Learning from GABAa Dysfunction in Huntington’s Disease – useful ideas for Autism therapies?

Today’s post is really for the regular readers of this blog who are interested in the GABA switch and Bumetanide. It is not light reading.  We see how advanced some Taiwanese researchers are in their understanding of GABA_A dysfunctions in Huntington’s Disease.

Taipei 101, briefly the world’s tallest building

It is an excellent paper and much of it is applicable to autism. There are some omissions, but you will struggle to find a more complete paper.

They even go into the detail of altered the sub-unit expression of GABA_A receptors that occurs as the disease progresses. I think that correcting sub-unit miss-expression has great potential in treating some autism.

Huntington’s is an inherited brain disorder that first manifests itself around the age of 40 and then progresses for the next 15 to 20 years.

Much autism is present prior to birth but there is a progression that occurs as the brain develops in early childhood. Some people do seem to be entirely typical at birth and only around 2 years old develop symptoms. After 5 years old you cannot really develop “autism”, just the symptoms might not get noticed till later in life.

Schizophrenia only develops in early to mid-adulthood.

It is surprising to many people that such varied disorders share some similar aspects of biology.

In terms of practical interventions, in today’s paper these include:       

·        Inhibition of NKCC1 (bumetanide)

·        Activation of KCC2 (N-Ethylmaleimide)

·        Enhancer of CKB (creatine)

·        Inhibitor of WNK/SPAK

·        Activation of extra-synaptic GABAa receptors (taurine, progesterone)

·        Activation of synaptic GABAa receptors (zolpidem, alprazolam)

·        Inhibition of GABA transport mechanism (Tiagabine)

One thing to note is that activating GABAa receptors may well have a negative effect in some people.

Sub-unit specific therapies, like very low dose clonazepam targeting α3, are not mentioned in this paper, nor is the role of GABAb on NKCC1/KCC2 expression.

We are familiar with Bumetanide as an NKCC1 blocker intervention in autism, but looking at the list there are other common autism therapies (creatine and taurine) and the female hormone progesterone. We come upon the beneficial effect of female hormones on a regular basis in this blog (estradiol, pregnenalone, progesterone …).  We even saw how a sub-SSRI dose of Prozac increases the amount of the neurosteroid 3α-hydroxy-5α-pregnan-20-one (Allo) that potently, positively, and allosterically modulates GABA action at GABA_A receptors. Progesterone is converted to Allo in the body.

 

Here is the excellent paper on Huntington’s:-

Insights into GABAAergic system alteration in Huntington’s disease

                                                                                                               

An overview of the g-aminobutyric acid (GABA) signalling system. (a) GABA homeostasis is regulated by neurons and astrocytes. GABA is synthesized by GAD65/67 from glutamate in neurons, while astrocytic GABA is synthesized through MAOB. The release of GABA is mediated by membrane depolarization in neurons and Best1 in astrocytes. The reuptake of GABA is mediated through GAT1 in neurons and GAT3 in astrocytes. The metabolism of GABA is mediated by GABA-T in neurons and astrocytes. The reuptake of GABA in astrocytes is further transformed into glutamine via the TCA cycle and glutamine synthetase (GS). The glutamine is then transported to neurons and converted to glutamate for regeneration of GABA.

(b) GABAA receptors are heteropentameric complexes assembled from 19 different subunits. The compositions of different subunits determines the subcellular distributions and functional properties of the receptors. Phasic inhibition is mediated via the activation of synaptic GABAA receptors following brief exposure to a high concentration of extracellular GABA. Tonic inhibition is mediated via the activation of extrasynaptic GABAA receptors by a low concentration of ambient GABA.

c) The excitatory inhibitory response of GABA is driven by the chloride gradient across cell membranes, which can be determined via two cation–chloride cotransporters (NKCC1 and KCC2). The high expression of NKCC1 during the developmental stage maintains higher intracellular [Cl2] via chloride influx to the cell. The activation of GABAA receptors at an early developmental stage results in an outward flow of chloride and an excitatory GABAergic response. As neurons mature, the high expression of KCC2 maintains lower intracellular [Cl2] via chloride efflux out of the cell. The activation of GABAA receptors on mature neurons results in the inward flow of chloride and an inhibitory GABAergic response.

An excerpt showing data on sub-unit misexpression in different parts of the brain at different stages of the disease

