Showing posts with label EPA. Show all posts
Showing posts with label EPA. Show all posts

Thursday 21 February 2019

Clemastine and Depression – Myelin, Microglia or Both? Microglia in Psychiatry, Plus LPC EPA and LPC DHA as promising future therapies

Nibbling on Neurons – This is synaptic pruning

Microglia (red) "nibble" a bit of filopodia (green), the membranous protrusions found primarily on dendritic stretches of developing neurons. Video from Weinhard, et al., Nature Communications, "Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction,"

Today it is the post anticipated by Valentia in Uruguay, where it is currently hot and sunny. For Roger it is still winter and he may not be a fan today’s deep dive into the science, but I think he has already got the message about Clemastine’s potential for myelination, including, and importantly for him, in the peripheral nervous system (i.e. for myelination outside the brain). 
If my posts get too complicated just skip down to the conclusion. I do go a little off-topic looking at EPA and DHA. We also take a deep look at microglia in psychiatric disorders.
Today we see how Clemastine’s benefit in some people may be related to its effect on microglia. Rather strangely, you can very logically justify Clemastine’s amelioration of depression by its effect on myelin and also Clemastine’s known effect on microglia – which is the effect that really matters?
If you take a step back you may recall that Clemastine’s effect on myelination is by making special cells called oligodendrocytes work harder and add myelin to more axons (a nice graphic  of this follows later in this post). Oligodendrocytes are actually in the same class as microglia, they are both glial cells.

Glial Cells
Glial cell have four main functions: (1) to surround neurons and hold them in place; (2) to supply nutrients and oxygen to neurons; (3) to insulate one neuron from another; (4) to destroy pathogens and remove dead neurons. They also play a role in neurotransmission and synaptic connections. 
So glial cells are like little housekeeping cells with added room service. 
Perhaps we should just consider Clemastine as having a therapeutic effect on glial cells.
In the central nervous system (CNS), glial cells include oligodendrocytes, astrocytes, ependymal cells, and microglia, and in the peripheral nervous system (PNS) glial cells include Schwann cells and satellite cells. 
We saw in a previous post that Clemastine also affects those Schwann cells in the peripheral nervous system (PNS) and this is why it might help Roger with myelination outside the brain. Schwann cells do the myelination outside the brain/CNS; the hard to pronounce/remember oligodendrocytes do the myelination inside the brain/CNS.
P2X7 receptors are mainly found in glial cells and immune cells. They play a role in mast cell degranulation. They play a role in neuropathic pain, which is not understood. Neuropathic pain, like bumetanide responsive autism involves the over-expression of NKCC1 and the under-expression of KCC2 leading to elevated intracellular chloride. This suggests that future neuropathic pain drugs could be repurposed for bumetanide responsive autism
Clemastine is known to activate P2X7, so in effect it is an agonist.
Perhaps equally strange, and worth noting, is that when it comes to P2X7, oftentimes Antagonists behaves like an Agonists, so take care before drawing premature conclusions with this receptor. You may need more P2X7 signaling, you might less, or things might well be just fine how they are. This actually applies to most receptors implicated in autism. Different people with autism may respond to the opposite therapy, so best be cautious about making making judgements.
Even with oxidative stress, for some people it is bad thing, while for others it is actually brings benefits (athelites, cancer sufferers etc). In the same person as they age what was good may become bad, and indeed vica versa.  This is why we all stand to benefit from personalized medicine. 

When an Antagonist behaves like an Agonist – (don’t speak too soon about Clemastine/Berberine, Fatherof2)

P2X7 Receptor Antagonists Display Agonist-like Effects on Cell Signaling Proteins

Several agents used as P2X7R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.

This means your P2X7 blocking natural supplement may actually be having the opposite effect to the one you believe.
As I keep repeating in this blog, for all kinds of reasons, it is very easy to get things the wrong way around. So instead of forwards, try reverse – just like if you get your car stuck in snow.
If Clemastine has a bad effect, try Oxatomide, and vica versa. This of course just applies to their effect on P2X7, both of these allergy drugs affect numerous other receptors as well. In both cases we are talking about a fraction of the allergy dose, so not exactly a risky exercise.

Back to Nibbling on Neurons
Since we have looked at synaptic pruning in earlier posts, I have included recent research that, for the first time, actually shows how microglia prune synapses. It is not quite the way researchers had previously expected.
Synaptic pruning goes awry in autism and we know that microglia are activated rather than being ramified/resting.
Our reader Maja wants to put her daughter’s microglia back to work doing the synaptic pruning; if your microglia are in the activated M1 state, this may be impaired.  

Human beings are born with a wealth of synapses, functional contact points between brain cells. Post-mortem studies have shown the brains of newborns show a veritable boom of synaptic connections as they enter childhood. The number of those connections will dramatically decline during adolescence, and researchers think this pruning process is critical to healthy brain development. When the process goes awry, it can lead to incapacitating neurodevelopmental disorders like schizophrenia, as well as neurodegeneration later in life (See “Tracking Neuroinflammation in Development, Neurodegenerative Disease”)

The story of synaptic pruning really rests on post-mortem data. We have tools that can show us evidence that synaptic density is abnormal but that’s not quite the same as being able to say you have evidence that the developmental process of synaptic pruning is the explanation for that. We’ve lacked the tools to properly investigate.”
Cornelius Gross and his team at EMBL hoped to find a way to provide such tools to image what he calls “eating events.”
“The prevailing theory is that microglia were literally eating whole synapses,” he says. “Synapses are so small, and microglia are so dynamic, that most in vivo imaging techniques have not had the time or spatial resolution to see these things.”

