenotype, the Shank3 point mutations in particular were often observed in non-symptomatic siblings77,78. Thus, these mutations may only increase the chance of autism, but not actually cause autism.

Second, the same mutations can be associated with quite different phenotypes in different people. For example, a microdeletion in Nlgn4 was found to cause severe autism in one brother, but Tourette’s syndrome in the other26. This raises the issue whether the ‘autism’ observed in patients with mutations in these genes is actually autism, an issue that could also be rephrased as the question of whether autism is qualitatively distinct from other cognitive diseases, as opposed to a continuum of cognitive disorders. In support of the latter idea, two different deletions of Nrxn1α have also been observed in families with schizophrenia19,20, indicating that there is a continuum of disorders that involves dysfunctions in synaptic cell adhesion and manifests in different ways. Conversely, very different molecular changes may produce a similar syndrome, as exemplified by the quite different mutations that are associated with ASDs84.

At present, the relation between the Nrxn/Nlgn synaptic cell-adhesion complex and ASDs is tenuous. On one hand, many of the mutations observed in familial ASD are clearly not polymorphisms but deleterious, as evidenced by the effect of these mutations on the structure or expression of the corresponding genes, and by the severe autism-like phenotypes observed in Nlgn3 and Nlgn4 mutant mice8587. On the other hand, the nonlinear genotype/phenotype relationship in humans, evident from the only 70–80% heritability and from the occasional presence of mutations in non-symptomatic individuals, requires explanation. Elucidating the underlying mechanisms for this incomplete genotype/phenotype relationship is a promising avenue to insight into the genesis of autism. Furthermore, in addition to the link of Nrxn1α mutations to schizophrenia19,20, linkage studies have connected Nrxn3 to different types of addiction88,89. It is possible that because of the nature of their function, mutations in genes encoding Nrxns and Nlgns constitute hotspots for human cognitive diseases.

As you will have seen from the above paper, whose author seems to be very well informed of the broader picture (a continuum of disorders that involves dysfunctions in synaptic cell adhesion, and even the link to addiction), neuroligins and neurexins are very relevant to autism and other cognitive disease.

Let’s get back on subject and focus on Neuroligin 2 
The very recent paper below mentions sensory processing defects and NLG2 alongside what we already have figured out so far.


Neuroligins are post-synaptic, cellular adhesion molecules implicated in synaptic formation and function. NLGN2 is strongly linked to inhibitory, GABAergic signaling and is crucial for maintaining the excitation-inhibition balance in the brain. Disruption of the excitation-inhibition balance is associated with neuropsychiatric disease. In animal models, altered NLGN2 expression causes anxiety, developmental delay, motor discoordination, social impairment, aggression, and sensory processing defects. In humans, mutations in NLGN3 and NLGN4 are linked to autism and schizophrenia; NLGN2 missense variants are implicated in schizophrenia. Copy number variants encompassing NLGN2 on 17p13.1 are associated with autism, intellectual disability, metabolic syndrome, diabetes, and dysmorphic features, but an isolated NLGN2 nonsense variant has not yet been described in humans. Here, we describe a 15-year-old male with severe anxiety, obsessive-compulsive behaviors, developmental delay, autism, obesity, macrocephaly, and some dysmorphic features. Exome sequencing identified a heterozygous, de novo, c.441C>A p.(Tyr147Ter) variant in NLGN2 that is predicted to cause loss of normal protein function. This is the first report of an NLGN2 nonsense variant in humans, adding to the accumulating evidence that links synaptic proteins with a spectrum of neurodevelopmental phenotypes

After some investigation I learned that both estradiol/estrogen and progesterone increase expression of neuroligin 2, at least in rats.
Increasing neuroligin 2/NLGN2/NL2 looks a promising strategy.

In addition, neuroligin 2 mRNA levels were increased by both 17beta-oestradiol (E(2)) and P(4), although P(4) administration upregulated gene expression to a greater extent than injection of E(2). These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E(2) and P(4), which are secreted periodically during the oestrous cycle.[1]

So a female steroid-regulated gene is down-regulated in male-dominated autism.  Another example of the protective nature of female hormones?  I think it is.

Estrogens Suppress a Behavioral Phenotype in Zebrafish Mutants of the Autism Risk Gene, CNTNAP2


·         Zebrafish mutants of the autism risk gene cntnap2 have GABAergic neuron deficits

·         High-throughput behavioral profiling identifies nighttime hyperactivity in mutants

·         cntnap2 mutants exhibit altered responses to GABAergic and glutamatergic compounds

·         Estrogenic compounds suppress the cntnap2 mutant behavioral phenotype


Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.

