Inflammation Leading to Cognitive Dysfunction

Today’s post highlights a paper with some very concise insights into how microglial cells become “activated” resulting in the “exaggerated inflammatory response” that many people with autism experience on a daily basis.  

This is very relevant to treatment, which is not usually the objective of much autism research.

I recall reading a comment from John’s Hopkins about neuroinflammation/activated microglia in autism; they commented that no known therapy currently exists and that, of course, common NSAIDs like ibuprofen will not be effective.  But NSAIDs are effective.

As we see in today’s paper, there a least 4 indirect cytokine-dependent pathways leading to the microglia, plus one direct one.

NSAIDs most definitely can reduce cytokine signaling and thus, indirectly, reduce microglial activation.

The ideal therapy would act directly at the microglia, and as Johns Hopkins pointed out, that does not yet exist with today's drugs.  If you read the research on various natural flavonoids you will see that “in vitro” there are known substances with anti-neuroinflammatory effects on microglial activation.  The recurring “problem” with such substances is low bioavailability and inability to cross the blood brain barrier.

Back to Today’s Paper

It was a conference paper at the 114th Abbott Nutrition Research Conference - Cognition and Nutrition

The full conference proceedings can be downloaded

The paper is not about autism, it is about more general cognitive dysfunction.  It is from mainstream science (I checked).

It explains how inflammation anywhere in the body can be translated across the BBB (Blood Brain Barrier) to activate the microglia.  This of course allows you to think of ways to counter these mechanisms.

It also raises the issue of whether or not anti-inflammatory agents really need to cross the BBB.  While you might think that ability to cross the BBB is a perquisite to mitigate the activated microglia, this may not be the case.  Much can be achieved outside the BBB, and we should not rule out substances that cannot cross the BBB.

Very many known anti-inflammatory substances do not cross the BBB.   

  

From Inflammation to Sickness and Cognitive Dysfunction: When the Immune System Subjugates the Brain

extracts from the above paper ...

Example – Influenza and Cognition

Neurological and cognitive effects associated with influenza infection have been reported throughout history.

The simplest explanation for these neurocognitive effects is that influenza virus makes its way to the brain, where it is detected by neurons.

However, most influenza strains, including those responsible for pandemics, are considered non-neurotropic, neurological symptoms associated with influenza infection are not a result of direct viral invasion into the CNS.

Moreover, neurons do not have receptors to detect viruses (or other pathogens) directly.

Cells of the immune system do, however, as the immune system’s primary responsibility is to recognize infectious pathogens and contend with them. For example, sentinel immune cells such as monocytes and macrophages are equipped with toll-like receptors (TLR) that recognize unique molecules associated with groups of pathogens (i.e., pathogen-associated molecular patterns). Stimulation of TLRs that recognize viruses (TLR3 and TLR7) and bacteria (TLR4) on immune sentinel cells can have profound neurological and cognitive effects, suggesting the immune system conveys a message to the brain after detecting an infectious agent. This message is cytokine based.

Macrophages and monocytes produce inflammatory cytokines (e.g., interleukin [IL]-1β, IL-6, and tumor necrosis factor-α [TNF-α]) that facilitate communication between the periphery and brain.

Cytokine-dependent Pathways to the Brain

Several cytokine-dependent pathways that enable the peripheral immune system to transcend the blood-brain barrier have been dissected.

Inflammatory cytokines present in blood can be actively transported into the brain.

But there are also four indirect pathways:-

1.     Cytokines produced in the periphery need not enter the brain to elicit neurocognitive changes. This is because inflammatory stimuli in the periphery can induce microglial cells to produce a similar repertoire of inflammatory cytokines. Thus, brain microglia recapitulates the message from the peripheral immune system.

2.     in a second pathway, inflammatory cytokines in the periphery can bind receptors on blood-brain barrier endothelial cells and induce perivascular microglia or macrophages to express cytokines that are released into the brain

3.     In a third pathway, cytokines in the periphery convey a message to the brain via the vagus nerve. After immune challenge, dendritic cells and macrophages that are closely associated with the abdominal vagus have been shown to express IL-1β protein; IL-1 binding sites have been identified in several regions of the vagus as well. When activated by cytokines, the vagus can activate specific neural pathways that are involved in neurocognitive behavior. However, activation of the vagus also stimulates microglia in the brain to produce cytokines via the central adrenergic system 

4.     A fourth pathway provides a slower immune-to-brain signaling mechanism based on volume transmission.  In this method of immune-to-brain communication, production of IL-1β by the brain first occurs in the choroid plexus and circumventricular organs—brain areas devoid of an intact blood-brain barrier. The cytokines then slowly diffuse throughout the brain by volume transmission, along the way activating microglia, neurons, and neural pathways that induce sickness behavior and inhibit cognition.

Can Flavonoids Reduce Neuroinflammation and Inhibit Cognitive Aging?

Flavonoids are naturally occurring polyphenolic compounds present in plants. The major sources of flavonoids in the human diet include fruits, vegetables, tea, wine, and cocoa.  Significant evidence has emerged to indicate that consuming a diet rich in flavonoids may inhibit or reverse cognitive aging

Flavonoids may improve cognition in the aged through a number of physiological mechanisms, including scavenging of reactive oxygen and nitrogen species and interactions with intracellular signaling pathways. Through these physiological mechanisms, flavonoids also impart an anti-inflammatory effect that may improve cognition. This seems likely for the flavone luteolin, which is most prominent in parsley, celery, and green peppers.

Whereas luteolin inhibits several transcription factors that mediate inflammatory genes (e.g., nuclear factor kappa B [NF-κB]and activator protein 1 [AP-1]), it is a potent activator of nuclear factor erythroid 2-related factor 2 (Nrf2), which induces the expression of genes encoding antioxidant enzymes. A recent study of old healthy mice found improved learning and memory and reduced expression of inflammatory genes in the hippocampus when luteolin was included in the diet. Thus, dietary luteolin may improve cognitive function in the aged by reducing brain microglial cell activity.

Hence, the flavonoid luteolin is a naturally occurring immune modulator that may be effective in reducing inflammatory microglia in the senescent brain.