They used a combination of light sheet fluorescence microscopy with correlative light and electron microscopy (CLEM) and were able to see that microglia weren’t eating whole connections but “nibbling” off bits and pieces at the point of contact. They were able to visualize microglia send out a thin projection called a filopodia to make contact with a synapse, before doing said “nibbling.”
“This is a selective process. And we saw that the synapse wasn’t fully eliminated—it stayed behind even after it was nibbled. So the microglia eat, but don’t eliminate,” Gross says. “This means that we have to rethink the theory of what microglia are doing when it comes to remodeling these circuits. We believe, instead of removing synapses, they are actually helping to form them. By removing those bits and pieces, they aren’t weakening synapses, they are making space to allow new connections to form.”
“Synaptic pruning, in many ways, is still a big mystery. Microglia are moving all the time. We need to do more studies where we have the time and spatial resolution to see what they are actually doing,” he says. “Now, what we need to find out is what is it about the synapses that get nibbled and those that don’t. What is the ‘eat me’ or ‘don’t eat me’ signal? Once we can better understand these signals, and the logic behind the pruning, we have the possibility of understanding how these cells help guide the brain as it develops.”

Our findings confirm the hypothesis that microglia directly engulf and eliminate synaptic material. However, contrary to previous assumptions, we found no evidence for the phagocytosis of entire synapses. Instead, we observed microglia trogocytosis—or nibbling—of synaptic structures. Importantly, microglia trogocytosis was restricted to presynaptic boutons and axons, with no evidence for elimination of postsynaptic material. Intriguingly, microglia contacts at postsynaptic sites frequently elicited transient filopodia, most of which originated from mature spines. These data support the current hypothesis that microglia can “eat” synaptic material, but point to a more nuanced role for microglia in synapse remodeling that may explain the diverse synaptic alterations observed following the disruption of microglial function.

Instead, our data show that only small fragments (250 nm average diameter, Fig. 3) of the presynaptic compartment are engulfed by microglia. This partial elimination, or trogocytosis (from the Greek trogo: to nibble) has been previously described in immune and amoeboid cells18,26,27 that ingest small parts (< 1 µm) of their targets within a few minutes, a timeframe compatible with our observations (Fig. 4)

BHB and Benfotiamine
I did write about the Chinese research showing how the ketone BHB causes the ramification of microglia, this means switching them from an activated state to a resting (ramified state).
We also saw on earlier occasions of how the antibiotic minocycline has a well known  of switching microglia back to a resting state.
Our guest blogger Seth Bittker wrote this post below in 2016. 

Benfotiamine is a derivative of vitamin B1 that is used as a drug to treat neuropathy in Eastern Europe, Russia and Asia. It is also sold as a supplement.
As Agnieszka has highlighted Benfotiamine has a known effect on calming activated microglia. 

Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.

Depression, Myelin, Mice and Clemastine

Here is the case for Clemastine improving symptoms of depression by enhancing myelination. It seems to work for mice at least.

Altered myelin structure and oligodendrocyte function have been shown to correlate with cognitive and motor dysfunction and deficits in social behavior. We and others have previously demonstrated that social isolation in mice induced behavioral, transcriptional, and ultrastructural changes in oligodendrocytes of the prefrontal cortex (PFC). However, whether enhancing myelination and oligodendrocyte differentiation could be beneficial in reversing such changes remains unexplored. To test this hypothesis, we orally administered clemastine, an antimuscarinic compound that has been shown to enhance oligodendrocyte differentiation and myelination in vitro, for 2 weeks in adult mice following social isolation. Clemastine successfully reversed social avoidance behavior in mice undergoing prolonged social isolation. Impaired myelination was rescued by oral clemastine treatment, and was associated with enhanced oligodendrocyte progenitor differentiation and epigenetic changes. Clemastine induced higher levels of repressive histone methylation (H3K9me3), a marker for heterochromatin, in oligodendrocytes, but not neurons, of the PFC. This was consistent with the capability of clemastine in elevating H3K9 histone methyltransferases activity in cultured primary mouse oligodendrocytes, an effect that could be antagonized by cotreatment with muscarine. Our data suggest that promoting adult myelination is a potential strategy for reversing depressive-like social behaviour. 

Clemastine is a leading candidate for myelin formation, identified from a high-throughput screening using a library containing Food and Drug Administration-approved small compounds (Mei et al., 2014). It has been shown to promote OPC differentiation in vitro and remyelination after demyelinating lesions in mice (Deshmukh et al., 2013; Mei et al., 2014; Li et al., 2015). A recent study has shown a beneficial effect in restoring spatial working memory in mice treated with clemastine following 6 weeks of cuprizone diet, a diet that induces demyelination (Li et al., 2015). Here we hypothesized that enhancing myelination could be beneficial for rescuing social withdrawal behavior in socially isolated mice. The effects of oral clemastine treatment in mice after 8 weeks of social isolation were assessed by social interaction and myelination in the PFC. We also examined epigenetic modifications in both neuron and oligodendrocyte populations and detected a specific effect of repressive histone methylation in oligodendrocytes, but not neurons, by clemastine treatment. The study below suggests a positive effect of enhanced myelination and oligodendrocyte differentiation in reversing depressive-like behavior in adult mice.

