Primary and Secondary Dysfunctions in Autism - plus Candesartan

Sometimes the secondary event can completely overshadow the primary event.  
The above relates to dust explosions (in large silos containing grain, sugar etc.) rather than autism.

As we continue to investigate the science behind autism and associated possible therapies, it is becoming necessary to introduce some further segmentation.

I have referred to autism “flare-ups” many times, but even that term means very different things to different people.

We now have many examples of autism treatments (NAC, Bumetanide etc), once effective, suddenly stopping working in certain people.  This needs explaining.

We know from the research that in most cases, autism is caused by multiple “hits”, only when taken together do they lead to autism.

We also see the “double tap” variety of autism, when relatively mild autism later develops into something more serious, following some event, or trigger.  

Thanks to the internet, we know have numerous n=1 examples of certain drugs showing a positive effect in some people.  You do have to discount all those people trying to sell you something, or support the cause of others trying to sell you something.  We also have full access to all those people who have patented their clever ideas, although 99% never develop them.

Within all this information there are some very useful insights, which can help further our understanding of autism

Candesartan

A case in point is Candesartan, which one reader of this blog brought to my attention, in the comment below.  This drug is used to treat high blood pressure and is often combined with a diuretic.

A very recent study relating to neurodegenerative disease and Parkinson's especially:

http://www.sciencedaily.com/releases/2015/05/150512150022.htm

discusses the use of a new drug as well as another blood pressure drug sometimes used in conjunction with Bumetanide called Candesartan. Their goal in this study was to explore how to attenuate chronic microglial activation (a hallmark of autism) by targeting toll-like receptors TLR1 and TLR2 via these two drugs.

Candesartan also modulates NKCC2 activity:

http://www.ncbi.nlm.nih.gov/pubmed/18305093

which is interesting considering the original cited research above deals with attenuating microglial activation, rather than modulating the chloride levels within GABA inhibitory neurons as Bumetanide does.

Note that Bumetanide affects both NKCC1 and NKCC2 transporters.  NKCC1 is present in the brain at birth, but should not be present in the adult brain.  However, it appears to remain in a large sub-group of those with autism, causing GABA to remain excitatory.  NKCC2 is found specifically in the kidney, where it serves to extract sodium, potassium, and chloride from the urine so that they can be reabsorbed into the blood

This drug is, along with Minocycline, is one of the few that is known to have an effect on microglial activation.

In a clinical trial, Minocycline was shown to have no effect on autism.

I do feel this kind of assessment is too simplistic; so I was interested to see the actual effect of Candesartan in autism, albeit with n=1.

Conveniently somebody has filed a patent for the use of Candesartan in autism.  Within the document is the n=1 case report of its effect.

Example of treatment with Candersartan in autism

[00047] A 16 year old boy with autism was evaluated for behavioral management. He was frequently aggressive, primarily directed to himself but to others as well. These episodes were usually unprovoked but would also occur when his parents attempted to re direct him. The child was essentially non verbal except for echolalia. His comprehension to verbal re direction was limited, making non pharmacological interventions to his aggression limited.

[00048] His neurological exam was otherwise normal.

[00049] An MRI, EEG were normal. Routine studies, including examination for fragile x and other metabolic disorders were negative.

[00050] Prior medication trials included anti convulsants which were without benefit and atypical neuroleptics, which resulted in weight gain and unsatisfactory effects on behavior.

[00051] After obtaining consent from his parents, Candesartan was started. An initial dose of 8 mg resulted in significant attenuation of aggressive behavior. Blood pressure remained stable. After 2 weeks, the dose was raised to 16 mg. Further improvement in aggression was noted with no adverse lowering of blood pressure.

[00052] The patient has remained on Candesartan with beneficial anti aggression effects being maintained over one year.

[00053] A preferred dose found by the inventor to treat autism is approximately O.lmg/kg. In children, a liquid form may be used.

So we can conclude from this that in a non-verbal 16 year old boy with autism, with significant aggressive tendencies, this drug successfully reduced aggression.  Since he was on the drug for a year, there were no other major changes, such as language or cognitive function, otherwise they would surely be mentioned to support the patent.