5.2. Modulation of chloride homeostasis via cation – chloride cotransporters

Emerging evidence suggests that chloride homeostasis is a therapeutic target for HD. Pharmacological agents that target cation–chloride cotransporters (i.e. NKCC1 or KCC2) therefore might be used to treat HD (figure 3b). Of note, dysregulation of cation–chloride cotransporters and GABA polarity was associated with several neuropsychiatric disorders [70,134–139] (reviewed in [27,140]). Such abnormal excitatory GABAA receptor neurotransmission can be rescued by bumetanide, an NKCC1 inhibitor that decreases intracellular chloride concentration. Bumetanide is an FDA-approved diuretic agent that has been used in the clinic. It attenuates many neurological and psychiatric disorders in preclinical studies and some clinical trials for traumatic brain injury, seizure, chronic pain, cerebral infarction, Down syndrome, schizophrenia, fragile X syndrome and autism (reviewed in [141]). Daily intraperitoneal injections of bumetanide also restored the impaired motor function of HD mice. The effect of bumetanide is likely to be mediated by NKCC1 because genetic ablation of NKCC1 in the striatum also rescued the motor deficits in R6/2 mice. This study uncovered a previously unrecognized depolarizing or excitatory action of GABA in the aberrant motor control in HD. In addition, chronic treatment with bumetanide also improved the impaired memory in R6/2 mice [69], supporting the importance of NKCC1 in HD pathogenesis. Owing to the poor ability of bumetanide to pass through the blood–brain barrier, further optimization of bumetanide and other NKCC1 inhibitors is warranted [142,143]. Disruption of KCC2 function is detrimental to inhibitory transmission and agents to activate KCC2 function would be beneficial in HD. However, no agonist of KCC2 has been described until very recently [144,145]. A new KCC2 agonist (CLP290) has been shown to facilitate functional recovery after spinal cord injury [145]. It would be of great interest to evaluate the effect of KCC2 agonists on HD progression. Another KCC2 activator, CLP257, was found to increase the cell surface expression of KCC2 in a rat model of neuropathic pain [146]. Post-translational modification of KCC2 by kinases may modulate the function of KCC2. The WNK/ SPAK kinase complex, composed of WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase), is known to phosphorylate and stimulate NKCC1 or inhibit KCC2 [147]. Thus, compounds that inhibit WNK/SPAK kinases will result in KCC2 activation and NKCC1 inhibition. Some compounds have been noted as potential inhibitors of WNK/SPAK kinases and need to be further tested for their effects on cation –chloride cotransporters [148–150]. An alternative mechanism to activate KCC2 is manipulation of its interacting proteins (e.g. CKB [65,66]). Because CKB could activate the function of KCC2 [65,66], CKB enhancers may increase the function of KCC2. In HD, reduced expression and activity of CKB is associated with motor deficits and hearing impairment [68,88]. Enhancing CKB activity by creatine supplements ameliorated the motor deficits and hearing impairment of HD mice. It is worthwhile to further investigate the interaction of KCC2 and CKB in GABAergic neurotransmission and motor deficits in HD. The depolarizing GABA action with altered expression levels of NKCC1 or KCC2 is associated with neuroinflammation in HD brains [32,69]. Blockade of TNF-a using Xpro1595 (a dominant negative inhibitor of soluble TNF-a) [151] in vivo led to significant beneficial effects on disease progression in HD mice [152] and reduced the expression of NKCC1. It would be of great interest to test the effect of other anti-inflammatory agents [153] on the function and expression of NKCC1 and GABAergic inhibition. Neuroinflammation is implicated in most neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease [154,155], and the interaction of cation–chloride cotransporters and neuroinflammation in GABAergic neurotransmission may also play a critical role in other neurodegenerative diseases.

Figure 2. Molecular mechanism(s) underlying the abnormal GABAAergic system in HD. (a) In the normal condition, adult neurons express high KCC2 and few NKCC1 to maintain the lower intracellular chloride concentration, which results in an inward flow of chloride when GABAA receptors are activated. Astrocytes function normally for the homeostasis of glutamate, potassium and glutamate/GABA-glutamine cycle. (b) In Huntington’s disease, reduced GABAA receptor-mediated neuronal inhibition is associated with enhanced NKCC1 expression and a decreased expression in KCC2 and membrane localized GABAA receptors. The dysregulated GABAAergic system might be caused by mutant HTT, excitotoxicity, neuroinflammation or other factors. Mutant HTT in neurons alters the transcription of genes (GABAAR and KCC2) through interactions with transcriptional activators (SP1) and repressors (REST/NRSF). Mutant HTT in neurons also disrupts the intracellular trafficking of GABAARs to the cellular membrane. HD astrocytes have impaired homeostasis of extracellular potassium/glutamate (due to deficits of astrocytic Kir4.1 channel and glutamate transporters, Glt-1) and cause neuronal excitability, which might be related to the changes of KCC2, NKCC1 and GABAAR. The activity of KCC2 could be affected through its interacting proteins, such as CKB and mHTT. Neuroinflammation, which is evoked by the interaction of HD astrocyte and microglia, enhances NKCC1 expression in neurons at the transcriptional level through an NF-kB-dependent pathway. HD astrocytes also have compromised astrocytic metabolism of glutamate/GABA–glutamine cycle that contributes to lower GABA synthesis.

Notably, neuroinflammation and the GABA neurotransmitter system are reciprocally regulated in the brain (reviewed in [104,105]). Specifically, neuroinflammation induces changes in the GABA neurotransmitter system, such as reduced GABAA receptor subunit expression, while activation

of GABAA receptors likely antagonizes inflammation.

TNF-a, a proinflammatory cytokine, induces a downregulation of the surface expression of GABAARs containing a1, a2, b2/3 and g2 subunits and a decrease in inhibitory synaptic strength in a cellular model of hippocampal neuron culture [106]. The same group further demonstrated that protein phosphatase 1-dependent trafficking of GABAARs was involved in the TNF-a evoked downregulation of GABAergic neurotransmission [107]. Upregulation of TNF-a also negatively impacts the expression of GABAAR a2 subunit mRNA and thus decreases the presynaptic inhibition in the dorsal root ganglion in a rat experimental neuropathic painmodel [108]. Conversely, blockade of central GABAARs in mice by aGABAAR antagonist increased both the basal and restraint stress-induced plasma IL-6 levels [109]. Inhibition of GABAAR activation by picrotoxin increased the nuclear translocation of NF-kB in acute hippocampal slice preparations [110]. Collectively, neuroinflammation

weakens the inhibitory synaptic strength in neurons, at least partly, through the reduction of GABAARs.

The reduced expression and function of GABAARs may further increase inflammatory responses. It remains elusive whether the same mechanism occurs in the inflammatory environment in HD brains.

hyperexcitability resulting from deficiency of astrocytic Kir4.1 might have also contributed to neuronal NKCC1 upregulation and altered GABAergic signalling in HD brains.

