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Showing posts with label GSK-3. Show all posts
Showing posts with label GSK-3. Show all posts

Tuesday 28 January 2020

Piperine/Resveratrol/Sunitinib for Rett’s and indeed much Autism? Or, R-Baclofen to raise KCC2 expression in Bumetanide-responsive autism.



Piperine/Pepper             Resveratrol/Red wine          Sunitinib/Sutent
  

This post is all about lowering chloride within neurons, by increasing the expression of the transporter that lets it leave, called KCC2.


Today’s post is one I never finished writing from last year; I looked up the price of Sutent/Sunitinib and then I remembered why. It does again highlight how cancer drugs, when they become cheap generics, will provide interesting options for autism treatment. It also shows again how Rett Syndrome is getting attention from researchers.

It also highlights that really clever Americans are looking for bumetanide alternatives, in the false belief that bumetanide has troubling side effects that cannot be managed/mitigated.

The study is by some clever guys in Cambridge Massachusetts.

Another group of clever guys from MIT burned through $40 million dollars a few years ago trying to develop R-Baclofen for Fragile-X and autism.  After that Roche-funded clinical trial failed, R-Baclofen has now been resurrected and a new trial is planned, with different end points (measures of success).

Today we see why many people should indeed respond positively to R-Baclofen, but the mode of action is entirely different to the one originally targeted by the clever guys from MIT.

Tucked away in the supplementary material of today’s paper we see that R-Baclofen increases the expression of the transporter (KCC2) that takes chloride out of neurons. So, R-Baclofen is doing the same thing as Bumetanide, just to a lesser extent and in a different way.  Both lower intracellular chloride.

That means that people responsive to bumetanide should get a further boost from R baclofen, but you might need a lot of it.

Clever they may be, but these researchers do not know how to communicate their findings.  I had to dig through the supplementary tables to extract the good stuff, which is a list of what substances increase KCC2 in regular brains (Table S1) and specifically in Rett Syndrome brains (Table S2).

This blog does rather bang on about blocking/inhibiting NKCC1 that lets chloride into neurons, you can of course alternatively open up KCC2 to let the chloride flood out. This latter strategy is proposed by the MIT researchers.

What really matters is the ratio KCC2/NKCC1.  In people with bumetanide-responsive autism, which pretty clearly will include girls with Rett Syndrome, you want to increase KCC2/NKCC1. So, block/down-regulate NKCC1 and/or up-regulate KCC2.

·        NKCC1

·        KCC2


The researchers identified 14 compounds.  To be useful as drugs these compounds have to be able to cross the blood brain barrier to be of much use, many do not.

In the paper they call KCC2 expression-enhancing compounds KEECs.

We have five approved drugs to add to the list that are functionally the same to primary hit compounds. 

·        Sunitinib
·        Crenolanib
·        Indirubin Monoxiome
·        Cabozantinib
·        TWS-119


The researchers went on to test just two compounds in Rett syndrome mice; they picked piperine (from black pepper) and KW 2449 (a leukemia drug)


Even R-baclofen pops up, with a “B score” of 6.65 (needs to be >3 to increase KCC2 expression).



Abstract
Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. There are currently no approved treatments for RTT. The expression of K+/Cl- cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression-enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration-approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3β (GSK3β) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2.





Table S1. KEECs identified from screening with WT human KCC2 reporter neurons.






Table S2. KEECs identified from screening with RTT human KCC2 reporter neurons


Note Baclofen, Quercetin, Luteolin etc

















Fig. 3. Identification of KEECs that increase KCC2 expression in human RTT neurons
B score >3 indicates compounds potentially increasing KCC2 expression

In cultured RTT neurons, treatment with KEECs KW-2449 and BIO restored the impaired KCC2 expression and rescued deficits in both GABAergic and glutamatergic neurotransmissions, as well as abnormal neuronal morphology. Previous data suggested that disrupted Cl− homeostasis in the brainstem causes abnormalities in breathing pattern (64), consistent with breathing abnormalities seen in mice carrying a conditional Mecp2 deletion in GABAergic neurons (67). The reduction in locomotion activity observed in the Mecp2 mutant mice has also been attributed to abnormalities in the GABAergic system (65). Therefore, treatment with the KEEC KW-2449 or piperine may ameliorate disease phenotypes in MeCP2 mutant mice through restoration of the impaired KCC2 expression and GABAergic inhibition.

Most KEECs that enhanced KCC2 expression in WT neurons, including KW-2449, BIO, and resveratrol, also induced a robust increase of KCC2 reporter activity in RTT neurons (Fig. 3, A and B; a complete list of hit compounds is provided in table S2). The increase in KCC2 signal induced by KEECs was higher in RTT neurons than in WT neurons,


Our results establish a causal relationship between reduced FLT3 or GSK3 signaling activity and increased KCC2 expression.

Two hit compounds, resveratrol and piperine, act on different pathways than the kinase inhibitors, activating the SIRT1 signaling pathway (50) and the TRPV1 (51), respectively

Thus, our data demonstrate that activation of the SIRT1 pathway or the TRPV1 channel enhances KCC2 expression in RTT human neurons.