SIGNIFICANCE STATEMENT Oligodendrocyte development and myelination are highly dynamic processes influenced by experience and neuronal activity. However, whether enhancing myelination and oligodendrocyte differentiation is beneficial to treat depressive-like behavior has been unexplored. Mice undergoing prolonged social isolation display impaired myelination in the prefrontal cortex. Clemastine, a Food and Drug Administration-approved antimuscarinic compound that has been shown to enhance myelination under demyelinating conditions, successfully reversed social avoidance behavior in adult socially isolated mice. This was associated with enhanced myelination and oligodendrocyte differentiation in the prefrontal cortex through epigenetic regulation. Thus, enhancing myelination may be a potential means of reversing depressive-like social behaviour.

Here we reported that enhanced myelination and OPC differentiation are beneficial for reversing depressive-like behavior in adult mice. Clemastine successfully enhanced myelination and OPC differentiation, and was sufficient to rescue social avoidance behavior in socially isolated mice. Clemastine and other muscarinic antagonists have been recently identified from drug-screening assays to promote OPC differentiation and myelination (Deshmukh et al., 2013; Mei et al., 2014; Li et al., 2015), although the underlying mechanism remains undefined. Subtypes of muscarinic receptors have been shown to be expressed in OPCs and mature oligodendrocytes (De Angelis et al., 2012). One possibility is that clemastine directly acts on these receptors in OPCs by favoring chromatin compaction, which has been shown to play a critical role in OPC lineage progression (Liu et al., 2012, 2015). In fact, clemastine is capable of promoting OPC differentiation (Mei et al., 2014) and activating H3K9 HMTs in cultured primary oligodendrocytes (Fig. 3) in the absence of neuronal or astrocytic signals. Levels of H3K9me3, the outcome of activated HMTs, were specifically enhanced in oligodendrocytes, but not neurons, in the PFC of socially isolated mice, thereby supporting a direct and cell-autonomous effect on oligodendrocyte differentiation. A potentially alternative mechanism of action of clemastine, as a muscarinic receptor antagonist, was suggested by the activation of Akt/mammalian target of rapamycin (mTOR) pathways induced in synaptoneurosomes by scopolamine, a nonselective muscarinic antagonist (Voleti et al., 2013; Navarria et al., 2015). The Akt/mTOR pathway is a well characterized positive regulator of oligodendrocyte differentiation and myelination (Wood et al., 2013; Wahl et al., 2014). Therefore, similar signaling pathways could be activated in oligodendrocytes following clemastine treatment, which directly results in OPC differentiation and myelination.

Lack of social experience has been shown to induce impaired myelination in the PFC of juvenile and adult mice (Liu et al., 2012; Makinodan et al., 2012). It is widely accepted that exposure to stress in rodents altered neuronal activity in the PFC and resulted in depressive-like behavior (Covington et al., 2005, 2010; Veeraiah et al., 2014). Selective activation of neurons in PFC through optogenetic stimulation provided an antidepressant effect in a mouse model of depression (Covington et al., 2010). Increased neuronal activity through optogenetic manipulation in the mouse premotor cortex has been shown to promote myelination and oligodendrogenesis (Gibson et al., 2014). Therefore, a potential explanation for the impaired myelination detected in socially isolated mice could be diminished neuronal activity in the PFC. Here we demonstrated that clemastine-induced adult myelination is sufficient to rescue the depressive-like behavior in socially isolated rodents, thereby potentially overcoming the lack of activity of specific circuitry involving PFC and consequent behavioral deficits. However, one caution should be made when interpreting our results as we cannot exclude the possibility that clemastine-induced myelination and OPC differentiation could be a secondary effect of the drug on the neuronal circuitry. Systemic as well as local infusion of scopolamine had been previously shown to increase neuronal activity in the PFC by altering glutamate transmission and provided an antidepressant effect in the forced swim test of mice (Voleti et al., 2013; Navarria et al., 2015). Therefore, although specific PFC circuitry was not activated by “exogenous” social stimuli, neuronal activity could be altered through clemastine uptake, which in turn resulted in myelination enhancement. The precise mechanism of clemastine-mediated enhanced myelination and OPC differentiation in our model remains to be identified.

In summary, we propose that enhancing oligodendrocyte differentiation and myelination in the adult brain contributes to reverse depressive-like behaviors in mice. Thus, our findings provide a new insight into the role of myelination and oligodendrocyte function in modulating emotional behavior, and might be helpful for designing novel strategies to ameliorate psychiatric symptoms in mental disorders.