I can of course look further into why Candesartan might have been effective.

Our blog reader suggested this research:-

Unraveling mystery of a-synuclein inneurodegenerative disease and reversing its course

"The real job of microglia is to keep the brain healthy by getting rid of pathogens as well as cellular debris," says Maguire-Zeiss, "However, in a diseased state microglia can become chronically activated, leading to a continuous onslaught of inflammation which is damaging to the brain."

In this study, the Maguire-Zeiss lab found that only a certain size structures of misfolded α-synuclein can activate microglial cells -- normal protein and even smaller forms of misfolded α-synuclein cannot. Then the researchers sought to discover precisely how microglia responded to misfolded α-synuclein; that is, which of its many "pattern recognition receptors" reacted to the toxic protein.

Microglia use many different pattern recognition proteins, called toll-like receptors (TLR), to recognize potential threats. The investigators found that misfolded α-synuclein caused TLR1 and TLR2 to come together into one complex (receptor), creating TLR1/2. They traced the entire molecular pathway from the protein's engagement of TLR1/2 at the cell surface to the production of inflammatory molecules.

Then Maguire-Zeiss and her team tested a drug, developed by researchers at the University of Colorado, which specifically targets TLR1/2. They also tested the hypertension drug candesartan, which can target TLR2. Both agents significantly reduced inflammation.

I found some other possible explanations:-

Angiotensin II AT1Receptor Blockade Ameliorates Brain Inflammation

Brain inflammation has a critical role in the pathophysiology of brain diseases of high prevalence and economic impact, such as major depression, schizophrenia, post-traumatic stress disorder, Parkinson's and Alzheimer's disease, and traumatic brain injury. Our results demonstrate that systemic administration of the centrally acting angiotensin II AT₁ receptor blocker (ARB) candesartan to normotensive rats decreases the acute brain inflammatory response to administration of the bacterial endotoxin lipopolysaccharide (LPS), a model of brain inflammation. The broad anti-inflammatory effects of candesartan were seen across the entire inflammatory cascade, including decreased production and release to the circulation of centrally acting proinflammatory cytokines, repression of nuclear transcription factors activation in the brain, reduction of gene expression of brain proinflammatory cytokines, cytokine and prostanoid receptors, adhesion molecules, proinflammatory inducible enzymes, and reduced microglia activation. These effects are widespread, occurring not only in well-known brain target areas for circulating proinflammatory factors and LPS, that is, hypothalamic paraventricular nucleus and the subfornical organ, but also in the prefrontal cortex, hippocampus, and amygdala. Candesartan reduced the associated anorexic effects, and ameliorated associated body weight loss and anxiety. Direct anti-inflammatory effects of candesartan were also documented in cultured rat microglia, cerebellar granule cells, and cerebral microvascular endothelial cells. ARBs are widely used in the treatment of hypertension and stroke, and their anti-inflammatory effects contribute to reduce renal and cardiac failure. Our results indicate that these compounds may offer a novel and safe therapeutic approach for the treatment of brain disorders.

However the underlying mechanism may indeed be (yet again) activating PPAR γ.

Involvement of PPAR-γ in the neuroprotective and anti-inflammatory effects of angiotensin type 1 receptor inhibition: effects of the receptor antagonist telmisartan and receptor deletion in a mouse MPTP modelof Parkinson's disease


This paper suggests that the effect of Candesartan on microglia is :-


"Several recent studies have shown that angiotensin type 1 receptor (AT1) antagonists such as candesartan inhibit the microglial inflammatory response and dopaminergic cell loss in animal models of Parkinson's disease. However, the mechanisms involved in the neuroprotective and anti-inflammatory effects of AT1 blockers in the brain have not been clarified. A number of studies have reported that AT1 blockers activate peroxisome proliferator-activated receptor gamma (PPAR γ). PPAR-γ activation inhibits inflammation, and may be responsible for neuroprotective effects, independently of AT1 blocking actions."