Conclusion

In light of the recent evidence suggesting microglial cells become dysregulated due to aging and cause neuroinflammation, which can disrupt neural structure and function, it is an interesting prospect to think dietary flavonoids and other bioactives can be used to constrain microglia. But how can flavonoids impart this anti-inflammatory effect? Although in vitro studies clearly indicate that several flavonoids can act directly on microglial cells to restrict the inflammatory response, results from in vivo studies thus far do not prove that dietary flavonoids access the brain to interact with microglia in a meaningful way. This is a complicated question to dissect because flavonoids reduce inflammation in the periphery and microglia seem to act like an “immunostat,” detecting and responding to signals emerging from immune-to-brain signaling pathways. Thus, whether dietary flavonoids enter the brain and impart an anti-inflammatory effect on microglia is an interesting question but one that is more theoretical than practical because what is most important is how the immunostat is adjusted, whether that is via a direct or indirect route. However, because flavonoids are detectable in the brain they most likely affect microglia both directly and by dampening immune-to-brain signaling.

Interesting Natural Substances

In no particular order, these are several very interesting flavonoids/carotenoids.  In the lab, they all do some remarkable things.

In humans, they also do some interesting things; how helpful they might be in autism remains to be seen.

Being “natural” does not mean they are good for you and have no side-effects.

Some of the following are very widely used and so you can establish if there are issues with long term use.  It also makes them accessible.

Quercetin (found in many fruits, numerous interesting effects)

and two Quercetin-related flavonoids:-

Kaempferol (widely used in traditional medicine)

Myricetin (has good and bad effects)

Lycopene  (from tomatoes, potent anti-cancer, does not cross the BBB)

  

Luteolin(in many vegetables, like broccoli) 

Apigenin (from chamomile, stimulates neurogenesis, PAM of GABA_A, block NDMA receptors, antagonist of opioid receptors …)

Tangeretin (from tangerines, does cross the BBB, has potent effects in vitro)

Nobiletin (from tangerines)

Hesperidin (from tangerines)

Naringin (from Grapefruit, contraindicated with many prescription drugs)

Epicatechin/Catechin  (the chocolate/cocoa flavonoids, do cross the BBB, well researched)

Nystatin in autism - a potent Potassium Channel Kv1.3 blocker (anti-inflammatory) or an antifungal/candida treatment?

Today’s post will go against some people’s understanding of autism and inflammatory bowel disease.

Just as there is a belief that heavy metals are a problem in autism there is another is another belief that candida is involved in autism and indeed inflammatory bowel disease (IBD).  Various types of IBD are highly comorbid with autism, but most people with IBD do not have autism.

The most common treatment for candida is an antifungal medicine called nystatin.  This drug is a cheap and widely available.

But nystatin has another property, it is a highly effective blocker of the potassium channel Kv1.3.

Regular readers will recall that this ion channel is key mediator in the inflammatory process, it is a target in many inflammatory conditions such as IBD and indeed autism.  Those little helminths (TSO) parasites that are being researched for both autism and IBD were found to reduce inflammation by releasing their own Kv1.3 blocker which stops the host (human or animal) from rejecting them.

Immunomodulatory Therapy in Autism - Potassium Channel Kv1.3, Parasitic Worms, and their ShK–related peptides

Candida Albicans Infection in Autism

Abstract: Background: Autism children were reported to have gastrointestinal problems that are more frequent and more severe than in children from the general population. Although many studies demonstrate that GI symptoms are common in autism, the exact percentage suffering from gastrointestinal (GI) problems is not well known, but there is a general consensus that GI problems are common in autism. The observation that antifungal medications improve the behavior of autism children, encourage us to investigate their intestinal colonization with yeasts. Aim of the work: The purpose of this work was to investigate the intestinal colonization with yeasts in autistic patients and to assess the role of yeast as a risk factor to cause autism behavior. Patients and methods: The study included 83 cases diagnosed as autistic children referred from the neuro-pediatric clinic and 25 normal children as a control group. All children under the study came to Phoniatric clinic, during the period from 2010 to 2012, complaining of delayed language development with autistic features. Children in this study were classified into 2 groups; control and study groups. All children were subjected to interview, E.N.T examination, language assessment, Childhood Autistic Rating Score (CARS), stool culture for Candida albicans, complete audiological and psychometric evaluation. Results: There was significant relation between the autistic children and heavy growth of Candida albicans in stool culture. Conclusion: The high rate of Candida albicans intestinal infection in autistic children may be a part of syndrome related to immune system disorders in these patients.

Conclusion: Candida albicans infection may be a part of syndrome related to the immune system and depends on genetic basis of autism, or Candida albicans may be etiological factor lead to excessive ammonia in gut which is responsible of autistic behavior in children. More researches are needed to clarify the exact mechanism by which Candida albicans affects autistic children.

  

In another study the results were not so clear:-

Gastrointestinal flora and gastrointestinal status in children with autism-- comparisons to typical children and correlation with autism severity

This study was done by James Adams (of the Autism Research Institute, former home of DAN).  According to Wikipedia, Adams' research has been described as "a laundry list of autism woo"; I think he is well intentioned.

You would have expected him to find Candida, but he did not. 

Note that they did not find any parasites either, although they did give up testing after the first 20 results were negative (not very scientific, I think).  Regular readers will know that some “holistic doctors” insist that parasites are the cause of autism.

  

Yeast

The presence of yeast was determined by both culture and by microscopic observation. Yeast was only rarely observed by culture in the autism or typical groups, and the difference between the two groups was not significant, as shown in Table ​Table5.5. Yeast was more commonly observed microscopically, but again the difference between the two groups was not significant.

Parasitology

The parasitology test was used on the first 20 autism samples only, which were all negative. It was then decided to do no additional testing on other samples

  

The finding that yeast levels were similar in both the autistic and control group is interesting, as there has been a great deal of speculation that yeast infections are a major problem in autism. Our data indicates that yeast is present at normal levels in the stool of this group of children with autism. A study by Horvath and Perman [21] reported that 43% of children with autism undergoing endoscopies had a positive fungal culture for yeast in their duodenal juice, vs. 23% of age-matched controls with other gastrointestinal problems requiring endoscopies. Since their study involved children with severe enough symptoms to warrant endoscopies, the greater symptom severity may explain some of the difference with our study. Since the survey by the Autism Research Institute of over 25,000 parents' reports that parents find antifungals to be one of the most effective medications for improving behavior [44], our findings are puzzling. It is possible that children with autism are more sensitive to even a normal level of yeast. Also, it is possible that antifungals have other effects, such as reducing inflammation.