Now “Take 2”, for Microglia as the mode of action

Depression, Microglia, Mice and Clemastine

Backgrounds: Abundant reports indicate that neuroinflammatory signaling contributes to behavioral complications associated with depression and may be related to treatment response. The glial cells, especially microglia and astrocytes in brain regions of hippocampus and medial prefrontal cortex (mPFC), are major components of CNS innate immunity. Moreover, purinergic receptor P2X, ligand-gated ion channel 7 (P2X7R) was recently reckoned as a pivotal regulator in central immune system. Besides, it was pointed out that clemastine, a first-generation histamine receptor H1 (HRH1) antagonist with considerable safety profile and pharmacological effect, may suppress immune activation through modulating P2X7R. Herein, we investigated the potential anti-neuroinflammatory effects of clemastine on chronic unpredictable mild stress (CUMS)-induced depressive-like behavior in a mouse model.

Methods: Male BALB/c mice were subjected to CUMS for 4 weeks, some of them were injected with clemastine fumarate solution. After the stress procedure, behavioral tests including Sucrose Preference Tests (SPTs), Tail Suspension Tests (TSTs) and locomotor activities were performed to evaluate depressive-like phenotype. Subsequently, expression of cytokines and microglia-related inflammatory biomarkers were assessed.
Results: In the present research, we found that clemastine significantly reversed both the declination of SPT percentage and the extension of TST immobility durations in depression mouse model without affecting locomotor activity. Also, we observed that clemastine regulated the imbalance of pro-inflammatory cytokines including interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in the hippocampus and serum of depressive-like mice. Additionally, clemastine significantly suppressed microglial M1-like activation specifically in the hippocampus, and also improved hippocampal astrocytic loss. Furthermore, clemastine downregulated hippocampal P2X7R without interfering with the expression of HRH1.
Conclusion: As a safe and efficient anti-allergic agent, clemastine could impressively alleviate stress-related depressive-like phenotype in mice. Further evidence supported that it was because of the potential function of clemastine in modulating the expression of P2X7 receptor possibly independent of HRH1, therefore suppressing the microglial M1-like activation and pro-inflammatory cytokines release in brain regions of hippocampus rather than mPFC.

Totally, we conclude the central immune imbalance which resulted from the activation of microglia and astrocytes may be responsible for depressive-like behavior induced by chronic unpredictable mild stress. When it shifts towards a M1-like proinflammatory polarization, various of deleterious cytokines would be secreted and then interfered with the normal function of central neurons and glial cells, leading to specific behavioral changes. To our knowledge, P2X7 receptor and downstream signaling might be a vital regulator in maintaining the aforementioned equilibration. As a safe and efficient anti-allergic agent, clemastine could significantly ameliorate stress-related depressive-like phenotype in mice. Further evidence supported that it was because of the potential function of clemastine in downregulating P2X7R independent of histamine H1 receptor, therefore suppressing the M1-like microglial activation and inflammatory cytokines release in brain region of hippocampus other than mPFC.  

Now for Microglia in other human psychiatric disorders
This subject is complex; you could consider it as a balancing act as in the graphic below between M1 and M2 states of microglia.  It is more complex because within M2 are various sub-states.
The resting (or ramified, in the jargon) microglia is called M0.


Psychiatric disorders such as schizophrenia and major depressive disorder were thought to be caused by neurotransmitter abnormalities. Patients with these disorders often experience relapse and remission; however the underlying molecular mechanisms of relapse and remission still remain unclear. Recent advanced immunological analyses have revealed that M1/M2 polarization of macrophages plays an important role in controlling the balance between promotion and suppression in inflammation. Microglial cells share certain characteristics with macrophages and contribute to immune-surveillance in the central nervous system (CNS). In this review, we summarize immunoregulatory functions of microglia and discuss a possible role of microglial M1/M2 polarization in relapse and remission of psychiatric disorders and diseases. M1 polarized microglia can produce pro-inflammatory cytokines, reactive oxygen species, and nitric oxide, suggesting that these molecules contribute to dysfunction of neural network in the CNS. Alternatively, M2 polarized microglia express cytokines and receptors that are implicated in inhibiting inflammation and restoring homeostasis. Based on these aspects, we propose a possibility that M1 and M2 microglia are related to relapse and remission, respectively in psychiatric disorders and diseases. Consequently, a target molecule skewing M2 polarization of microglia may provide beneficial therapies for these disorders and diseases in the CNS.

Classically activated microglia (M1 polarized microglia) can produce pro-inflammatory cytokines, reactive oxygen species (ROS), and nitric oxide (NO), implying their contribution to neural network dysfunction in the CNS. On the other hand, alternatively activated microglia (M2 polarized microglia) can express cytokines and receptors that are implicated in inhibiting inflammation and restoring homeostasis.


Hypothetical model of relationship between M1/M2 microglia activities and symptom severity in schizophrenia. (A) In the early stage of schizophrenia, symptoms may be followed by microglial M1 polarization which is induced by neuronal hyperactivation in insula, inferior frontal gyrus, and hippocampus, possible initiating brain regions of the disorder. M1 microglia can produce pro-inflammatory cytokines and remove the damaged nerve fibers by phagocytosis, whereas M2 microglia down-regulate M1 microglial function and restore tissue homeostasis with consequent attenuation of symptoms. (B) If M2 polarization of microglia is insufficient, M1 microglial functions are maintained and induce neural network dysfunctions continuously. Symptom severity may gradually become high according to the frequency of M1 polarization.