Figure 3. Strategy to target (a) GABAAR and (b) cation–chloride cotransporters as potential therapeutic avenues. (a) The GABAergic system is influenced directly by agents that (1) target synaptic GABAAR, (2) increase tonic GABA current or interfere with synaptic GABA concentrations via a reduction of GABA reuptake (3), and (4) block GABA metabolism.

5.1. Modulating the GABAA receptor as a therapeutic target

In view of the presently discovered HD-related deficit in the GABA system, the question arises whether HD patients can benefit from drugs that stimulate the GABA system (figure 3a). HD patients suffer from motor abnormalities and

non-motor symptoms, including cognitive deficits, psychiatric symptoms, sleep disturbance, irritability, anxiety, depression and an increased incidence of seizures [74,77,116,117].

Seizures are a well-established part of juvenile HD but no more prevalent in adult-onset HD than in the general population [73,74,118]. Several pharmacological compounds can enhance inhibitory GABAergic neurotransmission by targeting GABAAR and thereby producing sedative, anxiolytic, anticonvulsant and muscle-relaxant effects. A recent study demonstrated that zolpidem, a GABAAR modulator that enhances GABA inhibition mainly via the a1-containing GABAA receptors, corrected sleep disturbance and electroencephalographic abnormalities in symptomatic HD mice (R6/2) [119]. Alprazolam, a benzodiazepine-activating GABA receptor, reversed the dysregulated circadian rhythms and improved cognitive performance of HD mice (R6/2) [120].

In addition, progesterone, a positive modulator of GABAAR, significantly reversed the behavioural impairment in a 3-nitropropionic acid (3-NP)-induced HD rat model [121]. Apart from modulating the activity of the GABAergic system by interfering directly with the receptor, pharmacological agents can also interfere with synaptic GABA concentrations. Tiagabine, a drug that specifically blocks the GABA transporter (GAT1) to increase synaptic GABA level,was found to improve motor performance and extend survival inN171-82Q and R6/2 mice [122]. It is also worth evaluating whether vigabatrin, a GABA-T inhibitor that blocksGABAcatabolismin neurons and astrocytes [123], plays a role in the compromised astrocytic glutamate–GABA–glutamine cycling [56]. Interestingly, taurine exerted GABAA agonistic and antioxidant activities in a 3-NP HD model and improved locomotor deficits and increased GABA levels [124]. However, several early studies failed to provide the expected benefits of GABA analogues in slowing disease progression in HD patients [125–127]. For example, gaboxadol, an agonist for the extrasynaptic d-containing GABAA receptor, failed to improve the decline in cognitive and motor functions of five HD patients during a short two-week trial, but it caused side effects at the maximal dose [125]. Interestingly, although treatment with muscimol (a potent agonist of GABA receptors) did not improve motor or cognitive deficits in 10HDpatients, it did ameliorate chorea in the most severely hyperkinetic patient [126]. The therapeutic failure of GABA stimulation in early clinical trials does not argue against the importance of GABAergic deficits in HD pathogenesis. The alteration of GABAergic circuits plays a primary role or is a compensatory response to excitotoxicity, and it may contribute to HD by disrupting the balance between the excitation and inhibition systems and the overall functions of neuronal circuits. Because the subunits of the GABAA receptor are brain region- or neuron subtypespecific, the choice of drugs may have distinct effects on the brain region or neuronal population targeted [128–130]. For example, the expression of GABAAR subunits is differentially altered in MSNs and other striatal interneurons in HD 54,60]. The early involvement of D2-expressing MSNs can cause chorea [131], while dysfunctional PV-expressing interneurons can cause dystonia in HD patients [132]. Specific alteration in neuronal populations and receptor subtypes during HD progression needs to be taken into consideration when treating the dysfunction of GABAergic circuitry.

Notably, striatal tonic inhibition mediated by the dcontaining GABAARs may have neuroprotective effects against excitotoxicity in the adult striatum [63]. Because the reductions in d-containing GABAARs and tonic GABA currents in D2-expressing MSNs have been observed in early HD [32,39,40,54,61], it would be of great interest to evaluate the effects of several available compounds, such as alphaxalone and ganaxolone [133], that target d-containing GABAARs, in animal models of HD.

(b) GABAAR-mediated signalling in HD neurons is depolarizing due to the high intracellular chloride concentration caused by high NKCC1 expression and low KCC2 expression. Rescuing the function of cation–chloride cotransporters can occur via (1) inhibition of NKCC1 activity using bumetanide, (2, 3) increase in KCC2 function using a KCC2 activator or CKB enhancer, and (4) inhibitors of WNK/SPAK kinases.