  

Which Study to believe?

I have to say that I give more credence to the first study, which is from Egypt.

I think that autism in Egypt is likely to be the “real deal”.  People with severe autism will likely have associated auto-immune/inflammatory conditions and this will include abnormal GI conditions.

Also, the more severe the autism, the more restrictive the diet is likely to be, which will affect what grows inside the intestines.   

   

Ion Channels and Channelopathies

Ion channels are complex, but fortunately there are not that many of them, unlike genes.

A good source of information is provided by École polytechnique fédérale de Lausanne, on the banks of lake Geneva.  On their Channelpedia site you can see a nice entry on the potassium channel Kv1.3.  It may all look rather too complicated, but there under the Scorpion toxin, is a very common drug, Nystatin.

Kv1.3

Interactions

---

PAP-1

MbCD and MbCD/C

Zn

Leukocyte Subunits effect Kv1.3

Cluster at C-terminus

Kv1.3 associates with Kv1.5

Kv1.3 forms heteromeric channels

Scorpion toxin ADWX-1 is a pore blocker of Kv1.3 channel without affecting its kinetics

Nystatin

The concentrations for nystatin and its structural analog, amphotericin B, required to produce half maximal inhibition (IC50) of the current were estimated to be about 3 and 60 microM, respectively. The effects of nystatin on the amplitude and inactivation of Kv1.3 currents were not voltage-dependent. In inside-out patches, tetraethylammonium (TEA) produced a rapid block of Kv1.3 currents upon the onset of a voltage pulse, while the inhibition by nystatin developed slowly. When co-applied with TEA, nystatin potentiated the extent of the TEA-dependent block, and the kinetic effect of nystatin was slowed by TEA. In summary, nystatin, a compound frequently used in perforated patch recordings to preserve intracellular dialyzable components, specifically inhibited the potassium channel Kv1.3 at concentrations well below those required for perforation

KCa3.1 is related to acute immune responses and Kv1.3 is related to chronic immune responses, the combined administration with Kv1.3 and KCa3.1 inhibitors is likely to enhance their effects in autoimmune disorders or graft rejection

We know that Kv1.3 is widely expressed in the brain, but is it expressed in the intestines of people with inflammatory/auto-immune conditions?

We do not have far to look and since we know that ulcerative colitis is comorbid with autism, we can stick with that

Expression of T-cell KV1.3 potassium channel correlates with pro-inflammatory cytokines and disease activity in ulcerative colitis

Abstract

BACKGROUND AND AIMS:

Potassium channels, KV1.3 and KCa3.1, have been suggested to control T-cell activation, proliferation, and cytokine production and may thus constitute targets for anti-inflammatory therapy. Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by excessive T-cell infiltration and cytokine production. It is unknown if KV1.3 and KCa3.1 in the inflamed mucosa are markers of active UC. We hypothesized that KV1.3 and KCa3.1 correlate with disease activity and cytokine production in patients with UC.

METHODS:

Mucosal biopsies were collected from patients with active UC (n=33) and controls (n=15). Protein and mRNA expression of KV1.3 and KCa3.1, immune cell markers, and pro-inflammatory cytokines were determined by quantitative-real-time-polymerase-chain-reaction (qPCR) and immunofluorescence, and correlated with clinical parameters of inflammation. In-vitro cytokine production was measured in human CD3(+) T-cells after pharmacological blockade of KV1.3 and KCa3.1.

RESULTS:

Active UC KV1.3 mRNA expression was increased 5-fold compared to controls. Immunofluorescence analyses revealed that KV1.3 protein was present in inflamed mucosa in 57% of CD4(+) and 23% of CD8(+) T-cells. KV1.3 was virtually absent on infiltrating macrophages. KV1.3 mRNA expression correlated significantly with mRNA expression of pro-inflammatory cytokines TNF-α (R(2)=0.61) and IL-17A (R(2)=0.51), the mayo endoscopic subscore (R(2)=0.13), and histological inflammation (R(2)=0.23). In-vitro blockade of T-cell KV1.3 and KCa3.1 decreased production of IFN-γ, TNF-α, and IL-17A.

CONCLUSIONS:

High levels of KV1.3 in CD4 and CD8 positive T-cells infiltrates are associated with production of pro-inflammatory IL-17A and TNF-α in active UC. KV1.3 may serve as a marker of disease activity and pharmacological blockade might constitute a novel immunosuppressive strategy.

So now we have some evidence that Kv1.3 is involved in the inflammatory response within the intestines of people with inflammatory bowel disease (IBD).

Now we just need to look at what happens when you give Nystatin to people with IBD.

Since we do have to link all this back to Candida, let us look for people with IBD claiming that the problem was all about Candida.

If you google Crohns disease (a type of ulcerative colitis/IBD) you will find numerous reference to the benefit of Nystatin and again the assumption that “yeast overgrowth” is somehow the cause of the disease.  Lots of "holistic" doctors etc.

Why do so many people with autism benefit from Nystatin?

We have seen why some people with GI inflammation should find Nystatin very helpful, it will act locally as an immuno-suppressant.  

By reducing this inflammation there will be a reduction in inflammatory cytokines like IL-6.  But the whole idea of Nystatin being safe for children with autism is that it does not enter the blood stream, in stays inside the intestines.

Leaky Gut

Many people subscribe to the notion of the “leaky gut” in autism.  If indeed the gut was leaky, the Nystatin might leak out.  It would then act as a Kv1.3 blocker elsewhere in the body.  It may, or may not, be able to cross the blood brain barrier.

There is now some scientific evidence to show that  “leaky gut” is a real phenomenon.

In people with ulcerative colitis, of course the gut is leaking.  Blood is coming in and therefore other things can flow the other way.

In healthy people, Nystatin will stay almost entirely where it should, within the intestines.  In people with “leaks” it would seem likely that some will leak out.  In these people we might expect a greater effect.