Primary Autism Dysfunctions

I define Primary Autism Dysfunctions as those core dysfunctions that are always present.

So in the case of Monty, aged 11 with ASD, the primary dysfunctions include:-

·        GABA_A dysfunction, due to over expression of NKCC1,  leading to excitatory imbalance

·        Oxidative stress

In some other people the primary dysfunctions are quite different:-

·        Mitochondrial disease

·        etc...

I think that most aggressive behavior resulting from these dysfunctions can be traced back to communication problems and frustration.  So if the person is non-verbal and cannot get what he/she wants, aggression may follow; or if the person has pain and cannot understand it or seek help he may lash out at his care giver.

Secondary Autism Dysfunctions

I define Secondary Autism Dysfunctions as additional dysfunctions that can appear and disappear over time, these are my "flare-ups".

These dysfunctions can be more disabling that the Primary Autism Dysfunctions and it appears these are the dysfunctions that may trigger un-prompted self-injury and other random aggression.

These secondary dysfunctions can be so strong that they completely outweigh the primary dysfunction, giving the effect that the treatment for the primary dysfunction has “stopped working”.

It appears that many  Secondary Autism Dysfunctions are linked to an “over activated immune system”.  It does appear that from the research that activated microglia is an expression of this immune state and we saw one researcher calling the microglia the brain's “immunostat”. 

So in the case of Monty, aged 11 with ASD, the secondary dysfunctions are:-

·        over activated immune system / activated microglia

·        mast cell degranulation as a trigger

·        Il-6 from dissolving milk teeth as a trigger

·        Emotional distress (aged 8, when his long-time assistant left) as trigger (Emotional distress is known to cause oxidative stress)

In other people the secondary dysfunctions may be similar or quite different, for example:-

·        over activated immune system / activated microglia

·        leaky gut with GI problems as a trigger

·        food intolerance as a trigger

·        bacterial infection, with remission while on antibiotics, as a trigger

·        etc …

So I think the trial of Minocycline may have failed because the subjects were only affected by Primary Autism Dysfunctions.

I think the 16 year old aggressive boy in the Candersartan patent most likely had big Secondary Autism Dysfunctions.  The drug reduced microglial activation and so damped the effect of whatever his particular triggers were.

So probably Minocycline should be trialed again, but only in people with autism and regular SIB and aggression.  Success would be measured as a reduction in violent events.

Drugs targeting Primary Autism Dysfunctions should show things like:-

·        Cognitive improvement

·        Increased speech

·        Improved social interactions

·        Reduction in stereotypy

·        Reduction in anxiety (in higher functioning cases)

So I could classify my own interventions as

Primary

·        Bumetanide

·        Low dose Clonazepam

·        NAC

·        Sulforaphane (broccoli)

·        Atorvastatin

·        Potassium

Secondary

·        Verapamil

·        Sytrinol/Tangeretin PPAR-γ agonist for microglia

·        Occasional use of Ibuprofen (anti IL-6 therapy)

·        Quercetin/Azelastine/ Fluticasone Propionate for mast cells

The over activated immune system/activated microglia needs a trigger

Just like a modern plastic explosive is completely harmless to touch and needs the combination of extreme heat and shock wave from a detonator, it appears that the activated microglia, commonly found in autism, is in itself harmless, like Play-Doh, without a trigger.

But with a trigger, you probably know what can happen next.

What about all those failed clinical trials? False Negatives?

So now you not only need to match the trial therapy with the correct sub-type of autism, but you also cannot reliably trial a drug for a Primary Dysfunction, if there is an "active" Secondary Dysfunction.

This is indeed the reason why I do not try new therapies during the summer pollen season.

Perhaps this partly explains why clinical trials in autism always seem to fail.

Diamox & Bumetanide, Ion Channels Nav1.4 and Cav1.1, HypoPP, Autism and Seizures

So you’re going to spend some time at high altitude, climbing mountains or trekking, don’t forgetto pack your Diamox before you leave home, it can save your life.  She is right.

Today’s post links together subjects that have been covered previously.