The group of KEECs reported here may help to elucidate the molecular mechanisms that regulate KCC2 gene expression in neurons. A previous study conducted with a glioma cell line showed that resveratrol activates the SIRT1 pathway and reduces the expression of NRSF/REST (50), a transcription factor that suppresses KCC2 expression (52). Our results demonstrate that resveratrol increases KCC2 expression by a similar mechanism, which could contribute to the therapeutic benefit of resveratrol on a number of brain disease conditions (68, 69). We also identified a group of GSK3 pathway inhibitors as KEECs. Overactivation of the GSK3 pathway has been reported in a number of brain diseases (70). Thus, our results suggest that GSK3 pathway inhibitors could exert beneficial effects on brain function through stimulating KCC2 expression. Another major KEEC target pathway, the FLT3 kinase signaling, has been investigated as a cancer therapy target (71, 72). Although FLT3 is expressed in the brain (73), drugs that target FLT3 pathway have not been extensively studied as potential treatments for brain diseases. Our results provide the first evidence that FLT3 signaling in the brain is critical for the regulation of key neuronal genes such as KCC2. Therefore, this work lays the foundation for further research to repurpose a number of clinically approved FLT3 inhibitors as novel brain disease therapies

Our results are valuable for the development of novel therapeutic strategies to treat neurodevelopmental diseases through rectification of dysfunctional neuronal chloride homeostasis. Because of the lack of pharmaceutical reagents that enhance KCC2 expression, bumetanide, a blocker of the inward chloride transporter NKCC1 that counteracts KCC2, has been used as an alternative (74). Bumetanide treatment has shown benefits in treating symptoms in mouse models of fragile X syndrome (75) and Down’s syndrome (76) and was shown to confer symptomatic benefit to human patients with autism or fragile X syndrome (77, 78). These findings strongly suggest that pharmacological restoration of disrupted chloride homeostasis may provide symptomatic treatment for various neurodevelopmental and neuropsychiatric disorders. However, NKCC1 lacks the neuron- restricted expression pattern of KCC2 and is also expressed in nonbrain tissue including kidney and inner ears (79), consistent with knockout of Nkcc1 in mouse model leading to deafness and imbalance (30). Therefore, bumetanide treatment may trigger undesirable side effects, thus severely limiting its therapeutic application. In contrast, the expression of KCC2 is restricted to neurons, and a number of the KEECs identified in this study that enhance KCC2 expression in neurons are Food and Drug Administration–approved and have not elicited any severe adverse effects in clinical trials (80–83). The promising efficacy of KEECs demonstrated in this study and the known safety of the KCC2 target warrant further preclinical and clinical studies to investigate these drugs and their derivatives as potential therapies for neurodevelopmental diseases.

In summary, in this work, we investigated the efficacy of KEECs to rescue a number of well-documented cellular and behavior phenotypes of RTT, including impaired GABA functional switch, reductions in excitatory synapse number and strength, immature neuronal morphology (53, 54), as well as an increase in breathing pauses and a decrease in locomotion (84). It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases (17, 85) including epilepsy (86–88), schizophrenia (19, 20, 89), brain and spinal cord injury (21, 90), stroke and ammonia toxicity conditions (91–93), as well as the impairments in learning and memory observed in the senile brain (23). Thus, a phenotypically diverse array of brain diseases may benefit from enhancing the expression of KCC2. The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders



Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. There are currently no approved treatments for RTT. The expression of K+/Cl− cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression– enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration–approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3 (GSK3) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2.


By screening these KCC2 reporter human neurons, we identified a number of hits KCC2 expression–enhancing compounds (KEECs) from ~900 small-molecule compounds. Identified KEECs were validated by Western blot and quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments on cultured human wild-type (WT) and isogenic RTT neurons, as well as on organotypic mouse brain slices. Pharmacological and molecular biology experiments showed that identified KEECs act through inhibition of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3b (GSK3b) kinases, or activation of the sirtuin 1 (SIRT1) or transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment of RTT neurons with KEECs rescued disease-related deficits in GABA functional switch, excitatory synapses, and neuronal morphological development. Last, injection of the identified KEEC KW-2449 or piperine into a Mecp2 mutant mice ameliorated behavioral phenotypes including breathing pauses and reduced locomotion, which represent important preclinical data, suggesting that the KEECs identified in this study may be effective in restoring impaired E/I balance in the RTT brain and provide symptomatic treatment for patients with RTT.





Fig. 2. KEEC treatment–induced enhancement of KCC2 protein and mRNA expression in cultured organotypic mouse brain slices and a hyperpolarizing EGABA shift in cultured immature neurons.

(E to G) KCC2 and NKCC1 mRNA expression induced by FLT3 inhibitors including sunitinib (n = 4), XL-184 (n = 6), crenolanib (n = 4), or a structural analog of BIO termed indirubin monoxime (n = 6). The calculated ratios of KCC2/NKCC1 mRNA expression are shown in (G). A.U., arbitrary units