We do know that inflammatory activity within the gut can transmitted elsewhere in the body via the vagus nerve.  This means that reducing inflammation within the GI will reduce the pro-inflammatory signalling sent around the body via the vagus nerve, even with no "leaky gut".  

This may indeed sound very odd, but very promising results are now being found in treating people with arthritis (an inflammatory condition, where IL-6 plays a key role) using implanted electrical devices that affect the vagus nerve.  Vagus nerve stimulation is not pseudoscience, even though it does sound like it should be.

  

My conclusion

The “father” of ARI and the DAN movement, Dr Bernard Rimland, a research psychologist, suggested that a small proportion of people diagnosed with autism had nothing more than an overgrowth of candida, caused by the frequent use of antibiotics.

It does seem that very many things can lead to “autism” and this diagnosis is now equally applied to people with very mild symptoms and those with debilitating ones.  I imagine that Bernie may indeed have been right; in a small number of people the problem may indeed be yeast.  However, given the relatively large number of people with autism (and IBD) who find Nystatin very helpful, I think the real issue is inflammation and  K_V1.3.  The people who respond to Nystatin would very likely also respond to those TSO helminths, and even Stichodactyla toxin (see later).

One problem with regular use of antifungal medication is that you are going to kill off not just the candida.  A healthy gut is supposed have all sorts of things living in it.   

For me, the conclusion is to go back to the ion channels and look not just for KV1.3 blockers but also KCa3.1.  There are plenty of people doing just this, but not for autism, for example:-

The K+ channels KCa3.1 and Kv1.3 as novel targets for asthma therapy

Kv1.3 blockers do exist and they include:-

·        Curcumin (problem is low bioavailability)

·        Acacetin (rarely studied and mainly used by bodybuilders)

Acacetin attenuates neuroinflammation via regulation the response to LPS stimuli in vitro and in vivo.

Abstract

Under normal conditions in the brain, microglia play roles in homeostasis regulation and defense against injury. However, over-activated microglia secrete proinflammatory and cytotoxic factors that can induce progressive brain disorders, including Alzheimer's disease, Parkinson's disease and ischemia. Therefore, regulation of microglial activation contributes to the suppression of neuronal diseases via neuroinflammatory regulation. In this study, we investigated the effects of acacetin (5,7-dihydroxy-4'-methoxyflavone), which is derived from Robinia pseudoacacia, on neuroinflammation in lipopolysaccharide (LPS)-stimulated BV-2 cells and in animal models of neuroinflammation and ischemia. Acacetin significantly inhibited the release of nitric oxide (NO) and prostaglandin E(2) and the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV-2 cells. The compound also reduced proinflammatory cytokines, tumor necrosis factor-α and interleukin-1β, and inhibited the activation of nuclear factor-κB and p38 mitogen-activated protein kinase. In an LPS-induced neuroinflammation mouse model, acacetin significantly suppressed microglial activation. Moreover, acacetin reduced neuronal cell death in an animal model of ischemia. These results suggest that acacetin may act as a potential therapeutic agent for brain diseases involving neuroinflammation.

·        Progesterone (as a hormone, has many other effects)

·        Verapamil (already in the PolyPill)

The most unusual/interesting comes from Cuba:-

Stichodactyla toxin

Stichodactyla toxin (ShK) is a peptide toxin that blocks the voltage-gated potassium channels: K_v1.1, K_v1.3, K_v1.6, K_v3.2 and K_Ca3.1.

Selective Kv1.3 channel blocker as therapeutic for obesity and insulin resistance

In humans, a polymorphism in the Kv1.3 promoter is associated with impaired glucose tolerance and with lower insulin sensitivity (11). These results suggest that selective Kv1.3 blockers might have use in the management of obesity and insulin resistance

Because pancreatic beta cells, which have K_v3.2 channels, are thought to play a role in glucose-dependent firing, ShK, as a K_v3.2 blocker, might be useful in the treatment of type-2 diabetes.

  

You may recall we already saw in this blog the older people taking Verapamil (for heart problems) did not develop type 2 diabetes. According to the table below, ShK toxin is a K_v3.2 blocker in humans, but Verapamil only works in rats.

Since it looks like selective Kv1.3 blockers may prevent/treat obesity, you can expect them to be attractive targets for pharmaceutical companies.  This is a disease of the 21^st century.

The spin-off might later be a cost-effective treatment for inflammatory conditions like IBD and autism.

The clever new arthritis treatments, that could be used in autism, are hugely expensive.

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

This post does get a little complicated, so here is a summary.

Key points

·        High doses of oral Pregnenolone are shown to help Schizophrenia, particularly in females.  (these are all adults)

·        High dose oral Pregnenolone has also been shown to help adults with autism.

·        Low doses of transdermal progesterone (and likely Pregnenolone), anecdotally, reduce anxiety in Asperger’s and ADHD

·        Unusual levels of various hormones are a hallmark of autism, this can directly affect neurotransmitters like GABA

·        Hormones are produced in the brain as well as elsewhere in the body and so supplementing them may have unintended side effects.  Some hormones do not cross the blood brain barrier.

·        Side effects should be less likely after puberty, so research is done on adults

·        Some people regularly give very young children hormones, like melatonin

·        It may be possible to get the benefit of the hormones affecting GABA_A at low doses

·        Changes in certain hormone levels actually change the structure (and hence the effect) of the GABA_A receptor

·        Modulating the GABA_A receptor via the neurosteroid site then changes how the Benzo site of the same receptor responds to modulation (hence changes the effect, and side effects of Benzodiazepines)

Today’s post has a very odd tittle.

It will explain some of the odd things that we have been seeing in seizure drugs having potent effects at tiny doses.  It really is a case where “less is more”.

We will see that modulating the GABA_A receptor using the neurosteroid binding site (not the usual Benzo site) has potential for many neurological conditions.  There are some interesting interventions possible today and some are OTC.  We will also see that the structure of the GABA_A receptor is itself dynamic and some drugs affecting it are actually changing it.

This is all very relevant because it appears that GABA_A dysfunction is at the very core of the common autism variants and a key factor in schizophrenia.

   

I did say in a recent post that  GABA_A receptor is rather complicated and best left to Professors Sigel and Catterall and their mice, but then I came across the explanation myself.  As usual, the answer is there in the science, you just have to know where to look for it; or just stumble upon it.