5.2. Modulation of chloride homeostasis via cation–chloride cotransporters

Emerging evidence suggests that chloride homeostasis is a therapeutic target for HD. Pharmacological agents that target cation–chloride cotransporters (i.e.NKCC1 orKCC2) therefore might be used to treat HD (figure 3b). Of note, dysregulation of cation–chloride cotransporters and GABA polarity was associated with several neuropsychiatric disorders [70,134–139] (reviewed in [27,140]). Such abnormal   receptor neurotransmission can be rescued by bumetanide, an NKCC1 inhibitor that decreases intracellular chloride concentration. Bumetanide is an FDA-approved diuretic agent that has been used in the clinic. It attenuates many neurological and psychiatric disorders in preclinical studies and some clinical trials for traumatic brain injury, seizure, chronic pain, cerebral infarction, Down syndrome, schizophrenia, fragile X syndrome and autism (reviewed in [141]). Daily intraperitoneal injections of bumetanide also restored the impaired motor function ofHDmice (R6/2, Y-T Hsu,Y-GChang, Y-CLi, K-YWang, H-MChen, D-J Lee, C-HTsai, C-C Lien,YChern 2018, personal communication). The effect of bumetanide is likely to be mediated by NKCC1 because genetic ablation of NKCC1 in the striatum also rescued the motor deficits in R6/2 mice (Y-T Hsu, Y-G Chang, Y-C Li, K-Y Wang, H-M Chen, D-J Lee, C-H Tsai, C-C Lien, Y Chern 2018, personal communication). This study uncovered a previously unrecognized depolarizing or excitatory action of GABA in the aberrant motor control in HD. In addition, chronic treatment with bumetanide also improved the impaired memory in R6/2 mice [69], supporting the importance of NKCC1 in HD pathogenesis. Owing to the poor ability of bumetanide to pass through the blood–brain barrier, further optimization of bumetanide and other NKCC1 inhibitors is warranted [142,143].

Disruption of KCC2 function is detrimental to inhibitory transmission and agents to activate KCC2 function would be beneficial in HD. However, no agonist of KCC2 has been described until very recently [144,145]. A new KCC2 agonist (CLP290) has been shown to facilitate functional recovery after spinal cord injury [145]. It would be of great interest to evaluate the effect of KCC2 agonists on HD progression. Another KCC2 activator, CLP257, was found to increase the cell surface expression of KCC2 in a rat model of neuropathic pain [146]. Post-translational modification of KCC2 by kinases may modulate the function of KCC2. The WNK/SPAK kinase complex, composed of WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase), is known to phosphorylate and stimulate NKCC1 or inhibit KCC2 [147]. Thus, compounds that inhibit WNK/SPAK kinases will result in KCC2 activation and NKCC1 inhibition.

Some compounds have been noted as potential inhibitors of WNK/SPAK kinases and need to be further tested for their effects on cation–chloride cotransporters [148–150]. An alternative mechanism to activate KCC2 is manipulation of its interacting proteins (e.g. CKB [65,66]). Because CKB could activate the function of KCC2 [65,66], CKB enhancers may increase the function of KCC2. In HD, reduced expression and activity of CKB is associated with motor deficits and hearing impairment [68,88]. Enhancing CKB activity by creatine supplements ameliorated the motor deficits and hearing impairment of HD mice. It is worthwhile to further investigate the interaction of KCC2 and CKB in GABAergic neurotransmission and motor deficits in HD. The depolarizing GABA action with altered expression levels of NKCC1 or KCC2 is associated with neuroinflammation in HD brains [32,69]. Blockade of TNF-a using Xpro1595 (a dominant negative inhibitor of soluble TNF-a) [151] in vivo led to significant beneficial effects on disease progression in HD mice [152] and reduced the expression of NKCC1It would be of great interest to test the effect of other anti-inflammatory agents [153] on the function and expression of NKCC1 and GABAergic inhibition. Neuroinflammation is implicated in most neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease [154,155], and the interaction of cation–chloride cotransporters and neuroinflammation in GABAergic neurotransmission may also play a critical role in other neurodegenerative diseases.

Chloride extrusion enhancers as novel therapeutics for neurological diseases

Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline–alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na⁺-Cl⁻ cotransporter (NCC) and Na⁺-K⁺-2Cl⁻ cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.

Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy.

WNKs (with-no-lysine kinases) are the causative genes of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), and form a signal cascade with OSR1 (oxidative stress-responsive 1)/SPAK (STE20/SPS1-related proline/alanine-rich protein kinase) and Slc12a (solute carrier family 12) transporters. We have shown that this signal cascade regulates blood pressure by controlling vascular tone as well as renal NaCl excretion. Therefore agents that inhibit this signal cascade could be a new class of antihypertensive drugs. Since the binding of WNK to OSR1/SPAK kinases was postulated to be important for signal transduction, we sought to discover inhibitors of WNK/SPAK binding by screening chemical compounds that disrupt the binding. For this purpose, we developed a high-throughput screening method using fluorescent correlation spectroscopy. As a result of screening 17000 compounds, we discovered two novel compounds that reproducibly disrupted the binding of WNK to SPAK. Both compounds mediated dose-dependent inhibition of hypotonicity-induced activation of WNK, namely the phosphorylation of SPAK and its downstream transporters NKCC1 (Na/K/Cl cotransporter 1) and NCC (NaCl cotransporter) in cultured cell lines. The two compounds could be the promising seeds of new types of antihypertensive drugs, and the method that we developed could be applied as a general screening method to identify compounds that disrupt the binding of two molecules.

N-Ethylmaleimide increases KCC2 cotransporter activity by modulating transporter phosphorylation

K⁺/Cl⁻ cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl⁻ levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABA_A) receptors, which are Cl⁻-permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.

                                                                  

Tiagabine (trade name Gabitril) is n anticonvulsant medication produced by Cephalon that is used in the treatment of epilepsy. The drug is also used off-label in the treatment of anxiety disorders and panic disorder.

Tiagabine is approved by U.S. Food and Drug Administration (FDA) as an adjunctive treatment for partial seizures in individuals of age 12 and up. It may also be prescribed off-label by physicians to treat anxiety disorders and panic disorder as well as neuropathic pain (including fibromyalgia). For anxiety and neuropathic pain, tiagabine is used primarily to augment other treatments. Tiagabine may be used alongside selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, or benzodiazepines for anxiety, or antidepressants, gabapentin, other anticonvulsants, or opioids for neuropathic pain.^[4]

Tiagabine increases the level of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, by blocking the GABA transporter 1 (GAT-1), and hence is classified as a GABA reuptake inhibitor (GRI).

