This blog is about noticing connections and making things a little simpler to understand. Today’s post is going to be a good example; all those odd sounding things like Purkinje cells and neuroligins all fitting nicely together.
Today we see how a central hormonal dysfunction (testosterone/estradiol) can lead to an ion channel dysfunction (NKCC1/KCC2) at one end of the chain and at the other explains the absence of many Purkinje cells in the autistic cerebellum, which leads to some of the observed features of autism.
I am calling it the Purkinje-RORa-Estradiol-Neuroligin-KCC2 axis, or Purkinje-KCC2 axis for short.
We also get to see how melatonin fits in here and see why disturbed sleeping patterns should be expected in someone affected by the Purkinje- KCC2 axis.
I should point out that not everyone with autism is likely affected by the Purkinje-NKCC1 axis, but I think it will apply to a majority of those with non-regressive, multigenic, strictly defined autism (SDA).
We saw in a recent post how the enzyme aromatase acts in the so-called testosterone – estradiol shunt.
I suggested that lack of aromatase was leading to too little estradiol which then affected neuroligin 2 (NL2) which then caused down-regulation of the KCC2 cotransporter that takes chloride out of neurons. This then caused neurons to remain in a permanent immature state.
Digging a little deeper we find recent research that shows how the control loops that balance aromatase act through RORA/RORα, RORa (retinoic acid-related orphan receptor alpha.
The schematic illustrates a mechanism through which the observed reduction in RORA in autistic brain may lead to increased testosterone levels through downregulation of aromatase. Through AR, testosterone negatively modulates RORA, whereas estrogen upregulates RORA through ER.
androgen receptor = AR
estrogen receptor = ER
RORα (retinoic acid-related orphan receptor alpha.)
RORα certainly has a long full name. Retinoic acid is a metabolite of vitamin A (retinol).
RORα does some clever things.
RORα is necessary for normal circadian rhythms
ROR-alpha is expressed in a variety of cell types and is involved in regulating several aspects of development, inflammatory responses, and lymphocyte development
RORα is involved in processes that regulate metabolism, development, immunity, and circadian rhythm and so shows potential as drug targets. Synthetic ligands have a variety of potential therapeutic uses, and can be used to treat diseases such as diabetes, atherosclerosis, autoimmunity, and cancer. T0901317 and SR1001, two synthetic ligands, have been found to be RORα and RORγ inverse agonists that suppress reporter activity and have been shown to delay onset and clinical severity of multiple sclerosis and other Th17 cell-mediated autoimmune diseases. SR1078 has been discovered as a RORα and RORγ agonist that increases the expression of G6PC and FGF21, yielding the therapeutic potential to treat obesity and diabetes as well as cancer of the breast, ovaries, and prostate. SR3335 has also been discovered as a RORα inverse agonist.
RORs are also called nuclear melatonin receptors. Many people with autism take melatonin to balance circadian rhythms and fall asleep.
The reduced estrogen levels in women during menopause likely caused them not to sleep due to the effect on RORα.
So it would appear that some of what is good for menopausal women may actually be helpful for some people with autism.
Many Genes affected by RORα
Most exciting, the researchers say, is that 426 of RORA’s gene targets are listed in AutismKB, a database of autism candidates maintained by scientists at Peking University in Beijing, and 49 in SFARI Gene.
Therapeutic Effect of a Synthetic RORα/γ Agonist in an Animal Model of Autism
Autism is a developmental disorder of the nervous system associated with impaired social communication and interactions as well excessive repetitive behaviors. There are no drug therapies that directly target the pathology of this disease. The retinoic acid receptor-related orphan receptor α (RORα) is a nuclear receptor that has been demonstrated to have reduced expression in many individuals with autism spectrum disorder (ASD). Several genes that have been shown to be downregulated in individuals with ASD have also been identified as putative RORα target genes. Utilizing a synthetic RORα/γ agonist, SR1078, that we identified previously, we demonstrate that treatment of BTBR mice (a model of autism) with SR1078 results in reduced repetitive behavior. Furthermore, these mice display increased expression of ASD-associated RORα target genes in both the brains of the BTBR mice and in a human neuroblastoma cell line treated with SR1078. These data suggest that pharmacological activation of RORα may be a method for treatment of autism.
For those who like natural substances, some research from Japan.
Abstract
The retinoic acid receptor-related orphan receptors α and γ (RORα and RORγ), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. In this study, we investigated the effects of isoflavones on RORα/γ activity and the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In doxycycline-inducible CHO stable cell lines, we found that four isoflavones, biochanin A (BA), genistein, formononetin, and daidzein, enhanced RORα- or RORγ-mediated transcriptional activity in a dose-dependent manner. In an activation assay of the Il17a promoter using Jurkat cells, these compounds enhanced the RORα- or RORγ-mediated activation of the Il17a promoter at concentrations of 1 × 10(-6)M to 1 × 10(-5)M. In mammalian two-hybrid assays, the four isoflavones enhanced the interaction between the RORα- or RORγ-ligand binding domain and the co-activator LXXLL peptide in a dose-dependent manner. In addition, these isoflavones potently enhanced Il17a mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin, but showed slight enhancement of Il17a gene expression in RORα/γ-knockdown EL4 cells. Immunoprecipitation and immunoblotting assays also revealed that BA enhanced the interaction between RORγt and SRC-1, which is a co-activator for nuclear receptors. Taken together, these results suggest that the isoflavones have the ability to enhance IL-17 gene expression by stabilizing the interactions between RORα/γ and co-activators. This also provides the first evidence that dietary chemicals can enhance IL-17 gene expression in immune cells.