It does suggest that there are multiple therapies that may be effective in the large sub-group of autism that is characterized by the neurotransmitter GABA being excitatory (E) rather than inhibitory (I).  The science was covered in the earlier very complicated post:-

GABA A Receptors in Autism – How and Why to Modulate Them

The growing list of potential therapies is:-

·        Bumetanide (awaiting funding for Stage 3 clinical trials in humans)

·        Micro-dose Clonazepam (trials in mouse models of autism)

·        Diamox (off-label use in autism)

·        Potassium Bromide  - to be covered in a later post (in use for 150 years)

Not surprisingly, all of these drugs also have an effect on certain types of seizure.

The optimal therapy in people with this E/I imbalance will likely be a combination of some of the above.

Periodic paralysis

Periodic paralysis (Hypokalemic periodic paralysis or HypoPP) is a rare condition that causes temporary paralysis that can be reversed by taking potassium.  A similar condition is hypokalemic sensory overload, when someone becomes overwhelmed by lights or sounds, but after taking potassium all goes back to normal. Autistic sensory overload, experienced by most people with autism, can also be reduced by potassium.

Though rare, we know that HypoPP is caused by dysfunction in the ion channels Na_v1.4 and/or Ca_v1.1.

For decades one of the treatments for HypoPP has been a diuretic called Diamox/Acetazolamide.

Other treatments include raising potassium levels using supplements or potassium sparing diuretics.

Bumetanide is a diuretic, but rather than raising potassium levels, it does the opposite.  So I always thought it was odd that bumetanide would have a positive effect on HypoPP.  But the research showed a benefit.

Autism and Channelopathies

We know that autism and epilepsy are associated with various ion channel and transporter dysfunctions (channelopathies).  In a recent post I was talking about Cav1.1 to Cav1.4.

Today we are talking about Cav1.1 and Nav1.4.

We know that Nav1.1 is associated with epilepsy and some autism (Dravet syndrome).

Nav1.4 is expressed at high levels in adult skeletal muscle, at low levels in neonatal skeletal muscle, and not at all in brain

Nav1.1 expression increases during the third postnatal week and peaks at the end of the first postnatal month, after which levels decrease by about 50% in the adult.

We saw with calcium channels that a dysfunction in one of Cav1.1 to Cav1.4 can cause a dysfunction in another dysfunction in another one of Cav1.1 to Cav1.4.

We also so that in autism the change in expression of NKCC1 and KCC2 as the brain matures failed to occur and so in effect they remain immature and therefore malfunction.

So it is plausible that sodium channels may also malfunction in a similar way. 

  

Acetazolamide efficacy in hypokalemic periodic paralysis and the predictive role of genotype

Hypokalemic periodic paralysis (hypoPP) is an autosomal dominant neuromuscular disorder characterized by episodes of flaccid skeletal muscle paralysis accompanied by reduced serum potassium levels. It is caused by mutations in one of two sarcolemmal ion channel genes, CACNA1S and SCN4A1-3 that lead to dysfunction of the dihydropyridine receptor or the alpha sub-unit of the skeletal muscle voltage gated sodium channel Nav1.4. Seventy to eighty percent of cases are caused by mutations of CACNA1S and ten percent by mutations of SCN4A4. 

There are no consensus guidelines for the treatment of hypoPP. Current pharmacological agents commonly used include potassium supplements, potassium sparing diuretics and carbonic anhydrase inhibitors (acetazolamide and dichlorphenamide). Dichlorphenamide is the only therapy for hypoPP to have undergone a randomized double blind placebo controlled cross over trial. This trial showed a significant efficacy of dichlorphenamide in reducing attack frequency but the inclusion criteria were based on clinical diagnosis of hypoPP and not genetic confirmation.

  

Ca_v1.1

Ca_v1.1 also known as the calcium channel, voltage-dependent, L type, alpha 1S subunit, (CACNA1S), is a protein which in humans is encoded by the CACNA1S gene

Na_v1.4

Sodium channel protein type 4 subunit alpha is a protein that in humans is encoded by the SCN4A gene.