Conclusion

Today’s post shows how you need to read well beyond the autism research, not to miss something useful.

Some of today’s suggested therapies for Huntington’s are likely to help some types of autism, but some will certainly have a negative effect in some people.  For example, increasing the amount of GABA in the CNS would do my son no good at all.

The emerging field of drugs that enhance KCC2 should be very beneficial to all those with autism who are bumetanide responders.

Enhancing CKB with creatine is interesting. Creatine is a muscle building supplement used by body builders and some DAN doctors. It does have interactions at high doses.

Arginine and its Derivatives in Cognitive Impairment

Source: Epiphany ASD Blog

Today’s post is very relevant to dementia, relevant to schizophrenia and diabetes and I believe some autism, including that of my son; agmatine is part of his Polypill therapy.

Arginine is highly versatile amino acid and you need the arginine metabolism to be working correctly, particularly in your brain.

Arginine is a widely available from diet and can be produced from citrulline and indirectly from glutamine; so you are unlikely to be deficient in arginine, except in your brain and particularly if you have Alzheimer’s.

In Alzheimer’s it has been shown that the microglia in effect destroy arginine in the brain and this may play a role in what initiates the disease.

Research has suggested that a deficiency in polyamines, another derivative of Arginine, is a feature of dementia.

A deficiency of arginine in the brain will likely cause a deficiency of polyamines.

Your body needs nitric oxide to maintain a healthy blood pressure and this requires arginine to follow the blue line in the above chart towards citrulline and be converted by eNOS.  In most older people this does not happen and oxidative stress appears to be a big part of the problem.

Agmatine – good 

Agmatine has been shown in research to have a benefit in Alzheimer’s.  

This could be due to increased eNOS improving blood flow, an increase in Polyamines, or by reducing insulin resistance in the brain. Recall those studies of intranasal insulin? We had "type 3 diabetes", which was a brain-specific blunting of insulin.

https://www.ncbi.nlm.nih.gov/pubmed/27810390 

"Agmatine administration rescued the reduction in insulin signalling, which in turn reduced the accumulation of Aβ and p-tau in the brain. Furthermore, agmatine treatment also reduced cognitive decline. Agmatine attenuated the occurrence of AD in T2DM mice via the activation of the blunted insulin signal"

Methylarginines – not good

Two by-products of arginine are bad for you in the way Agmatine is good for you.

Nitric Oxide is produced via iNOS, nNos and eNOS. In simple terms we want nitric oxide to be produced in the endothelium, the name for cells that line the interior surface of blood vessels and lymphatic vessels, To achieve this we needs lots of the enzyme eNOS and not much iNOS or nNOS, this is one of Agmatine’s jobs.

Two derivatives of arginine/proteins in the body with very long names are abbreviated to NMMA and ADMA. They both inhibit eNOS and so will restrict blood flow and this will appear as elevated blood pressure.   

The methylarginines NMMA, ADMA, and SDMA are ubiquitous constituents of the main vegetables of human nutrition.

Endogenous methylarginines, N(G),N(G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), N(G)-N('G)-dimethyl-L-arginine (symmetric dimethylarginine; SDMA), and N(G)-monomethyl-L-arginine (monomethyl arginine; NMMA) are supposed to be produced in human body through the methylation of protein arginine residues by protein arginine methyltransferases (PRMT) and released during proteolysis of the methylated proteins. Micromolar concentration of ADMA and NMMA can compete with arginine for nitric oxide synthase (NOS) reducing nitric oxide (NO) formation, whereas SDMA does not. Indeed, increased ADMA and SDMA plasma levels or a decreased arginine/ADMA ratio is related with risk factors for chronic kidney disease and cardiovascular disease. To the best of our knowledge the exogenous presence of methylarginines, like that in fruits and vegetables, has never been described so far. Here, we report the finding that methylarginines are ubiquitous in vegetables which represent an important part of human daily diet. Some of these vegetables contain discrete amounts of ADMA, SDMA, and NMMA. Specifically, among the vegetables examined, soybean, rye, sweet pepper, broad bean, and potato contain the highest ADMA and NMMA mean levels. Our results establish that the three methylarginines, in addition to being produced endogenously, can also be taken daily through the diet in conspicuous amounts. We propose that the contribution of the methylarginines contained in the vegetables of daily diet should be taken into account when the association between vegetable assumption and their levels is evaluated in clinical studies. Furthermore, a comprehensive understanding on the role of the digestive breakdown process and intestinal absorption grade of the methylarginines contained in vegetables is now needed. 

ADMA

Asymmetric dimethylarginine (ADMA) is a naturally occurring chemical found in blood plasma. It is closely related to L-arginine. ADMA interferes with L-arginine in the production of nitric oxide (NO), a key chemical involved in normal endothelial function and, by extension, cardiovascular health. ADMA inhibits eNOS, which in simple terms is the good NOS, the other two being iNOS and nNOS.

ADMA is considered a marker for vascular disease. 

NMMA (NG-monomethyl-l-arginine, or just called Targinine) 

The following study is very interesting for your older relatives. As we already know oxidative stress is a feature of aging. Many people have high blood pressure in old age. Nitric Oxide (NO) is needed keep blood vessels wide open. In old age (>60) oxidative stress reduces NO availability to nothing. 

Since oxidative stress is reversible (in this study vitamin C was used) you wonder why more older people, particularly with high blood pressure, do not take entioxidants. 