Our results are valuable for the development of novel therapeutic strategies to treat neurodevelopmental diseases through rectification of dysfunctional neuronal chloride homeostasis. Because of the lack of pharmaceutical reagents that enhance KCC2 expression, bumetanide, a blocker of the inward chloride transporter NKCC1 that counteracts KCC2, has been used as an alternative (74). Bumetanide treatment has shown benefits in treating symptoms in mouse models of fragile X syndrome (75) and Down’s syndrome (76) and was shown to confer symptomatic benefit to human patients with autism or fragile X syndrome (77, 78). These findings strongly suggest that pharmacological restoration of disrupted chloride homeostasis may provide symptomatic treatment for various neurodevelopmental and neuropsychiatric disorders. However, NKCC1 lacks the neuron restricted expression pattern of KCC2 and is also expressed in nonbrain tissue including kidney and inner ears (79), consistent with knockout of Nkcc1 in mouse model leading to deafness and imbalance (30). Therefore, bumetanide treatment may trigger undesirable side effects, thus severely limiting its therapeutic application. In contrast, the expression of KCC2 is restricted to neurons, and a number of the KEECs identified in this study that enhance KCC2 expression in neurons are Food and Drug Administration–approved and have not elicited any severe adverse effects in clinical trials (80–83). The promising efficacy of KEECs demonstrated in this study and the known safety of the KCC2 target warrant further preclinical and clinical studies to investigate these drugs and their derivatives as potential therapies for neurodevelopmental diseases.


In summary, in this work, we investigated the efficacy of KEECs to rescue a number of well-documented cellular and behavior phenotypes of RTT, including impaired GABA functional switch, reductions in excitatory synapse number and strength, immature neuronal morphology (53, 54), as well as an increase in breathing pauses and a decrease in locomotion (84). It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases (17, 85) including epilepsy (86–88), schizophrenia (19, 20, 89), brain and spinal cord injury (21, 90), stroke and ammonia toxicity conditions (91–93), as well as the impairments in learning and memory observed in the senile brain (23). Thus, a phenotypically diverse array of brain diseases may benefit from enhancing the expression of KCC2. The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders.




The science-light version:-

Drug screen reveals potential treatments for Rett syndrome

An experimental leukemia drug and a chemical in black pepper ease breathing and movement problems in a mouse model of Rett syndrome, according to a new study.

Rett syndrome is a rare brain condition related to autism, caused by mutations in the MECP2 gene. Because the gene is located on the X chromosome, the syndrome occurs almost exclusively in girls. No drugs are available to treat Rett.
The team screened 929 compounds from three large drug libraries, including one focused on Rett therapies. They found 30 compounds that boost KCC2’s expression in the MECP2 neurons; 14 of these also increased the protein’s expression in control neurons.

The team tested two of the identified compounds in mice with mutations in MECP2: KW-2449, which is a small molecule in clinical trials for leukemia, and piperine, an herbal supplement and component of black pepper. These mice have several traits reminiscent of Rett. They are prone to seizures, breathing problems, movement difficulties and disrupted social behavior.
Injecting the mice with either drug daily for two weeks improved the animals’ mobility relative to untreated mice. The drugs also eased the mice’s breathing problems, decreasing the frequency of pauses in breathing (apnea). The findings appeared in July in Science Translational Medicine.


 

Piperine, Resveratrol and analogs thereof

Piperine and Resveratrol are commercially available supplements.

Resveratrol has been mentioned many times in this blog.  It has numerous beneficial properties, to which we can now add increasing KCC2 expression, but it is held back by its poor ability to cross the blood barrier.

The other natural substance highlighted in the study is piperine. Piperine is the substance that gets added to curcumin to increases its bioavailability and hopefully get its health benefits.

Piperine has been recently been found to be a positive allosteric modulator of GABAA receptors.

It may be that piperine has 2 different effects on GABA, or maybe it is just the same one?

The result is that people are trying to develop modified versions of piperine that could be patentable commercial drugs.

Piperine also activated TRPV1 receptors.

You might wonder what is the effect in humans of plain old piperine in bumetanide-responsive autism.

Invitro blood–brain-barrier permeability predictions for GABAA receptor modulating piperine analogs

The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. In order to reach their target sites of action, these compounds need to enter the brain by crossing the blood–brain barrier (BBB). We here evaluated piperine and five selected analogs (SCT-66, SCT-64, SCT-29, LAU397, and LAU399) regarding their BBB permeability. Data were obtained in three in vitro BBB models, namely a recently established human model with immortalized hBMEC cells, a human brain-like endothelial cells (BLEC) model, and a primary animal (bovine endothelial/rat astrocytes co-culture) model. For each compound, quantitative UHPLC-MS/MS methods in the range of 5.00–500 ng/mL in the corresponding matrix were developed, and permeability coefficients in the three BBB models were determined. In vitro predictions from the two human BBB models were in good agreement, while permeability data from the animal model differed to some extent, possibly due to protein binding of the screened compounds. In all three BBB models, piperine and SCT-64 displayed the highest BBB permeation potential. This was corroborated by data from in silico prediction. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human BBB models, and moderate to high in the animal BBB model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux transporters.


The alkaloid piperine, the major pungent component of black pepper (Piper nigrum L.), was recently identified as a positive allosteric γ-aminobutyric acid type A (GABAA) receptor modulator. The compound showed anxiolytic-like activity in behavioral mouse models, and was found to interact with the GABAA receptors at a binding site that was independent of the benzodiazepine binding site [1,2]. Given that the compound complied with Lipinski’s “rule of five” [1], it represented a new scaffold for the development of novel GABAA receptor modulators [1–3]. Given that piperine also activates the transient receptor potential vanilloid 1 (TRPV1) receptors [4] which are involved in pain signaling and regulation of the body temperature [5,6], structural modification of the parent compound was required to dissect GABAA and TRPV1 activating properties

For drugs acting on the central nervous system (CNS), brain penetration is required. This process is controlled by the blood-brain barrier (BBB), a tight layer of endothelial cells lining the brain capillaries that limits the passage of molecules from the blood circulation into the brain [10]. Since low BBB permeability can reduce CNS exposure [11], lead compounds should be evaluated at an early stage of the drug development process for their ability to permeate the BBB [12].