It also appears that the recent autism trial at Stanford of pregnenolone, may have left untold part of the story.  They gave increasing high doses of pregnenolone, which is converted in the body into allopregnanolone, a positive allosteric modulator of GABA_A receptors. 

At tiny doses, allopregnanolone stimulates GABA_A, at higher doses it inhibits it and then at very high doses again it stimulates it.  So the precise dosage of Pregnenolone, or indeed progesterone, which also produces allopregnanolone, would be critical in achieving the desired modulation of GABA.  The Stanford researcher is a psychiatrist, by the way, not a biochemist; he did not investigate the effect of small doses.

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

There are a whole raft of similar studies in the works, trialing Pregnenolone in Schizophrenia, Bipolar, TBI and even Gulf War Illnesses.

Proof-of-concept randomized controlled trial of pregnenolone in schizophrenia

   

Allopregnanolone

As usual the most up-to-date source is Wikipedia:-

Function

Allopregnanolone possesses a wide variety of effects, including, in no particular order, antidepressant, anxiolytic, stress-reducing, rewarding, prosocial, antiaggressive, prosexual, sedative, pro-sleep, cognitive and memory-impairing, analgesic, anesthetic, anticonvulsant, neuroprotective, and neurogenic effects.

Fluctuations in the levels of allopregnanolone and the other neurosteroids seem to play an important role in the pathophysiology of mood, anxiety, premenstrual syndrome, catamenial epilepsy, and various other neuropsychiatric conditions.

Increased levels of allopregnanolone can produce paradoxical effects, including negative mood, anxiety, irritability, and aggression. This appears to be because allopregnanolone possesses biphasic, U-shaped actions at the GABA_A receptor – moderate level increases (in the range of 1.5–2 nM/L total allopregnanolone, which are approximately equivalent to luteal phase levels) inhibit the activity of the receptor, while lower and higher concentration increases stimulate it. This seems to be a common effect of many GABA_A receptor positive allosteric modulators. In accordance, acute administration of low doses of micronized progesterone (which reliably elevates allopregnanolone levels), have been found to have negative effects on mood, while higher doses have a neutral effect.

Neurosteroids and GABA-A receptor function

Possible Explanations for the Paradoxical Effect of GABA-Steroids

In this section, possible mechanisms of the biphasic response curve of allopregnanolone on behavioral parameters are discussed. The basic idea of so called paradoxical effect where neurosteroids show one type of effect at low concentrations and another type at high concentrations is that an enhanced GABA_A-receptor activity may give an excitatory net effect in certain situations, instead of the usual inhibitory effect. The following hypotheses suggest several possible mechanisms how this can be achieved. In addition, there are no contradictions between the different hypothesis and they may very well act in parallel.

The Effect of Neurosteroids on the GABA_A-Receptor

The GABA_A-receptor can be modulated by a number of therapeutic agents, including benzodiazepines , barbiturates , anesthetics, ethanol , zinc , and neurosteroids . The effect of neurosteroids on the GABA_A-receptor depends on the type of steroids (agonist or antagonist), the type of receptors (synaptic of extrasynaptic), the subunit compositions, and the intrinsic structure of the steroid. Recent studies indicate that the existence of at least two neurosteroid actions on the GABA_A-receptor, namely an agonistic action and an antagonistic action by the sulfated and 3β-OH steroids. The agonistic action can further be divided into an allosteric enhancement of GABA-evoked Cl⁻ current and a direct activation of the GABA_A-receptor.

It is puzzling why an increase in allopregnanolone during the menstrual cycle is related to development of negative mood as allopregnanolone should be anxiolytic agent like benzodiazepines. The answer depends on the fact that all GABA_A-receptor agonists such as benzodiazepines, barbiturates, alcohol, and allopregnanolone have paradoxical anxiogenic effects in certain individuals. At low concentrations or doses they give severe adverse emotional reactions in a subset of individuals (3–6%) and moderate reactions in up to 20–30% of individuals. This paradoxical effect is induced by allopregnanolone  benzodiazepines , barbiturates , and ethanol . Symptoms induced by these GABA_A-receptor active drugs are depressive mood, irritability, aggression, and other symptoms known to occur during the luteal phase in women with PMS/PMDD. A biphasic effect was also observed for medroxyprogesterone (MPA) and natural progesterone in postmenopausal women taking hormone therapy. These women felt worse on a lower dosage of MPA or progesterone than on a higher dosage or placebo.

Thus allopregnanolone seems to have a biphasic effect on mood with an inverted U-shaped relationship between concentration and effect. In postmenopausal women receiving progesterone, a biphasic relation between the negative mood symptoms and the plasma concentrations of allopregnanolone was observed. The negative mood increased with the elevating serum concentration of allopregnanolone up to the maximum concentration seen at the luteal phase. With further increase in allopregnanolone concentration there was a decrease in symptom severity  An inverted U-shaped relation between allopregnanolone dosage and irritability/aggression has also been noted in rats 

Antagonist Neurosteroids on the GABA_A-Receptor

Neurosteroids may both enhance and inhibit GABAergic neurotransmission

Paradoxical effects of GABA-A modulators may explain sex steroid induced negative mood symptoms in some persons.

Abstract

Some women have negative mood symptoms, caused by progestagens in hormonal contraceptives or sequential hormone therapy or by progesterone in the luteal phase of the menstrual cycle, which may be attributed to metabolites acting on the GABA-A receptor. The GABA system is the major inhibitory system in the adult CNS and most positive modulators of the GABA-A receptor (benzodiazepines, barbiturates, alcohol, GABA steroids), induce inhibitory (e.g. anesthetic, sedative, anticonvulsant, anxiolytic) effects. However, some individuals have adverse effects (seizures, increased pain, anxiety, irritability, aggression) upon exposure. Positive GABA-A receptor modulators induce strong paradoxical effects including negative mood in 3%-8% of those exposed, while up to 25% have moderate symptoms. The effect is biphasic: low concentrations induce an adverse anxiogenic effect while higher concentrations decrease this effect and show inhibitory, calming properties. The prevalence of premenstrual dysphoric disorder (PMDD) is also 3%-8% among women in fertile ages, and up to 25% have more moderate symptoms of premenstrual syndrome (PMS). Patients with PMDD have severe luteal phase-related symptoms and show changes in GABA-A receptor sensitivity and GABA concentrations. Findings suggest that negative mood symptoms in women with PMDD are caused by the paradoxical effect of allopregnanolone mediated via the GABA-A receptor, which may be explained by one or more of three hypotheses regarding the paradoxical effect of GABA steroids on behavior: (1) under certain conditions, such as puberty, the relative fraction of certain GABA-A receptor subtypes may be altered, and at those subtypes the GABA steroids may act as negative modulators in contrast to their usual role as positive modulators; (2) in certain brain areas of vulnerable women the transmembrane Cl(-) gradient may be altered by factors such as estrogens that favor excitability; (3) inhibition of inhibitory neurons may promote disinhibition, and hence excitability.