Genistein is a common supplement. It is a pytoestrogen and unfortunately these substances lack potency in real life. In test tubes they have interesting properties, but they are poorly absorbed when taken orally and so unless they are modified they are likely to have no effect in the usual tiny doses used in supplements.
This is true with very many products sold as supplements.
Sometimes care is taken to improve bioavailability as with some expensive curcumin supplements, like Longvida.
Trehalose, a supplement referred to recently in comments on this blog, is another interesting natural substance that lacks bioavailablity. Analogs of this natural substance have been produced that are much better absorbed and are now potential drugs.
Purkinje Cells
Back in 2013 I wrote a post about Purkinje cells.
Pep up those Purkinje cells
These cells are some of the largest neurons in the human with an intricately elaborate dendritic arbor, characterized by a large number of dendritic spines. Purkinje cells are found within the Purkinje layer in the cerebellum. Purkinje cells are aligned like dominos stacked one in front of the other. Their large dendritic arbors form nearly two-dimensional layers through which parallel fibers from the deeper-layers pass. These parallel fibers make relatively weaker excitatory (glutamatergic) synapses to spines in the Purkinje cell dendrite, whereas climbing fibers originating from the inferior olivary nucleus in the medulla provide very powerful excitatory input to the proximal dendrites and cell soma. Parallel fibers pass orthogonally through the Purkinje neuron's dendritic arbor, with up to 200,000 parallel fibers[2] forming a Granule-cell-Purkinje-cell synapse with a single Purkinje cell. Each Purkinje cell receives ca 500 climbing fiber synapses, all originating from a single climbing fiber.[3] Both basket and stellate cells (found in the cerebellar molecular layer) provide inhibitory (GABAergic) input to the Purkinje cell, with basket cells synapsing on the Purkinje cell axon initial segment and stellate cells onto the dendrites.
Purkinje cells send inhibitory projections to the deep cerebellar nuclei, and constitute the sole output of all motor coordination in the cerebellar cortex.
In humans, Purkinje cells can be harmed by a variety causes: toxic exposure, e.g. to alcohol or lithium; autoimmune diseases; genetic mutations causing spinocerebellar ataxias, Unverricht-Lundborg disease, or autism; and neurodegenerative diseases that are not known to have a genetic basis, such as the cerebellar type of multiple system atrophy or sporadic ataxias.
Purkinje cells are some of the largest neurons in the human brain and the most important.
Neuronal maturation during development is a multistep process regulated by transcription factors. The transcription factor RORα (retinoic acid-related orphan receptor α) is necessary for early Purkinje cell maturation but is also expressed throughout adulthood.
The active form (T3) of thyroid hormone controls critical aspects of cerebellar development, such as migration of postmitotic neurons and terminal dendritic differentiation of Purkinje cells. T3 action on the early Purkinje cell dendritic differentiation process is mediated by RORα.
In autism we have seen that oxidative stress may lead to low levels of T3 in the autistic brain. We now see that low levels of RORα are also likely in autsim.
The combined effect would help explain the loss of Purkinje cells in autism.
Neuropathological studies, using a variety of techniques, have reported a decrease in Purkinje cell (PC) density in the cerebellum in autism. We have used a systematic sampling technique that significantly reduces experimenter bias and variance to estimate PC densities in the postmortem brains of eight clinically well-documented individuals with autism, and eight age- and gender-matched controls. Four cerebellar regions were analyzed: a sensorimotor area comprised of hemispheric lobules IV–VI, crus I & II of the posterior lobe, and lobule X of the flocculonodular lobe. Overall PC density was thus estimated using data from all three cerebellar lobes and was found to be lower in the cases with autism as compared to controls. These findings support the hypothesis that abnormal PC density may contribute to selected clinical features of the autism phenotype.
Estradiol – Neuroligin 2 to KCC2
We saw in a recent post how reduced levels of estradiol could lead to KCC2 underexpression via the action of neuroligin 2.
Conclusion
So in my grossly oversimplified world of autism, I think I have a plausible case for the Purkinje-KCC2 axis. I think that in addressing this axis numerous other issues would also be solved ranging from sleep issues to those hundreds of other genes whose regulation is at least partly governed by RORα.
The KCC2 end of the axis can be treated by bumetanide, diamox/acetazolamide, potassium bromide and possibly by intranasal IGF-1/insulin.
How to address the rest of the Purkinje-KCC2 axis?
· More RORα, or just a RORα agonist.
· More aromatase
· Genistein may help, but you would need it by the bucket load, due to bioavailability issues
· Estrogen receptor agonists
· Exogenous estradiol
The simplest is the last one and really should be trialed on adult males with autism. The dose would need to be much lower than the feminizing dose, so 0.2mg would seem a good starting dose for such a study.
Due to the feedback loops somethings may work short term, but not long term.