Conclusions

Piperine and five selected piperine analogs with positive GABAA receptor modulatory activity were screened in three in vitro cell-based human and animal BBB models for their ability to cross the BBB. Data from the three models differed to some extent, possibly due to protein binding of the piperine analogs. In all three models, piperine and SCT-64 displayed the highest BBB permeation potential, which could be corroborated by in silico prediction data. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human models, and moderate to high in the animal model. ER calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux. In addition to the early in vitro BBB permeability assessment of the compounds, further studies (such as PK and drug metabolism studies) are currently in progress in our laboratory. Taken together, these data will serve for selecting the most promising candidate molecule for the next cycle of medicinal chemistry optimization




Conclusion

My conclusions are a little different to the MIT researchers

“The newly identified KEECs are potential therapeutic agents for otherwise elusive neurological disorders.”

This assumes that you cannot safely use bumetanide/azosemide, which you can.  Open your eyes and look at France, where several hundred children with autism are safely taking bumetanide.

”It is possible, however, that KEECs may also be effective in treatment of conditions other than RTT, as impairment in KCC2 expression has been linked to many brain diseases”

We have copious evidence that elevated chloride is a feature of many conditions, not just Rett’s and an effective cheap therapy has been sitting in the pharmacy for decades.

In the clinical trial of R-Baclofen that failed, there were some positive effects on some subjects.  Were the positive effects just caused by the effect of Baclofen in increasing KCC2 expression?

Should R-Baclofen become a cheap generic, it might indeed become a useful add-on for those with bumetanide-responsive. Regular Baclofen (Lioresal) is an approved drug, but it does have some side effects, so most likely R-baclofen will have side effects in some.

Baclofen itself in modest doses has little effect on bumetanide-responsive autism.



A cheap side-effect free KCC2 enhancer would be a good drug for autism, although cheap, safe NKCC1 blockers already exist. 

I have no idea if piperine benefits bumetanide-responsive autism.  Piperine has long been used in traditional medicine.

The TRPV1 receptor also affected by piperine plays a role in pain and anxiety.

We saw in the post below that TRPV1 controls cortical microglia activation and that GABARAP modulates TRPV1 expression.

So, TRPV1 and GABAA receptors are deeply intertwined.

  

GABAa receptor trafficking, Migraine, Pain, Light Sensitivity, Autophagy, Jacobsen Syndrome,Angelman Syndrome, GABARAP, TRPV1, PX-RICS, CaMKII and CGRP ... Oh and the"fever effect"



Is Piperine going to make autism better, or worse?








Thursday 27 July 2017

Targeting Dendritic Spines to Improve Cognitive Function and Behavior in Autism; plus Hair Loss/Graying



I have written several posts about dendritic spines and their varying shapes (morphology).  This sounds like a rather obscure subject, but it looks like it may be a key area where both behavior and cognition can be modified, even later in life.



Homer Simson after using a Wnt Activator 

Dendritic spines

In a typical neuron (brain cell) you have dendrites at one end and so-called axon terminals at the other. When neurons connect with each other, an axon terminal connects with a dendritic spine from another close by neuron.  Axons transmit electrochemical signals from one neuron to the dendrites of other neurons.  The junction formed between a dendritic spine and an axon terminal is called a synapse.







One neuron can have as many as 15,000 spines, some of which are picking up signals from axon terminals of other neurons.
The number and shape of these spines is constantly changing and not surprisingly defects in this process affect both cognition and behavior.
The other end of the neuron, with the axon terminals is much less studied.  The myelin sheath deserves a mention. This protective coating is constantly being repaired in a process called remyelination. MS (Multiple Sclerosis) is caused by damage to the myelin coating that does not self repair. A newly identified feature of autism is an abnormally thin layer of myelin. A lack of insulation along the axon will affect the flow of electrical signals.
Many factors are involved in dendritic spine morphology and plasticity. Many of the same factors are known to be disturbed in autism and other related dysfunctions (schizophrenia, bipolar, ADHD etc).
Recall that within autism there are two broad groups; the larger group has “too many” dendritic spines and the smaller group has “too few”. I am writing about the larger group. My post is a simplification of a complex subject.
Factors that influence dendritic spine morphology and plasticity include:- 

·        BDNF  (want less)

·        Estrogen  (want more)

·        Reelin (want more)

·        BCL2 (want more)

·        PAK1 (want less)

·        GSK3 beta (want more)

·        PTEN (want more)

All the above seem to work via

·        Wnt signaling (want less) 