Allopregnanolone and mood disorders.

Abstract

Certain women experience negative mood symptoms during the menstrual cycle and progesterone addition in estrogen treatments. In women with PMDD increased negative mood symptoms related to allopregnanolone increase during the luteal phase of ovulatory menstrual cycles. In anovulatory cycles no symptom or sex steroid increase occurs. This is unexpected as positive modulators of the GABA-A receptor are generally increasing mood. This paradoxical effect has brought forward a hypothesis that the symptoms are provoked by allopregnanolone the GABA-A receptor system. GABA-A is the major inhibitory system in the brain. Positive modulators of the GABA-A receptor include the progesterone metabolites allopregnanolone and pregnanolone, benzodiazepines, barbiturates, and alcohol. GABA-A receptor modulators are known, in low concentrations to induce adverse, anxiogenic effects whereas in higher concentrations show beneficial, calming properties. Positive GABA-A receptor modulators induce strong paradoxical effects e.g. negative mood in 3-8% of those exposed, while up to 25% have moderate symptoms thus similar as the prevalence of PMDD, 3-8% among women in fertile ages, and up to 25% have moderate symptoms of premenstrual syndrome (PMS). The mechanism behind paradoxical reaction might be similar among them who react on positive GABA-A receptor modulators and in women with PMDD. In women the severity of these mood symptoms are related to the allopregnanolone serum concentrations in an inverted U-shaped curve. Negative mood symptoms occur when the serum concentration of allopregnanolone is similar to endogenous luteal phase levels, while low and high concentrations have less effect on mood. Low to moderate progesterone/allopregnanolone concentrations in women increases the activity in the amygdala (measured with fMRI) similar to the changes seen during anxiety reactions. Higher concentrations give decreased amygdala activity similar as seen during benzodiazepine treatment with calming anxiolytic effects. Patients with PMDD show decreased sensitivity in GABA-A receptor sensitivity to diazepam and pregnanolone while increased sensitivity to allopregnanolone. This agrees with findings in animals showing a relation between changes in alpha4 and delta subunits of the GABA-A receptor and anxiogenic effects of allopregnanolone.

CONCLUSION:

These findings suggest that negative mood symptoms in women with PMDD are caused by the paradoxical effect of allopregnanolone mediated via the GABA-A receptor.

Neurosteroids, GABAA receptors, and escalated aggressive behavior.

Abstract

Aggressive behavior can serve important adaptive functions in social species. However, if it exceeds the species-typical pattern, it may become maladaptive. Very high or escalated levels of aggressive behavior can be induced in laboratory rodents by pharmacological (alcohol-heightened aggression), environmental (social instigation), or behavioral (frustration-induced aggression) means. These various forms of escalated aggressive behavior may be useful in further elucidating the neurochemical control over aggression and violence. One neurochemical system most consistently linked with escalated aggression is the GABAergic system, in conjunction with other amines and peptides. Although direct stimulation of GABA receptors generally suppresses aggression, a number of studies have found that positive allosteric modulators of GABAA receptors can cause increases in aggressive behavior. For example, alcohol, benzodiazepines, and many neurosteroids are all positive modulators of the GABAA receptor and all can cause increased levels of aggressive behavior. These effects are dose-dependent and higher doses of these compounds generally shift from heightening aggressive behavior to being sedative and anti-aggressive. In addition, these modulators interact with each other and can have additive effects on the GABAA receptor and on behavior, including aggression. The GABAA receptor is a heteropentameric protein that can be constituted from various subunits. It has been shown that subunit composition can affect sensitivity of the receptor to some modulators and that subunit composition differentially affects the sedative vs anxiolytic actions of benzodiazepines. Initial studies targeting alpha subunits of the GABAA receptor point to their significant role in the aggression-heightening effects of alcohol, benzodiazepines, and neurosteroids.

The Do No Harm Principle (Primum non nocere)

A guiding principle in this blog is not to do any harm, while trying to do some good.

When I read that Hardan was trialing Pregnenolone at Stanford, I thought it was very interesting, but I thought his doses were very high and did not pass the above “first, no harm” principle.  Our pediatric endocrinologist thought the dose rising to 500mg was very unwise.

When I looked into this hormone precursor a year ago I remember thinking it odd that some people were saying 5mg was a big dose, while others were using 50mg and Hardan was going up to 500mg.

Now that I have understood about the mode of action is likely GABA_A, and that allopregnanolone possesses “biphasic, U-shaped actions at the GABA_A receptor”, I understand what may be going on.  The tiny dose might be as effective as the huge dose, but without the side effects caused by all the other accompanying hormonal changes.

The endocrinologist would likely not worry about 5mg of Pregnenolone.  Unlike most other known PAMs of GABA_A, pregnenolone does not need a prescription.

I did look for reports of people trying it themselves for schizophrenia/autism, but did not find anything useful.

Depression and  anxiety, and are frequently-seen side effects of 5α-reductase inhibitors such as finasteride, and are thought to be caused, in part, by interfering with the normal production of allopregnanolone.

Experiments in Humans

The doctors/scientist amongst you will have realized the potential therapeutic value of these paradoxical behaviors at GABA_A receptors.

Instead of using the usual right hand side of the curve, where high doses are effective but may risk tolerance and side effects, we may in some cases be able to use the left hand side.  This means low doses and far less chance of any side effects.

You do of course need some data on the U curve itself and the existing levels, if any, of the chosen GABAA modulator.