Age-Related Reduction of NO Availability and Oxidative Stress in Humans

A novel finding of the present study is that in normotensive subjects, the reduction in endothelial function associated with aging seems to be mediated by a progressive reduction of NO availability, inasmuch as the inhibiting effect of L-NMMA on acetylcholine-induced vasodilation was progressively impaired by advancing age. It is worth noting that after the age of 60 years, the inhibiting effect of L-NMMA on response to acetylcholine was very weak, suggesting that in aged individuals NO availability is almost totally compromised. To assess the possible role exerted by oxidative stress, we tested the antioxidant vitamin C.¹⁹ Up to the age of 60 years, despite the evident decline in endothelium-dependent vasodilation, vitamin C did not modify the response to acetylcholine. In contrast, in the oldest individuals (age >60 years) characterized by a profound alteration in NO availability, vitamin C not only enhanced the response to the endothelial agonist but also restored the inhibiting effect of L-NMMA on vasodilation to acetylcholine. Thus, in the present study, the use of L-NMMA and vitamin C, never tested before in investigating the mechanisms responsible for the previously demonstrated age-related endothelial dysfunction in humans,¹⁷ seems to indicate that the progressive impairment in endothelium-dependent vasodilation is caused by a progressive alteration of the l-arginine-NO pathway. Only in old age (after ≈60 years) does the production of oxidative stress appear, leading to the complete compromise of NO availability.  

Arginase

Arginase is an enzyme that acts as the catalyst for the reaction.

 arginine + H₂O → ornithine + urea 

People with schizophrenia and also people with diabetes tend to have high levels of Arginase. This will affect how arginine is metabolized. If arginase is increased there is less arginine that can go towards creatine, citrulline or agmatine. 

Going towards citrulline involves the production of nitric oxide NO. Now in schizophrenia we see a reduction in the good type of NO, that produced in the endothelium, the cells that line the interior surface of blood vessels and lymphatic vessels. As a result, we vascular dysfunction in schizophrenia.

Agmatine is also elevated in schizophrenia, which may be one of those feedback loops since agmatine will inhibit iNOS, nNOS while increasing eNOS

So where is there a reduction in Arginine in schizophrenia?

Well it looks like it is creatine which takes the hit.

CREATINE ABNORMALITIES IN SCHIZOPHRENIA AND BIPOLAR DISORDER 

“Patients with schizophrenia had a statistically significant reduction in Cr levels as compared with controls; bipolar disorder patients showed no difference in Cr as compared with controls”

In people with elevated arginase a useful strategy might be to use an arginase inhibitor.

Arginase Inhibitors: A Rational Approach Over One Century.

The next paper highlights the arginase inhibitor I favour, which is L-norvaline. The paper is from Kursk university. Kursk gave its name to the nuclear-powered submarine that was lost in the Barents Sea in 2000 and triggered a new international cooperation to rescue stricken submarines. The Battle of Kursk was the largest tank battle of all time and the final major offensive by the Germans against the Russians in World War 2, where Hitler wanted to cut off a large bulge in the front line and trap a lot of Russians. Thanks to some clever English mathematicians, encrypted German communications were readable and the Russians repositioned their forces in advance, allowing them to counter attack. The Allies then invaded Sicily and that was the end for the Germans in Russia. 

Arginase Inhibitor in the Pharmacological Correction of Endothelial Dysfunction

The present research shows expressed endothelium-protective property of arginase inhibitor, L-norvaline, characterized by decrease of coefficient of endothelial dysfunction and the approached its application to a group of intact animals. In other words, L-norvaline prevents the development of systemic endothelial dysfunctions in L-NAME- and methionine-induced NO deficiency.

Age-induced memory impairment (AMI)

Now we move to Polyamines that are on the bottom left my graphic at the start of this post. Spermidine and Spermine are very beneficial derivatives of arginine that most older people will be lacking. Autophagy is the cellular garbage disposal service that is dysfunction in many neurological disorders. We generally want more autophagy.

Spermidine-triggered autophagy ameliorates memory during aging 

The aging process drives the progressive deterioration of an organism and is thus subject to a complex interplay of regulatory and executing mechanisms. Our understanding of this process eventually aims at the delay and/or prevention of age-related pathologies, among them the age-dependent decrease in cognitive performance (e.g., learning and memory). Using the fruit fly Drosophila melanogaster, which combines a generally high mechanistic conservation with an efficient experimental access regarding aging and memory studies, we have recently unveiled a protective function of polyamines (including spermidine) against age-induced memory impairment (AMI). The flies’ age-dependent decline of aversive olfactory memory, an established model for AMI, can be rescued by both pharmacological treatment with spermidine and genetic modulation that increases endogenous polyamine levels. Notably, we find that this effect strictly depends on autophagy, which is remarkable in light of the fact that autophagy is considered a key regulator of aging in other contexts. Given that polyamines in general and spermidine in particular are endogenous metabolites, our findings place them as candidate target substances for AMI treatment.  