BDNF is a growth factor within the brain, which tends to be elevated in most autism.
The female hormone estrogen seems to be reduced in male autism and this will have many effects via something called ROR alpha. There is also reduced expression of estrogen receptor beta.
Reelin is a protein that is critical in brain development and maintenance. Reelin is implicated in most brain diseases, including autism. It stimulates dendritic spine development. Reelin is found to be reduced in autism.
BCl2 is a very well-known cancer gene/protein. BCL2 is part of a broader family of genes/proteins that control cell growth/death. BCL2 is anti-apoptotic, meaning it encourages growth rather than cell death. You will find elevated BCL2 in cancers.  BCL2 is implicated in both schizophrenia and autism.
Bax is another key member of the BCL2 family. The BCL2 protein duels with Bax, its counteracting twin. When Bax is in excess, cells execute a death command. When BCL2 dominates, the program is inhibited and cells survive. In cancer you want more Bax.
Modulating BCL2/Bax has been proposed as an autism therapy in Japan.
BCL2 is found to be reduced in autism.
The Japanese proposed the use of Navitoclax, a drug responsible for inhibiting BCL2 production for the treatment of cancer. I think they want to activate BCL2 production. 
I covered PAK1 in some lengthy posts. This was what the Japanese Nobel Laureate at MIT was working on. In summary, a PAK1 inhibitor should be helpful in autism, schizophrenia and some cancer.  Some people with a condition called neurofibromatosis, where non-cancerous tumors grow, use a special kind of bee propolis that contains a substance called CAPE (caffeic acid phenethyl ester), that is a mild PAK1 inhibitor.


GSK3 beta plays a role in several key signaling pathways. Abnormal expression of GSK3 beta is associated with Bipolar disorder. One role played by GSK3 beta is in Wnt signaling, which then affects dendritic spines. A GSK3 beta inhibitor, like lithium, is a Wnt activator which will increase the number of dendritic spines.
PTEN is a tumor suppressor gene/protein that is also an autism gene.
PTEN deficiency results in abnormal arborization and myelination in humans. PTEN-deficient neurons in brains of animal models have increased synaptic spine density.
People with autism and PTEN mutations have large heads because they lacked enough PTEN to reign in cell growth (and head growth).  You would expect them to have increased synaptic spine density.
Note than in both autism/cancer genes (BCL2 and PTEN) the balance is shifted towards growth, which fits in with the broad concept of autism as a growth dysfunction.
Wnt signaling is a complex and only partially understood subject, that has been previously discussed in this blog.  The short version is that most people with autism and particularly the ones with large heads will likely have too much Wnt signaling as the result of their various metabolic “disturbances”. The best way to inhibit their Wnt signaling might be to counter their particular metabolic disturbances, so if you are one of the 2% of autism with a PTEN mutation, then increase your PTEN levels.  If this is not possible than any other way to inhibit Wnt might be effective.
In Bipolar, where GSK3 beta is a known risk gene, you want more dendritic spines and so you want a GSK3 beta inhibitor like lithium. 
I think lithium will have a negative effect on most autism. Within children diagnosed with autism, a minority may well better fit a diagnosis of bipolar.

OBJECTIVE:


Children with autism spectrum disorder (ASD) have higher rates of comorbid psychiatric disorders, including mood disorders, than the general child population. Although children with ASD may experience irritability (aggression, self-injury, and tantrums), a portion also experience symptoms that are typical of a mood disorder, such as euphoria/elevated mood, mania, hypersexuality, paranoia, or decreased need for sleep. Despite lithium's established efficacy in controlling mood disorder symptoms in the neurotypical population, lithium has been rarely studied in children with ASD.

METHODS:


We performed a retrospective chart review of 30 children and adolescents diagnosed with ASD by the Diagnostic and Statistical Manual of Mental Disorders, 4th ed., Text Revision (DSM-IV-TR) criteria who were prescribed lithium in order to assess target symptoms, safety, and tolerability. Clinical Global Impressions - Improvement (CGI-I) ratings were performed by two board-certified child psychiatrists with expertise in ASD. CGI-I scores were dichotomized into "improved" (CGI-I score of 1 or 2) or "not improved" (CGI-I score ≥3).

RESULTS:


Forty-three percent of patients who received lithium were rated as "improved" on the CGI-I. Seventy-one percent of patients who had two or more pretreatment mood disorder symptoms were rated as "improved." The presence of mania (p=0.033) or euphoria/elevated mood (p=0.041) were the pretreatment symptoms significantly associated with an "improved" rating. The mean lithium blood level was 0.70 mEq/L (SD=0.26), and the average length of lithium treatment was 29.7 days (SD=23.9). Forty-seven percent of patients were reported to have at least one side effect, most commonly vomiting (13%), tremor (10%), fatigue (10%), irritability (7%), and enuresis (7%).

CONCLUSIONS:


This preliminary assessment of lithium in children and adolescents with ASD suggests that lithium may be a medication of interest for those who exhibit two or more mood disorder symptoms, particularly mania or euphoria/elevated mood. A relatively high side effect rate merits caution, and these results are limited by the retrospective, uncontrolled study design. Future study of lithium in a prospective trial with treatment-sensitive outcome measures may be indicated.


Hair Growth and Graying 
One surprising observation is the apparent connection between dendritic spine modification and modifying growth/color of human hair.
The same pathway is involved in signaling growth and coloring in the hair on your head and growing the dendritic spines on the neurons inside your head. I have mentioned this once before in a previous post. It is relevant because if a substance is potent enough to affect your dendritic spines you would expect it also to have a visible effect on the hair, of at least some people.
For example one reader of this blog uses a PAK1 inhibitor to treat her case of autism and she found that it has a hair graying effect.

EdnrB Governs Regenerative Response of Melanocyte Stem Cells by Crosstalk with Wnt Signaling

Pigmented hair regeneration requires epithelial stem cells (EpSCs) and melanocyte stem cells (McSCs) in the hair follicle.