In the case of pregnenolone/ allopregnanolone/progesterone this seems to exist at a constant low level in males, but in cyclical low to high levels in females.

You would need to locate where you are on the curve, or perhaps to the left of the entire curve.  By adding a positive allosteric modulator (PAM) can only move to the right.  We know that 500mg of  pregnenolone in adults move to a “better” position on the allopregnanolone curve.

Circulating Levels of Allopregnanolone in Humans:

Gender, Age, and Endocrine Influences

In males 19-39 years old the level of Allopregnanolone is 0.8 nmol/l.

In Women  it varies from from 0.6 to 4.5 nmol/l during the month.

  

Allopregnanolone Elevations Following Pregnenolone Administration are Associated with Enhanced Activation of Emotion Regulation Neurocircuits

These results demonstrate that in response to emotional stimuli, allopregnanolone reduces activity in regions associated with generation of negative emotion. Furthermore, allopregnanolone may enhance activity in regions linked to regulatory processes. Aberrant activity in these regions has been linked to anxiety psychopathology. These results thus provide initial neuroimaging evidence that allopregnanolone may be a target for pharmacological intervention in the treatment of anxiety disorders, and suggest potential future directions for research into neurosteroid effects on emotion regulation neurocircuitry.

Pregnenolone is lipophilic and readily crosses the blood brain barrier. We have previously found that pregnenolone is preferentially metabolized to allopregnanolone, rather than other compounds such as cortisol or DHEA (43, 44); however these metabolites were also assayed. Allopregnanolone serum levels have been reported to triple two hours after oral administration of 400 mg pregnenolone (45). Thus, drug administration occurred two hours before neuroimaging to ensure elevated levels during the scan.

Pregnenolone administration reduced activity in neural circuits associated with the generation of negative emotions. Across all conditions and all face types, pregnenolone administration decreased right amygdala and right insula activity, and serum levels of pregnenolone and allopregnanolone were negatively correlated with amygdala and insula activation levels. The amygdala is a key region in threat detection (52), fear conditioning (53), and emotional salience (54). The insula is responsible for interoception (55), disgust (56), emotion processing (57), emotional recall (36), and anticipation of aversive stimuli (58). Both regions are associated with negative emotional response (57), and greater amygdala activation in response to the presentation of facial expressions is associated with greater magnitude of emotional response (59–63). Additionally, activation reductions in amygdala and insula are associated with down-regulation of negative emotions (64). Thus, allopregnanolone’s reduction of activity in amygdala and insula suggests that allopregnanolone may reduce emotional reactivity to aversive stimuli.

Allopregnanolone likely impacts emotion regulation neurocircuitry through GABAergic mechanisms, though it may also impact this circuitry through its enhancement of neurogenesis (78) myelination (79) or neuroprotection (80–83). Amygdala and mPFC are rich in GABA(A) receptors (28) and endogenous allopregnanolone (48), suggesting that allopregnanolone could feasibly have a direct impact on activity in these regions. Indeed, in our sample, allopregnanolone serum level was more strongly correlated to amygdala activity than activity in any other brain region. Preclinical research suggests that the amygdala may be a particular target of allopregnanolone’s anxiolytic effects (30). In rats, microinfusions of allopregnanolone directly into the amygdala produce anxiolytic (30) antidepressant (31) and anti-aggressive (32) effects. In previous neuroimaging studies, greater endogenous allopregnanolone has been reported to be associated with lower amygdala reactivity (33, 41) and greater coupling between amygdala and dmPFC (34). Though we did not directly test the GABAergic effect of our intervention, our findings illuminate potential neural pathways through which pregnenolone administration and resulting increases in allopregnanolone levels could feasibly impact GABAergic transmission in a manner that is relevant to pathological anxiety.

In conclusion, we demonstrate that pregnenolone administration (leading to increased downstream allopregnanolone levels) reduces activity in regions associated with the generation of negative emotion and enhances activity in regions linked to regulatory control over emotion, as well as increasing connectivity between two of these regions (dmPFC and amygdala). Considering the wealth of evidence that neurocircuits involving these regions are altered in anxiety disorders, our results invite further investigation into the brain basis for allopregnanolone’s use as an anxiolytic pharmacological intervention.

  

There is plenty of research into mood changes in females linked to the GABAA receptor.  So you could figure out what happens at what concentration of Allopregnanolone, in females.

The “problem” here is that plot thickens even further, in females it has been shown that the structure of the GABAA receptor actually changes.  When estrogen levels are higher than progesterone levels, the number of delta receptors decrease, increasing nerve cell activity, in turn increasing anxiety.

http://www.ncbi.nlm.nih.gov/pubmed/15895085

Here we demonstrate periodic alterations in specific GABA(A)R subunits during the estrous cycle in mice, causing cyclic changes of tonic inhibition in hippocampal neurons. In late diestrus (high-progesterone phase), enhanced expression of deltaGABA(A)Rs increases tonic inhibition, and a reduced neuronal excitability is reflected by diminished seizure susceptibility and anxiety. Eliminating cycling of deltaGABA(A)Rs by antisense RNA treatment or gene knockout prevents the lowering of excitability during diestrus

Since it appears the other GABAA receptors also exhibit the same U-shaped responses, there will be a choice.

I think a little experiment with very low doses of pregnenolone is worthwhile.

You may recall earlier mention in this blog of people with Asperger’s using a topical progesterone cream.  That would likely have exactly the same effect.  

There are other theories as to why progesterone cream might reduce anxiety, but it does seem to help some people.   Here is a comment on an Amazon forum:-

“My 9-year-old son is slightly autistic, suffers from severe anxiety as well as ADD, and although the medicine he takes works wonders for him, he still has residual anxiety. I rubbed 50 mg of natural progesterone cream into his chest every night for about three weeks and noticed unbelievable results. He just blossomed emotionally. We went in for his regular neuro checkup and even the neurologist immediately noticed a difference in my son, commenting on how happy and relaxed he seemed.”

There is also a pregnenolone cream.

It should be cautioned that these are all hormones and they do other things than modulate GABA_A receptors.  The effect/dosage will vary between males/females and with age.