Major Depressive Disorder

Possible roles of M1/M2 microglia in neural network functions, activities of monoamine neurons, and symptoms in major depressive disorder. In healthy individuals, prefrontal cortex regulates neural circuitry of mood including amygdala and dopamine neurons projecting from VTA (ventral tegmental area) to NAc (nucleus accumbens) (1, 2). In patients with major depressive disorder, hyperactivation of neural circuitry induces M1 polarization of microglia (3), resulting in dysfunction of nerve fibers between prefrontal cortex and the neural circuitry (4) and hypoactivation of 5-HT neurons projecting from raphe nucleus to prefrontal cortex (5). Dysfunction of prefrontal cortex can reduce activity of dopamine neurons projecting from VTA to NAc (6). Hypoactivation of prefrontal cortex and NAc are associated with depressed mood and loss of interest/pleasure, respectively. M2 microglia restore homeostasis of nerve fibers and 5-HT biosynthesis, recoverig dysfunction of prefrontal cortex and NAc (7, 8).

Molecules to Skew M2 Polarization of Microglia

7.1. Endocannabinoids and Cannabinoid Receptors

Based on these results, it is strongly suggested that 2-AG-CB1 axis contributes to polarization and maintenance of M1 microglia, while 2-AG-CB2 axis acts as a switch from M1 to M2 polarization of microglia (Figure 4). CB2 agonists are known to induce phosphorylation of AMP-activated protein kinase (AMPK), suggesting that the CB2 plays an important role in AMPK-mediated anti-oxidative and cytoprotective effects [119,120,121]. Furthermore, 2-AG is reported to activate PPAR-γ in M2 macrophages [122]. Thus, AMPK may be one of key signal molecules for the switch to M2 polarization. Besides endocannabinoids, adiponectin and ghrelin can induce down-stream signal transduction of their receptors via AMPK and therefore these molecules may be involved in skewing M2 polarization of microglia

7.2. Anti-Inflammatory and Pro-Resolving Lipid Mediators

The analyses of cellular and molecular mechanisms of the resolution of inflammation have revealed the key roles of anti-inflammatory and pro-resolving lipid mediators such as lipoxin A4, resolvin D1, resolvin E1, and protectin D1 [125]. These mediators are mainly biosynthesized from docosahexaenoic acid (DHA) or arachidonic acid by 15-lipoxygenase [125]. Resolvin D1 and lipoxin A4 are known to exhibit an agonistic activity at GPR32 and lipoxin A4 receptor/N‑formyl peptide receptor 2 (ALX/FPR2) [126]. Resolvin D1 up-regulates the levels of micro-RNAs (miR-208a and miR-219) and enhances IL-10 production by peritoneal exudate macrophages in zymosan-induced peritonitis in ALX/FPR2 transgenic mice [126]. Furthermore, it has been reported that resolvin D1 and DHA can induce M2 polarization of macrophages [127] and that ALX/FPR2 is expressed on macrophages and microglia [128]. A double-blind, placebo-controlled clinical studies revealed that the transition rate to psychotic disorder is significant lower in ARMS individuals received with capsules containing DHA and eicosapentaenoic acid (EPA) as compared with placebo-treated controls [129]. Furthermore, ethyl-EPA in combination with antipsychotics has been reported to improve PANSS scores significantly in schizophrenia patients [130]. From these results, it is strongly suggested that anti-inflammatory and pro-resolving lipid mediators such as resolvin D1 and lipoxin A4 play an important role in polarization and maintenance of M2 microglia (Figure 4).

Possible roles of the cannabinoid receptors in M1/M2 polarization of microglia. 2-AG released from M1 microglia promotes production of pro-inflammatory cytokines and mediators by M1 microglia via CB1 and then induces down-regulation of CB1. On the other hand, 2-AG stimulates M2 polarization of microglia via CB2. Subsequently, M2 microglia can produce IL-10 and anti-inflammatory/pro-resolving lipid mediators (resolvin D1 and lipoxin A4). 


Endocannabinoid system

It is found that the anti-inflammatory lipid lipoxin A4 is an endogenous allosteric enhancer of the CB1 cannabinoid receptor. Lipoxin A4 enhance the affinity of anandamide at this receptor to exert cannabimimetic effects in the brain, by allosterically enhancing AEA signaling and thereby potentiating the effects of this endocannabinoid both in vitro and in vivo. In addition to this, lipoxin A4 display a CB1 receptor-dependent protective effect against β-amyloid-induced spatial memory impairment in mice.

In this review, we provide a hypothesis that M1 and M2 phenotypes of microglia are closely related to relapse and remission, respectively, in psychiatric disorders and diseases. M1 polarization of microglia seems to induce dysfunction of the neural network in the CNS. Specifically, it is presumed that M1 microglia-induced dysregulation of prefrontal cortex for the neural circuitries of mood and pain results in symptoms of major depressive disorder, vascular depression, chronic pain, and migraine. M2 polarized microglia presumably attenuate M1 microglia-mediated neuroinflammation by production of anti-inflammatory cytokine, IL-10. On the other hand, further studies on M2 microglial functions are necessary to understand their exact roles in neuroinflammation, because M2 macrophages seem to induce Th2-type inflammatory conditions [131,132]. Since endocannabinoids, adiponectin, ghrelin, or anti-inflammatory/pro-resolving lipid mediators appear to skew M2 polarization of microglia, modulation of these molecules may afford favorable approaches for treatment of vascular depression to reduce a risk for neurocognitive disorders. Consequently, the molecules skewing M2 phenotype of microglia may provide a beneficial therapy to attenuate relapse of psychiatric disorders and diseases.