Thus far, only a handful of signals that regulate McSCs have been identified, including extrinsic signals, such as transforming growth factor beta (TGFB) and Wnts, which are provided by the epithelial niche. Wnt signaling induces activation of EpSCs to drive epithelial regeneration while coordinately inducing McSCs to proliferate and differentiate to pigment regenerating hair follicle


One known but uncommon side effect of my current favourite Wnt inhibitor, Mebendazole, is hair loss. Hair follicles require Wnt signaling and if there is too little Wnt signaling you will lose some hair.
BCL2 is a very important cancer gene/protein but it also plays a role in autism and in dendritic spine morphology.  Low levels of the protein BCl2 leads to premature graying.

The team then looked at what would happen if they 'knocked out' a gene in mice that is known to be important for cell survival.
Mice lacking this Bcl2 gene went grey shortly after birth.

The scientists believe the same principle might apply in humans, which would explain why some people - such as TV presenter Philip Schofield - go grey in their 20s, while others keep their dark locks into retirement.
  

BCL2 is known to be reduced in the reduced in the brains of people with autism, as is another substance called Reelin.  Both Reelin and Bcl-2 are needed for dendritic spines to develop correctly.  

Autism is a severe neurodevelopmental disorder with potential genetic and environmental causes. Cerebellar pathology including Purkinje cell atrophy has been demonstrated previously. We hypothesized that cell migration and apoptotic mechanisms may account for observed Purkinje cell abnormalities. Reelin is an important secretory glycoprotein responsible for normal layering of the brain. Bcl-2 is a regulatory protein responsible for control of programmed cell death in the brain. Autistic and normal control cerebellar corteces matched for age, sex, and post-mortem interval (PMI) were prepared for SDS-gel electrophoresis and Western blotting using specific anti-Reelin and anti-Bcl-2 antibodies. Quantification of Reelin bands showed 43%, 44%, and 44% reductions in autistic cerebellum (mean optical density +/- SD per 30 microg protein 4.05 +/- 4.0, 1.98 +/- 2.0, 13.88 +/- 11.9 for 410 kDa, 330 kDa, and 180 kDa bands, respectively; N = 5) compared with controls (mean optical density +/- SD per 30 microg protein, 7.1 +/- 1.6, 3.5 +/- 1.0, 24.7 +/- 5.0; N = 8, p < 0.0402 for 180 kDa band). Quantification of Bcl-2 levels showed a 34% to 51% reduction in autistic cerebellum (M +/- SD per 75 microg protein 0.29 +/- 0.08; N = 5) compared with controls (M +/- SD per 75 microg protein 0.59 +/- 0.31; N = 8, p < 0.0451). Measurement of beta-actin (M +/- SD for controls 7.3 +/- 2.9; for autistics 6.77 +/- 0.66) in the same homogenates did not differ significantly between groups. These results demonstrate for the first time that dysregulation of Reelin and Bcl-2 may be responsible for some of the brain structural and behavioral abnormalities observed in autism.  

Abstract

The development of distinct cellular layers and precise synaptic circuits is essential for the formation of well-functioning cortical structures in the mammalian brain. The extracellular protein Reelin through the activation of a core signaling pathway including the ApoER2 and VLDLR receptors and the adapter protein Dab1, controls the positioning of radially migrating principal neurons, promotes the extension of dendritic processes in immature forebrain neurons, and affects synaptic transmission. Here we report for the first time that the Reelin signaling pathway promotes the development of postsynaptic structures such as dendritic spines in hippocampal pyramidal neurons. Our data underscore the importance of Reelin as a factor that promotes the maturation of target neuronal populations and the development of excitatory circuits in the postnatal hippocampus. These findings may have implications for understanding the origin of cognitive disorders associated with Reelin deficiency.

While not everything relating to dendritic spines is variable, and hence potentially can be modified, much seems to be.
Rather like in this blog it took a few years to get a comprehensive view of the factors involved in neuronal chloride and extend the list of potential therapies, getting to the bottom of fine tuning dendritic spin morphology for improved behavior and cognition will be a complex task.
Much is already known.
Our reader AJ is busy looking at GSK3 beta inhibitors.
GSK3 beta is best known as a bipolar gene/protein, but it is becoming seen as an autism gene.


GSK3 is one of the few signaling mediators that play central roles in a diverse range of signaling pathways, including those activated by Wnts, hedgehog, growth factors, cytokines, and G protein-coupled ligands. Although the inhibition of GSK3-mediated β-catenin phosphorylation is known to be the key event in Wnt-β-catenin signaling, the mechanisms which underlie this event remain incompletely understood. The recent demonstration of GSK3 involvement in Wnt receptor phosphorylation illustrates the multifaceted roles that GSK3 plays in Wnt-β-catenin signaling. In this review, we will summarize these recent results and offer explanations, hypotheses, and models to reconcile some of these observations.
Recent advances indicate that GSK3 also plays a positive role in Wnt signal transduction by phosphorylating the Wnt receptors low density lipoprotein receptor-related protein (LRP5/6) and provide new mechanisms for the suppression of GSK3 activity by Wnt in β-catenin stabilization. Furthermore, GSK3 mediates crosstalk between signaling pathways and β-catenin-independent downstream signaling from Wnt.


it is known that glycogen synthase kinase 3β (GSK-3β) regulates both synaptic plasticity and memory. 
GSK-3β overexpression led to a general reduction in the number of dendritic spines. In addition, it caused a slight reduction in the percentage, head diameter and length of thin spines, whereas the head diameter of mushroom spines was increased.