  

Effects of modulating multiple binding sites of GABA_A

We did learn in an earlier post that the GABAA receptors have numerous different binding sites.  Since modulating some of these sites produces paradoxical results, you might wonder what happens if you really mix things up.  For example what happens if you modulate the benzo site and the neurosteroid site at the same time.

Neurosteroid coadministration prevents development of tolerance and augments recovery from benzodiazepine withdrawal anxiety and hyperactivity in mice

or

Mechanisms Underlying Tolerance after Long-Term Benzodiazepine Use: A Future for Subtype-Selective Receptor GABA_A Modulators?

4.4.3. Mechanism 6: Neurosteroids

There is ample and convincing evidence that neurosteroids are endogenous allosteric regulators that interact with GABA_A receptors to modulate both tonic (extrasynaptic) and phasic (synaptic) inhibition (for reviews, see [150, 151]). Also, acute or chronic neurosteroid treatment may change GABA_A receptor subunit expression, especially extrasynaptic α₄ and δ subunits [151]. In light of the plasticity-inducing actions of neurosteroids on inhibitory signaling, long-term enhancement of the GABA system with benzodiazepines may in turn evoke changes in the neurosteroids system such as changes in neurosteroid synthesis and metabolism, although classical benzodiazepines may differ in their potency to cause such changes [152]. In support, ovariectomy attenuated the development of tolerance to the anticonvulsant actions of diazepam [153]. Moreover, co-administration of the neurosteroids allopregnanolone or pregnenolone (but not dehydroepiandrosterone) prevented the development of tolerance after chronic treatment with either triazolam and diazepam [154]. Adding to the complexity of the putative involvement of neurosteroids in benzodiazepine tolerance, factors such as GABA_A receptor subunit composition, phosphorylation mechanisms, and ((extra)synaptic) localization—which are all factors that were already found to be involved in tolerance development—influence the specific dynamics of neurosteroid activity.

Ganaxolone – an altogether better Neurosteroid ?

Ganaxolone has the same chemical structure as allopregnanolone, with the addition of a methyl group designed to prevent conversion back to an active steroid, thereby eliminating the opportunity for unwanted hormonal effects while preserving its desired CNS activity.

We came across Ganaxolone in an earlier post, which looked at drugs being trialed in Fragile X.

I thought it was interesting that it is also being trialed in epilepsy and indeed PTSD (post-traumatic stress disorder)

I think that PTSD and TBI (Traumatic Brain Injury) can provide insights for understanding autism.

It is odd that nobody has trialed Ganaxolone for schizophrenia, to see if it shares the positive effects of Pregnenolone.  Then you might expect someone to think of trialing it in autism.

Conclusion

It would seem that, depending on your own natural level of allopregnanolone, supplementing it with pregnenolone or progesterone could have very different effects, depending on the dosage.

In addition, we have the fact that in autism the GABA_A receptor is dysfunctional and some aspects may work in reverse.

Having established that very large doses of Pregnenolone (which produces allopregnanolone) seems to be helpful in autism and schizophrenia, it would be well worth measuring the level of allopregnanolone, produced by different doses and of course investigating the effect of tiny doses of Pregnenolone.

Anecdotally, in typical people, tiny doses of Pregnenolone do have an effect.

It might turn out that because of where you are actually on the curve, you might actually need a low dose Negative Allosteric Modulator (NAM), to make you move to the left.  Interestingly there is such a NAM, Pregnenolone Sulfate.

Pregnenolone sulfate  is an endogenous excitatory neurosteroid that is synthesized from pregnenolone. It is known to have cognitive and memory-enhancing, antidepressant, anxiogenic, and proconvulsant effects.^[2]

Mechanism

Pregnenolone sulfate is a neurosteroid with excitatory effects in the brain, acting as a potent negative allosteric modulator of the GABA_A receptor and a weak positive allosteric modulator of the NMDA receptor.^] To a lesser extent, it also acts as a negative allosteric modulator of the AMPA, kainate, and glycine receptors, and may interact with the nACh receptors as well. In addition to its effects on ligand-gated ion channels, pregnenolone sulfate is an agonist of the sigma receptor, as well as an activator of the TRPM1 and TRPM3 channels. It may also interact with potassium channels and voltage-gated sodium channels.

Pregnenolone and its sulfate, like DHEA and its sulfate and progesterone, belong to the group of neurosteroids that are found in high concentrations in certain areas of the brain, and are synthesized there. Neurosteroids affect synaptic functioning, are neuroprotective, and enhance myelinization. Pregnenolone and its sulfate ester are under investigation for their potential to improve cognitive and memory functioning.^[3] Pregnenolone is also being considered as a potential treatment for schizophrenia.^[1]

Role of pregnenolone, dehydroepiandrosterone and their sulfate esters on learning and memory in cognitive aging

“Studies in animals demonstrated that the neurosteroids pregnenolone (PREG) and dehydroepiandrosterone (DHEA), as sulfate derivatives (PREGS and DHEAS, respectively), display memory-enhancing properties in aged rodents. Moreover, it was recently shown that memory performance was correlated with PREGS levels in the hippocampus of 24-month-old rats. Human studies, however, have reported contradictory results. First, improvement of learning and memory dysfunction was found after DHEA administration to individuals with low DHEAS levels, but other studies failed to detect significant cognitive effects after DHEA administration. Second, cognitive dysfunctions have been associated with low DHEAS levels, high DHEAS levels, or high DHEA levels; while in other studies, no relationship was found.”

Another NAM at the GABAA receptor is DHEA, a steroid hormone produced in the body.  Regular exercise stimulates the production of DHEA.

Lowered DHEA-S plasma levels in adult individuals with autistic disorder

Faulty serotonin--DHEA interactions in autism: results of the 5-hydroxytryptophan challenge test.

We also learnt that structure of the GABA_A receptor can be modified.  It happens continuously in females.  So as well as clever ways to use allosteric modulators, you could also copy nature and change the structure of the GABA_A receptors themselves.  In fact the genetic research does suggest this structure has been disturbed in autism; you would just be correcting a defect.

It does look like high dose Pregnenolone really is effective in reducing the symptoms in schizophrenia.  Here is yet another study showing this:-

Proof-of-Concept Trial with the Neurosteroid Pregnenolone Targeting Cognitive and Negative Symptoms in Schizophrenia