Note the role of IL-4, Resolvins and Lipoxins in the graphic below

At the M0 state (top), resting microglia function in a surveillance and detection mode, which appears to be regulated by various nuclear receptor pathways and select miRNAs: miR-124, miR-689 and miR-711. Upon detection of a danger or pattern molecule, the resting status is disrupted and transitions to the M1 state (right). The M1 phenotype is the “classic activation” status and prominently induces canonical M1 marker genes, e.g. IL-1β, TNF-α and IL-6. miR-124 and miR-689 are critical in initiation of the transition from the M0 to the M1 state. The M1 phenotype appears to be fully mediated by miR-155, which targets the STAT3 pathway for enabling the M1-phenotype. Later, through transition from M1 to M2 or through direct IL-4 stimulation (dashed line), microglia may enter the M2a status, characterized as an anti-inflammatory and resolution phenotype. As observed with in M1, down-regulation in miR-124 and miR-711 appears to be important for release from the M0-phenotype and transition to the M2 status. The M2a-phenotype appears to rely on induction of miR-145, which may regulate the ETS1 pathway. Lastly, IL-4 signaling is dependent on STAT6, TRIM24, and CREB1 along with select nuclear receptor signaling: PPARα/γ and RARα.

Neuroinflammation is recognised as one of the potential mechanisms mediating the onset of a broad range of psychiatric disorders and may contribute to nonresponsiveness to current therapies. Both preclinical and clinical studies have indicated that aberrant inflammatory responses can result in altered behavioral responses and cognitive deficits. In this review, we discuss the role of inflammation in the pathogenesis of neuropsychiatric disorders and ask the question if certain genetic copy-number variants (CNVs) associated with psychiatric disorders might play a role in modulating inflammation. Furthermore, we detail some of the potential treatment strategies for psychiatric disorders that may operate by altering inflammatory responses.

Potential mechanism of microglia activation in psychiatric disorders. Neuroinflammation is one of the key components of the pathogenic mechanisms underlying several psychiatric disorders and is often associated with microglial activation/dysfunction. Accumulating evidence indicate that M1-like microglia (proinflammatory) are significantly increased in comparison to ramified microglia (resting) and elongated M2-like microglia (anti-inflammatory) phenotypes in disease states. The levels of M1-like microglia in brain predominate and potentially can be associated with the severity of the disease, suggesting an imbalance in M1/M2 phenotype. M1-like microglia are characterized by the expression of MHC class II antigens and by the production of proinflammatory cytokines and nitric oxide synthase (iNOS). Continued production of proinflammatory cytokines can lead to neuronal damage, astrogliosis, plasticity, and cognitive decline. Peripherally derived macrophages and monocytes also participate in the inflammatory response. It is likely that during early stage of disease onset microglia can have phenotypic switch to an alternative state knows as M2-like phenotype, which are characterized by presence of surface markers like Arginase 1 and mannose receptor CD206, leading to resolution of inflammatory response by secretion of anti-inflammatory cytokines. The efficacy of the anti-inflammatory drug targeting M1/M2 balance will significantly depend on therapeutic time window and severity of symptoms associated with the diseases.

5.7. Autism Spectrum Disorder

Little is found on anti-inflammatory treatment in autism spectrum disorder so far. Add-on of celecoxib (up to 300 mg/day) to risperidone treatment in a study on 40 children suffering from autism leads to significant improvements of scores in irritability, social withdrawal, and stereotypy [191]. However, an open-label study in 11 children on treatment with 1.4 mg/kg body weight of minocycline found no clinical improvements after 6 months (measured using Clinical Global Impression Severity Scale, Clinical Global Impression Severity Scale Improvement, and Vineland Adaptive Behavior Scales). IL-8 was found significantly decreased in serum and cerebrospinal fluid, while other cytokines, that is, TNF-α, CD40L, IL-6, IFN-γ, and IL-1β, were unchanged [192].

Treating Depression - Myelin, Microglia or Both?

It looks to me that the answer when treating depression with Clemastine, is likely both improved myelination and shifting microglia away from the M1 state play a key role.
It is amazing that all this is potentially possible from a small dose of Clemastine, an OTC antihistamine.

It is clear that clemastine might well be therapeutic in human schizophrena, where both activated microglia and poor myelination are implicated. We previously saw promise in mouse studies, like this one:-

What about autism?
Impaired myelination and activated microglia are a known feature of autism.

One of those popular autism “protocols” trending on social media, the Nemechek Protocol “was designed to shift the harmful inflammatory microglia into their healthy, repairing mode of behavior (called phenotypic shifting) and allow the brain's natural repair mechanisms to reverse the cumulative brain injury.” Nemecheck recommends a combination of Inulin, Omega 3 oil and olive oil which he says will treat SIBO (Small intestinal bacterial overgrowth), this will reduce the production of harmful propionic acid and shift activated microglia back to M0 and then the brain will repair itself.  I have no idea what percentage of people with autism, ADHD or Alzheimer’s actually respond.
It seems Nemechek recommends a high DHA omega 3 oil. 