Over the past 2 decades, neuroscientists have built a body of evidence that links not only bipolar disease, but other psychiatric disorders including autism and schizophrenia to abnormal brain development. In particular, they have found abnormalities in the numbers of synapses and in the shape of neurons at the points where they form synapses. Their studies have often implicated abnormal signaling in a brain pathway called Wnt, which is involved both in early brain development and later, more complex, refining of brain connections. The role of Wnt could help explain why lithium is effective: It blocks an enzyme called GSK-3 β, which is an inhibitor on the Wnt pathway. By boosting Wnt signaling, lithium could produce a therapeutic effect in psychiatric diseases in which the Wnt pathway is underpowered.

They then treated the mutant mice with lithium. Although the researchers acknowledge that rodents are an imperfect proxy for human mood disorders, they did observe that the animals’ symptoms markedly improved; studies of their brains also revealed normal numbers of spines. “That’s the key finding,” Cheyette says. “It suggests that lithium could have its well-known therapeutic effect on patients with bipolar disorder by changing the stability of spines in the brain.”







GSK3 has numerous effects.

Glycogen synthase kinase-3 (GSK-3) is a cytoplasmic serine/threonine protein kinase that phosphorylates and inhibits glycogen synthase, thereby inhibiting glycogen synthesis from glucose. However, this serine/threonine kinase is now known to regulate numerous cellular processes through a number of signaling pathways important for cell proliferation, stem cell renewal, apoptosis and development. Because of these diverse roles, malfunction of this kinase is also known to be involved in the pathogenesis of human diseases, such as nervous system disorders, diabetes, bone formation, inflammation, cancer and heart failure. Therefore, GSK-3 is recognized as an attractive target for the development of new drugs. The present review summarizes the roles of GSK-3 in the insulin, Wnt/β-catenin and hedgehog signaling pathways including the regulation of their activities. The roles of GSK-3 in the development of human diseases within the context of its participation in various signaling pathways are also summarized. Finally, the possibility of new drug development targeting this kinase is discussed with recent information about inhibitors and activators of GSK-3.  

Estradiol


The present study demonstrates that estradiol may trigger formation of new dendritic spines by activation of a cAMPregulated CREB phosphorylation. Induction of the CREB response requires activation of NMDA receptors, increased intracellularcalciumconcentrationsandcAMP-activatedPKA.These systems together then contribute to the CREB response, which in turn leads to the morphological changes seen with estradiol—i.e., spine formation. The biochemical and cellular routes leading from activated CREB to the morphological change in dendritic spine density are still uncharted.

Dendritic spines of the medial amygdala: plasticity, density, shape, and subcellular modulation by sex steroids.

The medial nucleus of the amygdala (MeA) is a complex component of the "extended amygdala" in rats. Its posterodorsal subnucleus (MePD) has a remarkable expression of gonadal hormone receptors, is sexually dimorphic or affected by sex steroids, and modulates various social behaviors. Dendritic spines show remarkable changes relevant for synaptic strength and plasticity. Adult males have more spines than females, the density of dendritic spines changes in the course of hours to a few days and is lower in proestrous and estrous phases of the ovarian cycle, or is affected by both sex steroid withdrawal and hormonal replacement therapy in the MePD. Males also have more thin spines than mushroom-like or stubby/wide ones. The presence of dendritic fillopodia and axonal protrusions in the MePD neuropil of adult animals reinforces the evidence for local plasticity. Estrogen affects synaptic and cellular growth and neuroprotection in the MeA by regulating the activity of the cyclic AMP response element-binding protein (CREB)-related gene products, brain-derived neurotrophic factor (BDNF), the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and the activity-regulated cytoskeleton-related protein (Arc). These effects on signal transduction cascades can also lead to local protein synthesis and/or rearrangement of the cytoskeleton and subsequent numerical/morphological alterations in dendritic spines. Various working hypotheses are raised from these experimental data and reveal the MePD as a relevant region to study the effects of sex steroids in the rat brain.

PTEN 


CNS deletion of Pten in the mouse has revealed its roles in controlling cell size and number, thus providing compelling etiology for macrocephaly and Lhermitte-Duclos disease. PTEN mutations in individuals with autism spectrum disorders (ASD) have also been reported, although a causal link between PTEN and ASD remains unclear. In the present study, we deleted Pten in limited differentiated neuronal populations in the cerebral cortex and hippocampus of mice. Resulting mutant mice showed abnormal social interaction and exaggerated responses to sensory stimuli. We observed macrocephaly and neuronal hypertrophy, including hypertrophic and ectopic dendrites and axonal tracts with increased synapses. This abnormal morphology was associated with activation of the Akt/mTor/S6k pathway and inactivation of Gsk3β. Thus, our data suggest that abnormal activation of the PI3K/AKT pathway in specific neuronal populations can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD.  