Spermine and spermidine reversed age-related cardiac deterioration in rats

Aging is the most important risk factor for cardiovascular disease (CVD). Slowing or reversing the physiological impact of heart aging may reduce morbidity and mortality associated with age-related CVD. The polyamines, spermine (SP) and spermidine (SPD) are essential for cell growth, differentiation and apoptosis, and levels of both decline with age. To explore the effects of these polyamines on heart aging, we administered SP or SPD intraperitoneally to 22- to 24-month-old rats for 6 weeks. Both treatments reversed and inhibited age-related myocardial morphology alterations, myocardial fibrosis, and cell apoptosis. Using combined proteomics and metabolomics analyses, we identified proteins and metabolites up- or downregulated by SP and SPD in aging rat hearts. SP upregulated 51 proteins and 28 metabolites while downregulating 80 proteins and 29 metabolites. SPD upregulated 44 proteins and 24 metabolites and downregulated 84 proteins and 176 metabolites. These molecules were mainly associated with immune responses, blood coagulation, lipid metabolism, and glutathione metabolism pathways. Our study provides novel molecular information on the cardioprotective effects of polyamines in the aging heart, and supports the notion that SP and SPD are potential clinical therapeutics targeting heart disease                                                               

Spermidine boosts autophagy to protect from synapse aging 

Figure 1. summarizes the suggestion that spermidine-triggered restoration of autophagy protects synapses from age-induced changes, and thus delays the normally occurring decline of memory formation. Given that spermidine is a physiologic, easy administrable substance, future research may consider its supplementation to counter age-dependent dementia.

Spermidine operates directly at presynaptic active zone scaffolds (composed of Brp/bruchpilot protein) to allow for an autophagy-dependent homeostatic regulation of these specializations. In effect, spermidine protects learning efficacy from aging-induced decline.                                      

 Spermidine in health and disease

 Having your longevity and eating too

Although caloric restriction has clear benefits for maximizing health span and life span, it is sufficiently unpleasant that few humans stick to it. Madeo et al. review evidence that increased intake of the polyamine spermidine appears to reproduce many of the healthful effects of caloric restriction, and they explain its cellular actions, which include enhancement of autophagy and protein deacetylation. Spermidine is found in foods such as wheat germ, soybeans, nuts, and some fruits and vegetables and produced by the microbiota. Increased uptake of spermidine has protective effects against cancer, metabolic disease, heart disease, and neurodegeneration. 

Although spermidine induces autophagy and autophagy inhibition curtails many of the health-promoting effects of spermidine, additional mechanisms have been proposed to explain the beneficial effects of spermidine on aging. These potentially autophagy-independent mechanisms include direct antioxidant and metabolic effects on arginine bioavailability and nitric oxide (NO) production. However, it has not been formally determined whether these routes act in a completely autophagy-independent manner or are interrelated with autophagy (in an additive or synergistic way) (see the figure), and it will be important to define actionable molecular targets that explain the beneficial effects of spermidine in diverse pathophysiological settings. In this sense, it will also be of interest to explore synergisms of spermidine with other CRMs that initially act through different mechanisms.

It is a surprise that those long-lived Japanese eat Natto? Also, it is a good source of vitamin K2 and importantly it is an estrogen and so an ERβ agonist.

Not all probiotics are helpful to produce polyamines and one well known probiotic, VSL#3, has been shown to reduce their level. Choose your bacteria very carefully. 

Here the probiotic strain Bifidobacterium animalis subsp. lactis LKM512 is used to increase polyamine production

Longevity in Mice IsPromoted by Probiotic-Induced Suppression of Colonic Senescence Dependent on Upregulation of Gut Bacterial Polyamine Production

Alzheimer’s and Arginine

In a fairly recent study it was suggested that the immune system in the brain is being suppressed and the microglia are slightly mutated along with the over-expression of arginase. Arginase is the enzyme that coverts arginine to ornithine plus urea.

So, in Alzheimer’s there will be a lack of arginine available for its other purposes. 

So, we would expect a lack of creatine, agmatine and citrulline. Along the way we should see less Nitric Oxide.

Based on my graphic above, it would seem that L-Norvaline should improve the outcome in Alzheimer’s mice.

We already know that Agmatine improves Alzheimer’s mice, as we now should expect.

So, my cocktail for an aging mouse would be: - 

·        L-Norvaline (used by body builders)

·        Agmatine (used by body builders)

·        Creatine (used by body builders)

·        Natto/wheatgerm/ LKM512 probiotic

·        Vitamin C or NAC

·        Citrulline (used by body builders)

·        Betanin (an approved food colour additive, see below)

Served with cheese, naturally.