The Na_v1.4 voltage-gated sodium channel is encoded by the SCN4A gene. Mutations in the gene are associated with hypokalemic periodic paralysis, hyperkalemic periodic paralysis, paramyotonia congenita, and potassium-aggravated myotonia.

Ranolazine

Ranolazine is an antianginal and anti-ischemic drug that is used in patients with chronic angina. Ranzoline blocks Na+ currents of Nav1.4. Both muscle and neuronal Na+ channels are as sensitive to ranolazine block as their cardiac counterparts. At its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na+ channels. Ranolazine block of open Na+ channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.

Muscle channelopathies:does the predicted channel gating pore offer new treatment insights for hypokalaemic periodic paralysis?

Beneficial effects of bumetanide in a CaV1.1-R528H mouse model of hypokalaemic periodic paralysis

Transient attacks of weakness in hypokalaemic periodic paralysis are caused by reduced fibre excitability from paradoxical depolarization of the resting potential in low potassium. Mutations of calcium channel and sodium channel genes have been identified as the underlying molecular defects that cause instability of the resting potential. Despite these scientific advances, therapeutic options remain limited. In a mouse model of hypokalaemic periodic paralysis from a sodium channel mutation (NaV1.4-R669H), we recently showed that inhibition of chloride influx with bumetanide reduced the susceptibility to attacks of weakness, in vitro. The R528H mutation in the calcium channel gene (CACNA1S encoding CaV1.1) is the most common cause of hypokalaemic periodic paralysis. We developed a CaV1.1-R528H knock-in mouse model of hypokalaemic periodic paralysis and show herein that bumetanide protects against both muscle weakness from low K⁺ challenge in vitro and loss of muscle excitability in vivo from a glucose plus insulin infusion. This work demonstrates the critical role of the chloride gradient in modulating the susceptibility to ictal weakness and establishes bumetanide as a potential therapy for hypokalaemic periodic paralysis arising from either NaV1.4 or CaV1.1 mutations.

Mode of action

The research does state that nobody knows why Diamox is effective in many cases of hypoPP.

My reading of the research has already taken me in a different direction.  While researching the GABA_A receptor that is dysfunctional in some autism, it occurred to me that in addition to targeting the NKCC1 receptor with bumetanide, another way of lowering chloride levels within the cells might well exist.

I suggested in an earlier post that Diamox could be used to target the AE3 exchanger.

What Diamox (acetazolamide) does is lower the pH of the blood in the following way.

Acetazolamide is a carbonic anhydrase inhibitor, hence causing the accumulation of carbonic acid Carbonic anhydrase is an enzyme found in red blood cells that catalyses the following reaction:

hence lowering blood pH, by means of the following reaction that carbonic acid undergoes

In doing so there will be an effect on both AE3 and NDAE, below.  This will change the intracellular concentration of Cl-, and hence give a similar result to bumetanide.

This would also explain the phenomenon cited below that pH affects the excitability of the brain.

Over excitability of the brain is the cause of some of the effects seen as autism and clearly Over excitability of the brain will be the cause of some people’s seizures/epilepsy.

Not surprisingly, then one of the uses of Diamox is to avoid seizures.

Acetazolamide in the treatment of seizures.

  

Anion exchanger 3 (AE3) in autism

Anion exchange protein 3 is a membrane transport protein that in humans is encoded by the SLC4A3 gene. It exchanges chloride for bicarbonate ions.  It increases chloride concentration within the cell.  AE3 is an anion exchanger that is primarily expressed in the brain and heart

Its activity is sensitive to pH. AE3 mutations have been linked to seizures

Characterization of an Epilepsy-associated variant of the AE3 Cl-/HCO3-exchanger

Bicarbonate (HCO₃^-) transport mechanisms are the principal regulators of pH in animal cells. Such transport also plays a vital role in acid-base movements in the stomach, pancreas, intestine, kidney, reproductive organs and the central nervous system.