Here is a good place to note that while the brain has plenty of DHA it has no EPA and neither standard supplements containing DHA nor EPA can pass through the blood brain barrier.
It is thought that in neurological conditions, including but not limited to Alzheimer’s, the presence of EPA and additional DHA might be therapeutic.  Only recently has the transport mechanism of DHA across the BBB been understood.

You might wonder why omega 3 is being used to treat conditions of the brain such as ADHD, chronic fatigue syndrome and indeed sometimes autism.

We should note that inflammation outside the brain does affect inflammatory markers inside the brain. So reducing omega 6 and increasing omega 3 levels in your blood might well give you some benefit to your brain even though none actually crossed the blood brain barrier.  
The next generation of EPA and DHA will cross the blood brain barrier and who knows what their effect will be. 

In diseases that features low DHA in the brain like Alzheimer’s you need LPC DHA, rather than any of the current products.

Docosahexaenoic acid (DHA) is uniquely concentrated in the brain, and is essential for its function, but must be mostly acquired from diet. Most of the current supplements of DHA, including fish oil and krill oil, do not significantly increase brain DHA, because they are hydrolyzed to free DHA and are absorbed as triacylglycerol, whereas the transporter at blood brain barrier is specific for phospholipid form of DHA.

LPC-EPA, but not free EPA, increased brain DHA 2-fold. Free EPA increased EPA in adipose tissue, and both supplements increased EPA and DHA in the liver and heart. Only LPC-EPA increased EPA and DHA in retina, and expression of BDNF, CREB, and 5-HT1A receptor in the brain. These novel results show that brain EPA can be increased through diet. Because LPC-EPA increased both EPA and DHA in the brain, it may help treat depression as well as neuroinflammatory diseases, such as Alzheimers disease.
Getting enough of the omega 3 fatty acids DHA and EPA into the brain to study their effects on conditions such as Alzheimer's and depression -- which they have been shown to help -- is no easy task. While supplements containing these fatty acids exist, there is scant evidence showing that these supplements actually increase DHA or EPA in the brain. To measurably increase levels of EPA in the brain, a person would have to consume a small glass of it each day, quite possibly with the side effect of smelling like fish.

Now researchers from the University of Illinois at Chicago report that adding a lysophospholipid form of EPA (LPC-EPA) to the diet can increase levels of EPA in the brain 100-fold in mice. The amount of LPC-EPA in the diet required for this increase is rather small for mice -- less than a milligram per day. The human equivalent would amount to less than a quarter of a gram per day.
He reports that providing EPA in the form of lysophospholipid, unlike the type present in fish oil supplements, escapes degradation by pancreatic enzymes which render it unable to pass into the brain.
"It seems that there is a transporter at the blood-brain barrier that EPA must pass through in order to get into the brain, but EPA in fish oil can't get through, whereas LPC-EPA can," Subbaiah said. "You don't have to consume all that much LPC- EPA to have significant increases of EPA show up in the brain, so this could be a way to do rigorous studies on the effects of EPA in humans," Subbaiah said.
Producing LPC-EPA is not difficult

So if you buy Omega 3 oil for brain function it would ideally be LPC (lysophosphatidylcholine). It may not be hard to produce, but nobody currently sells it.

Back to microglia
In simple terms unless you are sick, it is best to have microglia in the M0 state. The M2 state is OK, but the M1 state is bad.

Many immunomodulatory therapies will affect your Microglia.
Azithromycin will shift microglia from M1 to M2.

3.1. Minocycline. Minocycline is a second-generation tetracycline with a variety of nonantibiotic biological effects, such as neuroprotection in experimental models of TBI, ischemia, and neurodegenerative diseases [39]. The anti inflammation effect is the most well-known advantage of the neuroprotective effects of minocycline. A series of studies have demonstrated that minocycline can inhibit microglial activation, using pan-microglial markers in TBI, SCI, SAH, and cerebral ischemia [40–45].

3.2. Etanercept

3.3. Statins.

3.7. Rosiglitazone. As a peroxisome-proliferator-activated receptor- (PPAR-) 𝛾 agonist, rosiglitazone is not only an antidiabetic drug but also a neuroprotective agent, and it has shown various effects in treating brain ischemia [103], TBI [104], and SAH [105]. A study demonstrated rosiglitazone’s ability to attenuate microglia/macrophage activation and neuronal loss after TBI [104]. In mouse models of focal cerebral ischemia and progressive Parkinson’s disease, rosiglitazone showed the ability to promote microglial M2 polarization[103,106].Another PPAR-𝛾 agonist pioglitazone has also been reported to decrease the M1/M2 ratio in experimental Alzheimer’s disease

3.8. Azithromycin. Many macrolide antibiotics might have neuroprotective effects. Among them, azithromycin is an extraordinary drug with the effect of reducing infarct volume, decreasing brain edema, and increasing neurological deficit scores in acute ischemic damage [108]. Additionally, azithromycin had the effect of altering the macrophage phenotype from proinflammatory M1 to alternatively activated M2 cells

It looks like BHB/C8 and Clemastine should all help shift activated microglia back to the resting state, M0. Benfotiamine’s effect is slightly different, but if you had “over-activated microglia” it might reduce the damage they cause. The classic research treatment for activated microglia is the old antibiotic minocycline.  In a small trial at Johns Hopkins in exclusively regressive autism, a 6 month course of minocycline had no benefit.
Clemastine should promote myelination. A further boost might well come from a PDE4 inhibitor like Ibudilast, or indeed Ling’s Pterostilbene.