Mutations in phosphatase and tensin homolog deleted on chromosome ten (PTEN) are implicated in neuropsychiatric disorders including autism. Previous studies report that PTEN knockdown in neurons in vivo leads to increased spine density and synaptic activity. To better characterize synaptic changes in neurons lacking PTEN, we examined the effects of shRNA knockdown of PTEN in basolateral amygdala neurons on synaptic spine density and morphology using fluorescent dye confocal imaging. Contrary to previous studies in dentate gyrus, we find that knockdown of PTEN in basolateral amygdala leads to a significant decrease in total spine density in distal dendrites. Curiously, this decreased spine density is associated with increased miniature excitatory post-synaptic current frequency and amplitude, suggesting an increase in number and function of mature spines. These seemingly contradictory findings were reconciled by spine morphology analysis demonstrating increased mushroom spine density and size with correspondingly decreased thin protrusion density at more distal segments. The same analysis of PTEN conditional deletion in dentate gyrus demonstrated that loss of PTEN does not significantly alter total density of dendritic protrusions in the dentate gyrus, but does decrease thin protrusion density and increases density of more mature mushroom spines. These findings suggest that, contrary to previous reports, PTEN knockdown may not induce de novo spinogenesis, but instead may increase synaptic activity by inducing morphological and functional maturation of spines. Furthermore, behavioral analysis of basolateral amygdala PTEN knockdown suggests that these changes limited only to the basolateral amygdala complex may not be sufficient to induce increased anxiety-related behaviors. 


Aberrant regulation of WNT/β-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context. In melanoma, for example, models of the disease predict differing effects of the WNT/β-catenin pathway on metastatic progression. Understanding the processes that underpin the highly context-dependent nature of WNT/β-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds. In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/β-catenin signaling, correlating with differing metabolic profiles. This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy. Thus, WNT/β-catenin signaling is a regulator of catabolic processes in cancer cells, which varies depending on the metabolic requirements of tumors.

BDNF
A meta-analysis of blood BDNF in 887 patients with ASD and 901 control subjects demonstrated significantly higher BDNF levels in ASD compared to controls with the SMD of 0.47 (95% CI 0.07-0.86, p = 0.02). In addition subgroup meta-analyses were performed based on the BDNF specimen. The present meta-analysis study led to conclusion that BDNF might play role in autism initiation/ propagation and therefore it can be considered as a possible biomarker of ASD.

Dendritic spines are major sites of excitatory synaptic transmission and changes in their numbers and morphology have been associated with neurodevelopmental and neurodegenerative disorders. Brain-derived Neurotrophic Factor (BDNF) is a secreted growth factor that influences hippocampal, striatal and neocortical pyramidal neuron dendritic spine density. However, the mechanisms by which BDNF regulates dendritic spines and how BDNF interacts with other regulators of spines remain unclear. We propose that one mechanism by which BDNF promotes dendritic spine formation is through an interaction with Wnt signaling. Here, we show that Wnt signaling inhibition in cultured cortical neurons disrupts dendritic spine development, reduces dendritic arbor size and complexity, and blocks BDNF-induced dendritic spine formation and maturation. Additionally, we show that BDNF regulates expression of Wnt2, and that Wnt2 is sufficient to promote cortical dendrite growth and dendritic spine formation. Together, these data suggest that BDNF and Wnt signaling cooperatively regulate dendritic spine formation.


Other Wnt inhibitors

Yet another anti-parasite drug, Niclosamide,  turns out to be a Wnt inhibitor. 


Not surprisingly, Niclosamide is now a candidate drug to treat several different types of cancer.  It is also thought to have great potential in suppressing the metastatic process of prostate cancer. Another extremely cheap drug, not available in the US.
Even the flavonoid quercetin can inhibit Wnt. 

Therapeutic Avenues

There certainly are many potential ways to fine tune dendritic spine morphology.
Some readers of this blog are already doing just that, perhaps not all realizing it. 
·        BDNF  (want less - TrkB inhibitor)

·        Estrogen 

·        Reelin (want more – statin via RAS activation)

·        BCL2 (want more – statin)

·        PAK1 (want less – PAK inhibitor, BIO30)

·        GSK3 beta (want more – GSK3 activator)

·        PTEN (want more – statin)

All the above seem to work via

·        Wnt signaling (want less – Mebendazole/Niclosamide etc)

If you inhibit GSK3 beta you activate Wnt. You need get things the right way around. 
Statins promote RAS signaling which appears to increase Reelin expression. 


Conclusion

Fine tuning dendritic spine morphology seems like a good target for those with MR/ID and also those with any kind of neurological disorder.
There appear to be many ways to achieve this.
It seems a plausible idea and in many ways seems more credible than the idea of a diuretic (bumetanide) raising some people’s IQ.
The big issue is which substances have sufficient potency, once they have crossed the blood brain barrier, to do anything at all.  This is an issue with all therapies targeting the brain, including bumetanide.
At least substances that can affect hair growth and color are making it through to the bloodstream, which is a start.
Does this mean that tuning your dendritic spines will inevitably make your hair turn grey or begin to thin?  I don’t think so. I think this will happen in people who have low to normal Wnt signaling to start with.
Do some people with naturally premature graying, or thinning, hair have low levels of Wnt signaling? Quite possibly. Are they more likely to have traits of bipolar/creativity? Look for actors with gray or thinning hair.
Do people with autism tend to have full heads of thicker hair, as well as bigger heads?
Do the minority of people with autism and small heads have thinning hair?
Some readers of this blog are already using statins to treat autism. As has been pointed out in earlier posts, other than lowing cholesterol, statins have potent anti-inflammatory effects and they also affect expression of RAS, PTEN and BCL2, all of which are implicated in autism and all affect dendritic spines. It seems plausible that these readers are already modifying dendritic spine morphology.