Two types of chloride transporters are required for GABA_A receptor-mediated inhibition in C. elegans

Abstract

Chloride influx through GABA-gated Cl⁻ channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl⁻ gradient established in part by K⁺–Cl⁻ cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na⁺-driven Cl⁻–HCO₃⁻ exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K⁺–Cl⁻ cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl⁻ extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl⁻ gradient such that Cl⁻ flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl⁻ gradient. Our results show that Na⁺-driven Cl⁻–HCO₃⁻ exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.

NKCC1 and AE3 Appear to Accumulate Chloride in Embryonic Motoneurons

Abstract

During early development, γ-aminobutyric acid (GABA) depolarizes and excites neurons, contrary to its typical function in the mature nervous system. As a result, developing networks are hyperexcitable and experience a spontaneous network activity that is important for several aspects of development. GABA is depolarizing because chloride is accumulated beyond its passive distribution in these developing cells. Identifying all of the transporters that accumulate chloride in immature neurons has been elusive and it is unknown whether chloride levels are different at synaptic and extrasynaptic locations. We have therefore assessed intracellular chloride levels specifically at synaptic locations in embryonic motoneurons by measuring the GABAergic reversal potential (E_GABA) for GABA_A miniature postsynaptic currents. When whole cell patch solutions contained 17–52 mM chloride, we found that synaptic E_GABA was around −30 mV. Because of the low HCO₃⁻ permeability of the GABA_A receptor, this value of E_GABA corresponds to approximately 50 mM intracellular chloride. It is likely that synaptic chloride is maintained at levels higher than the patch solution by chloride accumulators. We show that the Na⁺-K⁺-2Cl⁻ cotransporter, NKCC1, is clearly involved in the accumulation of chloride in motoneurons because blocking this transporter hyperpolarized E_GABA and reduced nerve potentials evoked by local application of a GABA_A agonist. However, chloride accumulation following NKCC1 block was still clearly present. We find physiological evidence of chloride accumulation that is dependent on HCO₃⁻ and sensitive to an anion exchanger blocker. These results suggest that the anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons.

 

Conclusion

So the science does confirm that “chloride accumulation following NKCC1 block was still clearly present”.  This means that bumetanide is likely only a partial solution.

We also see that “anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons” and “that chloride accumulation that is dependent on HCO₃⁻”.

This is a subject of some research, but it is still early days.

Bicarbonate homeostasis in excitable tissues: role of AE3 Cl−/HCO3−exchanger and carbonic anhydrase XIV interaction

  

I suggest that Diamox, via its effect on HCO₃⁻, may affect anion exchanger AE3 and further reduce chloride accumulation within cells.  This may have a further cumulative effect on GABA.

As we saw earlier, bumetanide does indeed shift GABA from excitatory to inhibitory in people who neurons remain in an immature state (like those of a typical two week old baby).  To my surprise, the use of micro-dose Clonazepam, as proposed by Professor Catterall, but in addition to Bumetanide, has a further effect on GABA’s excitatory/inhibitory imbalance.

Taken together this would highlight the possible further benefit of Diamox.

Normal blood pH is tightly regulated between 7.35 and 7.45.  I do wonder if perhaps in some people with autism, the pH of their blood is slightly elevated (alkaline), this would contribute to excitability of the brain.

Since Diamox increases the oxygen carrying capacity of the blood, I further wonder if this additional oxygen may also be beneficial in some cases.  Since some people are adamant that hypobaric oxygen therapy has beneficial (although not sustained) effects in autism, surely a better treatment would be Diamox?

Since the body is controlled via so-called feedback loops, perhaps in a small subset of people with autism who respond to extra O₂, they actually have blood pH that is higher than 7.45.  In which case measuring blood pH would be a biomarker of who would respond to hypobaric oxygen therapy.  Not surprisingly then, trials of hypobaric oxygen therapy in autism fail, because most of the trial subjects do not have elevated blood pH.

  

So there are many reasons that Diamox should be trialed in autism.  I did find one (DAN) doctor currently using it, but they do not really explain why.

Biomedical Treatment of the Young Adult with ASD