I have to say that adding BHB/C8 and Clemastine to Monty’s Polypill does produce a cognitive and speech benefit. It does seem that Clemastine itself has an incremental effect.

What about Cannabinoid Receptors CB1 and CB2?
Today’s research does indeed suggest CB1 and CB2 receptors could be used to alter the M0, M1, M2 balance.

Clemastine as a potential intervention in children with autism
It is notable that among the “early adopters” of a clemastine trial are some of our doctor readers.  Why is that?

The key point for any therapy for children with autism has to be safety. If a decades-old drug has already been used long term in children there is going to be reliable safety information.
Many drugs are going to have some effect, be it positive or negative, on neurological disorders like autism, but that in itself is not sufficient. All drugs can have side effects and many prescription drugs have significant side effects, that is why you need a prescription.

Many supplements also can have negative effects, as recently highlighted by another reader of this blog.

Any thoughts on this new study? 'Cytotoxicity and mitochondrial dysfunction caused by the dietary supplement l-norvaline' Journal: Toxicology in Vitro

Some chronic conditions are treated with drugs that have proved themselves over decades to be well tolerated, but even then there can be people who encounter rare side effects. Some people even react to the colorant used on the outside of the pill.

The attraction of a PolyPill or indeed a PolyPowder, like SpectrumNeeds below, is that you can deliver a combination of different therapies in a convenient way.
The big disadvantage of a Generic PolyPill/PolyPowder is that there will most definitely be people for whom the overall product is beneficial, except for one ingredient to which that person has a negative reaction.

In the case of SpectrumNeeds, a group of MAPS doctors have sat down and created an all-in-one product. Note Dr Frye’s calcium folinate for example.
If you look at the ingredients you will see items for which we have seen in this blog there will be a negative reaction in a substantial number of people. Biotin and vitamin B12 are good examples.

High doses of B vitamins do seem to help some people, but quite often the effect later becomes negative.
Unfortunately, it is the case that you need a personalized pill/powder, however inconvenient that might be.

Having established that the drug does not cause harmful side effects in long term use and that it is beneficial therapeutically, we come to cost and availability.

Here Clemastine, at a modest dose, would be a big winner.
It is cheap, OTC in some countries, and even sold on eBay (from Latvia).

My 120 tablets of Clemastine cost me $20 and at my dose would last 8 months and at Maja’s dose 16 months. Even if marginally effective you might continue.
Years ago I tried BIO 30 propolis as a PAK1 inhibitor. It did seem to have a small positive effect on cognition, but it is expensive, comes from New Zealand and is a liquid. Some other people have found it beneficial for their “autism”. I did not continue with BIO 30 propolis.  We have to wait for Roche to commercialize FRAX 486 as a drug to inhibit PAK1.

SpectrumNeeds will cost $77 plus tax and shipping. For a teenager, that will last you 20 days.
If SpectrumNeeds significantly improves your case of “autism” that would be great. I think someone with severe autism is going to need more, much more.

The key elements of Monty’s PollyPill are cheap generic drugs. The most expensive part is the NAC supplement. The cheapest part is the tiny dose of Clonazepam which costs less than 1 cent a day.
My current PolyPill costs me about $2.20 a day (half of the total cost is NAC).

The daily cost of my 6+ months BHB/C8 trial is another $2 a day.
The Clemastine trial dose costs 17 cents a day.

If it can stay below $5 a day, the “final” version of my PolyPill will end up costing like a large cup of Starbucks coffee. Our Australian reader Liz has a daughter who fortunately responds well to the PolyPill and I recall, as well as being very frank about dealing with her doctor, she highlighted how “affordable” the PolyPill really is.
“Peter has made poly pill very affordable which makes it so accessible to many families.”
I did show her comments at home, at this point I recall being asked “Peter, what are you selling?”

Peter is actually not selling anything. Everything is already available. Your PolyPill is likely to be different to the one that fortunately works not just for Monty but Liz in Australia and Thomas in Greece etc. Thomas is indeed a common Greek name, Θωμάς; he was one of the 12 apostles after all.
The costs of interventions do matter, even though it is rarely a subject raised in the comments. I hate to think how much any future new drugs for autism will cost. A simple to produce, but patented, new drug for cystic fibrosis costs $150,000 a year and not surprisingly some government funded health systems refuse to pay. I wonder what Servier will charge for their bumetanide syrup for autism when it hits the market in Europe in about 3 years time. What do Curemark hope to charge for their CM-AT enzyme mixture?

SpectrumNeeds would cost $1,400 a year for a teenager, I suppose a drug maker would ask 3-10 times more for his patented new autism drug in the US, and less in the rest of the world. 
I think other than establishing the effective dose of Clemastine in at least some “autism”, enough has been said. 

The next post (“Who lives in Libya?”) will be on the effect of BHB salts, BHB esters and C8 on the level of BHB in your blood and some anecdotes of the effect of taking BHB/C8 + Clemastine. It will thankfully be brief, but I did learn a lot from today's deep dive into the research.