Estrogen is known to help protect premenopausal women from maladies such as stroke and impaired cognition. Exposure to high levels of the male hormone testosterone during early development has been linked to autism, which is five times more common in males than females.

The new findings of reduced expression of estrogen receptor beta as well as that of an enzyme that converts testosterone to estrogen could help explain the high testosterone levels in autistic individuals and higher autism rates in males, Pillai said.
It was the 5-to-1 male-to-female ratio along with the testosterone hypothesis that led Pillai and his colleagues to pursue whether estrogen might help explain the significant gender disparity and possibly point toward a new treatment.

"The testosterone hypothesis is already there, but nobody had investigated whether it had anything to do with the female hormone in the brain," Pillai said. "Estrogen is known to be neuroprotective, but nobody has looked at whether its function is impaired in the brain of individuals with autism. We found that the children with autism didn't have sufficient estrogen receptor beta expression to mediate the protective benefits of estrogen."



They also plan to give an estrogen receptor beta agonist -- which should increase receptor function -- to a mouse with generalized inflammation and signs of autism to see if it mitigates those signs. Inflammation is a factor in many diseases of the brain and body, and estrogen receptor beta agonists already are in clinical trials for schizophrenia.

The following trial was run by a psychiatrist; when I looked at why he thought estrogen might improve schizophrenia, there was no biological explanation.  He is trying to avoid the possible side effects by using of a selective estrogen receptor agonist.  I hope the trial successful.  The question is whether his subjects are starting out as extreme male or just male.

Several lines of investigation have supported the potential therapeutic effects of estrogen for negative and cognitive symptoms in schizophrenia. However, estrogen has had limited therapeutic application for male and premenopausal patients with schizophrenia because of tolerability concerns including uterine cancer liability, and heart disease and feminization effects in men. Selective Estrogen Receptor Beta (ER beta) agonists are a new class of treatments that are relatively free of estrogen's primary side effects and yet have demonstrated estrogen-like effects in brain including improvement in cognitive performance and an association to extremes in social behavior. Thus, these agents may have a therapeutic role for cognitive and negative symptoms in schizophrenia. The primary objectives of this application are to determine if the selective ER beta agonist LY500307 significantly improves negative and cognitive symptoms in patients with schizophrenia. Secondary aims include assessing LY500307 effects on cerebral blood flow during working and episodic memory tasks with fMRI, and electrophysiological indices of auditory sensory processing and working memory. A single seamless phase 1b/2a adaptive design will be used to evaluate two LY500307 doses (25 mg/day and 75 mg/day) in the first stage of the trial (year 1 of the application) to determine which dose should be advanced to stage 2 (years 2and 3 of the application) or if the trial should be discontinued.

More generally:-

Steroid hormones exert a considerable influence on several aspect of cognition.

Estrogens and androgens exert positive effects on cognitive functions.

Progesterone and allopregnanolone have variable effects on cognitive functions.

Glucocorticoids act to encode and store information of the emotional events.

Epigenetic modifications are a powerful mechanism of memory regulation.


More female hormones and less male hormones? Seems a good idea.

More of the aromatase enzyme ?  There are numerous drugs to reduce/inhibit aromatase but not specifically to increase it.

Insulin does increase aromatase, as does alcohol and being overweight.
The clever thing to do would be to just correct the reduced level of aromatase, or wait for a selective estrogen receptor beta agonist like LY500307 to come to the market.

In those who are extreme male, a little estradiol might be the simple solution, but not the amount that is currently taken by those that abuse it.  Yes people abuse estradiol – males who want to be females.
Antonio Hardan at Stanford did trial high dose pregnenolone, another hormone mainly found in females, that should increase progesterone.

Brief report: an open-label study of the neurosteroid pregnenolone in adults with autism spectrum disorder.

Overall, pregnenolone was modestly effective and well-tolerated in individuals with ASD.

This steroid should increase the level of progesterone and so might be expected to cause some side effects in males. You would expect it to have an effect on anxiety, but as we saw in an earlier post it should be quite dose specific.

Why Low Doses can work differently, or “Biphasic, U-shaped actions at the GABAa receptor”

So Hardan may have just picked the "wrong dose".

If he would like to trial 0.3mg of oral estradiol in adults with autism, I think he might find a positive response.