A New Potential Cause for Alzheimer’s: Arginine DeprivatiON

Alzheimer’s study suggests immune cells chew up an important amino acid 

Increasingly, evidence supports the idea that the immune system, which protects our bodies from foreign invaders, plays a part in Alzheimer’s disease. But the exact role of immunity in the disease is still a mystery. A new Duke University study in mice suggests that in Alzheimer’s disease, certain immune cells that normally protect the brain begin to abnormally consume an important nutrient: arginine. Blocking this process with a small-molecule drug prevented the characteristic brain plaques and memory loss in a mouse model of the disease. Published April 15 in the Journal of Neuroscience, the new research not only points to a new potential cause of Alzheimer’s but also may eventually lead to a new treatment strategy. “If indeed arginine consumption is so important to the disease process, maybe we could block it and reverse the disease,” said senior author Carol Colton, professor of neurology at the Duke University School of Medicine, and a member of the Duke Institute for Brain Sciences. The brains of people with Alzheimer’s disease show two hallmarks -- ‘plaques’ and ‘tangles’ -- that researchers have puzzled over for some time. Plaques are the build-up of sticky proteins called beta amyloid, and tangles are twisted strands of a protein called tau. In the study, the scientists used a type of mouse, called CVN-AD, that they had created several years ago by swapping out a handful of important genes to make the animal’s immune system more similar to a human’s. Compared with other mice used in Alzheimer’s research, the CVN-AD mouse has it all: plaques and tangles, behaviour changes, and neuron loss. In addition, the gradual onset of these symptoms in the CVN-AD mouse gave researchers a chance to study its brain over time and to focus on how the disease begins, said the study’s first author Matthew Kan, an MD/PhD student in Colton’s lab. Looking for immune abnormalities throughout the lifespan of the mice, the group found that most immune system components stayed the same in number, but a type of brain-resident immune cells called microglia that are known first responders to infection begin to divide and change early in the disease. The microglia express a molecule, CD11c, on their surface. Isolating these cells and analyzing their patterns of gene activity, the scientists found heightened expression of genes associated with suppression of the immune system. They also found dampened expression of genes that work to ramp up the immune system. “It’s surprising, because [suppression of the immune system is] not what the field has been thinking is happening in AD,” Kan said. Instead, scientists have previously assumed that the brain releases molecules involved in ramping up the immune system, that supposedly damage the brain. The group did find CD11c microglia and arginase, an enzyme that breaks down arginine, are highly expressed in regions of the brain involved in memory, in the same regions where neurons had died. Blocking arginase using the small drug difluoromethylornithine (DFMO) before the start of symptoms in the mice, the scientists saw fewer CD11c microglia and plaques develop in their brains. These mice performed better on memory tests. “All of this suggests to us that if you can block this local process of amino acid deprivation, then you can protect -- the mouse, at least -- from Alzheimer’s disease,” Kan said. DFMO is being investigated in human clinical trials to treat some types of cancer, but it hasn’t been tested as a potential therapy for Alzheimer’s. In the new study, Colton’s group administered it before the onset of symptoms; now they are investigating whether DFMO can treat features of Alzheimer’s after they appear. Does the study suggest that people should eat more arginine or take dietary supplements? The answer is ‘no,’ Colton said, partly because a dense mesh of cells and blood vessels called the blood-brain barrier determines how much arginine will enter the brain. Eating more arginine may not help more get into the sites of the brain that need it. Besides, if the scientists’ theory is correct, then the enzyme arginase, unless it’s blocked, would still break down the arginine. “We see this study opening the doors to thinking about Alzheimer’s in a completely different way, to break the stalemate of ideas in AD," Colton said. "The field has been driven by amyloid for the past 15, 20 years and we have to look at other things because we still do not understand the mechanism of disease or how to develop effective therapeutics

The full study: -

Arginine deprivation and immune suppression in a mouse model of Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) is a critical unsolved question; and although recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes, and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by proinflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c(+) microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.

So Arginine for Alzheimer’s? Not so simple

Eating more arginine is not an effective way to increase the level of arginine in your brain and also the high level of arginase might just soak it all up anyway.

Other science does suggest that there are other ways to increase the amount of arginine in your brain, such as L-citrulline.  We have already seen that we can inhibit arginase with L-norvaline among other things.

Betanin for Alzheimer’s

Since we are on Alzheimer’s, we might as well include another clever idea.

Our reader Tyler highlighted another interesting Alzheimer’s study, which suggests preventing/treating Alzheimer’s with Betanin, the pigment in beet root.

This might sound mad, but is deadly serious. The research showed that Betanin inhibits the formation of the trademark beta-amyloid plaques that define Alzheimer’s. No plaques, no Alzheimer’s.

Vegetable compound could have a key role in ‘beeting’ Alzheimer’s disease

Beetroot has already been featured in this blog; it has numerous health benefits.

To lower blood pressure and increase exercise endurance it is the nitrates that are helpful, but beetroot has numerous other effects; it even increases insulin sensitivity, so is a good choice for diabetics and pre-diabetics.

The Potential Benefits of Red Beetroot Supplementation in Health and Disease

Betanin without the beetroot?

Betanin has such a strong colour it is used commercially as a food colourant, it appears as E162 on the label. In Europe it is called Beetroot red E162 and is inexpensive.

Personally, I take my betanin with the rest of the beetroot. 

Vascular Dementia - before I forget

Vascular dementia is the easiest type of cognitive impairment to understand. Reduced blood flow to the brain, most likely due to reasons including a loss of endothelial nitric oxide, effectively starves the brain. We saw how cocoa flavanols improve blood flow and hence mild cognitive impairment, this is via an NO-dependent mechanism that nobody fully understands. In autism things get more complicated and we saw in earlier posts that we seem to have unstable blood flow rather than just reduced blood flow. Nonetheless, improving cerebral blood flow may well be useful for some people with autism; so more eNOS and not too much arginase, cocoa flavanols may well be beneficial. Antioxidants are hopefully already being taken.

Conclusion

I was surprised just how much in the post can be implemented today with no prescription medication.

It is no surprise that certain diets (Mediterranean/Okinawan) promote not only longevity but also an extended healthy life expectancy.

I think there are some tips here for fine tuning out of balance brains found in autism, schizophrenia and bipolar.

I hope someone trials my cocktail on an Alzheimer’s mouse and a regular older mouse. 

·        L-Norvaline and Citrulline

·        Agmatine

·        Creatine

·        Natto/wheatgerm/ LKM512 probiotic

·        Vitamin C or NAC

·        Betanin


I suspect this cocktail would be more effective than Donepezil or Memantine, neither of which address the underlying cause of Alzheimer's disease. In reality some of the above might not even be needed (e.g. creatine and citrulline).

Agmatine as an alternative for some people who respond to intranasal insulin is an interesting idea. Research seems to have stalled because the preservative in the insulin causes irritation inside the nose.

Note: Creatine deficiency is a known cause of MR/ID/Autism and some types are treatable  https://creatineinfo.org/. It is detectable by Magnetic Resonance Spectroscopy or by measuring creatine levels in plasma and urine. Babies born with creatine deficiency may exhibit hypotonia (floppy baby syndrome) due to weak muscles.