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Possible cause of HPPD: Hyperactive mTORC1


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Hello everyone!
The following is a bunch of research on how, through the modulating of mTOR, we could arrive at our present situation. my apologies for the lackluster organization. its a lot. 
"Social behavior (SB) is a fundamental hallmark of human interaction. Repeated administration of low doses of the 5-HT2A agonist lysergic acid diethylamide (LSD) in mice enhances SB by potentiating 5-HT2A and AMPA receptor neurotransmission in the mPFC via an increasing phosphorylation of the mTORC1, a protein involved in the modulation of SB. Moreover, the inactivation of mPFC glutamate neurotransmission impairs SB and nullifies the prosocial effects of LSD. Finally, LSD requires the integrity of mTORC1 in excitatory glutamatergic, but not in inhibitory neurons, to produce prosocial effects. This study unveils a mechanism contributing to the role of 5-HT2A agonism in the modulation of SB."
Why is this important? Well mTor is how our body regulates EVERYTHING.
  • The mammalian target of rapamycin (mTOR) is an evolutionary conserved serine/threonine kinase that is present in two complexes, mTORC1 and mTORC2. mTORC1 is the main energy and nutrient sensor of the cell: it senses the presence of amino acids, glucose, lipids and ATP to allow efficient activation of the network in response to growth factors, Toll-like receptor ligands and cytokines.
  • Activation of the mTOR pathway usually promotes an anabolic response that induces the synthesis of nucleic acids, proteins and lipids. In addition, it stimulates glycolysis as well as mitochondrial respiration. Emerging data suggest that this metabolic reconfiguration is required for specific effector functions in myeloid cells.
  • Translational control of gene expression in myeloid immune cells emerges as one way in which mTORC1 controls cellular processes such as migration, interferon and pro- or anti-inflammatory cytokine expression as well as metabolic reprogramming.
  • The mammalian target of rapamycin (mTOR) integrates the intracellular signals to control cell growth, nutrient metabolism, and protein translation. mTOR regulates many functions in the development of the brain, such as proliferation, differentiation, migration, and dendrite formation. In addition, mTOR is important in synaptic formation and plasticity. Abnormalities in mTOR activity is linked with severe deficits in nervous system development, including tumors, autism, and seizures. Dissecting the wide-ranging roles of mTOR activity during critical periods in development will greatly expand our understanding of neurogenesis.
  • Inhibition of mTORC1 in macrophages promotes autophagy, which is important for intracellular pathogen killing and clearance of ingested complex lipids such as LDL cholesterol.
lets start with autophagy


Soo... whats autophagy? and how does it relate to mTOR?


Autophagy and mTOR
As a key regulator of autophagy, the mTOR plays an important role in autophagy, translation, cell growth and survival (Hwang et al., 2017). Mammalian target of rapamycin and autophagy are tightly bound within cells, and defects of mTOR and autophagy process might lead to a variety of human diseases (Hoeffer and Klann, 2010). Studies have shown that mTOR is widely involved in autophagy activation and synaptic plasticity (Ryskalin et al., 2018). The mTOR modulates long-lasting synaptic plasticity, memory and learning via regulating the synthesis of dendritic proteins (Liu et al., 2018a). Macroautophagy can degrade organelles and long-lived proteins in case of mTOR inactivation. Synaptic plasticity is further modulated by mTOR and neurodegeneration occurs when macroautophagy is absent (Hernandez et al., 2012). Therefore, macroautophagy following mTOR inactivation at the presynaptic terminal rapidly changes the neural transmission and presynaptic structure (Hernandez et al., 2012). The mechanisms for the target of rapamycin have been involved in modulating neurodegeneration and synaptic plasticity, but the role of mTOR in regulating presynaptic function via autophagy has not been clarified clearly (Torres and Sulzer, 2012).
In summary, there is a close relationship among mTOR, brain plasticity and autophagy. The mTOR related pathways play important role in regulating the process of autophagy and brain plasticity.
Autophagy is a lysosome-reliant degradation mechanism that regulate many biological courses, such as neuroprotection and cellular stress reactions (Shen and Ganetzky, 2009). There are different kinds of autophagy in most mammalian cells, and each type of autophagy performs very specific tasks in the course of intracellular degradation (Tasset and Cuervo, 2016). The autophagy-lysosomal pathway is a main proteolytic pathway, which mainly embraces chaperone-mediated autophagy and macroautophagy in mammalian systems (Xilouri and Stefanis, 2010). Macroautophagy, as a lysosomal pathway in charge of the circulation of long-lived proteins and organelles, is mainly considered as the inducible course in neurons, which is activated in conditions of injury and stress (Boland and Nixon, 2006). Coupled with macro-autophagy, chaperone-mediated autophagy (CMA) is crucial for maintaining intracellular survival and homeostasis via selectively reducing oxidized, misfolded, or degraded cytoplasmic proteins (Cai et al., 2015).
The plasticity of the central nervous system(CNS) can be regarded as changes of functional interaction between different types of cells, astrocytes, neurons, and oligodendrocytes (Aberg et al., 2006). The mature brain, as a highly dynamic organ, constantly alters its structure via eliminating and forming new connections. In general, these changes are known as brain plasticity and are related to functional changes (Viscomi and D’Amelio, 2012). Brain plasticity can be divided into structure plasticity and function plasticity. The structural plasticity of the brain refers to the fact that the connections between synapses and neurons in the brain can be established due to the influence of learning and experience. It includes the plasticity of synapses and neurons. Synaptic plasticity refers to the changes of pre-existing relationship between two neurons including structure and function alteration (De Pitta et al., 2016). Synaptic plasticity is considered as the representative of cellular mechanisms of memory and learning. Mitochondria are related to the modulation of complicated course of synaptic plasticity (Todorova and Blokland, 2017). For a long period, synaptic plasticity has been considered as a neuronal mechanism under the regulation of neural network action (Ronzano, 2017). Recent data indicate that autophagy is a homeostatic mechanism which is compatible with the microenvironment of the synapse, with the purpose of serving local functions linked with synaptic transmission (Todorova and Blokland, 2017). Neuronal plasticity is maintained by the fine modulation of organelle biogenesis and degradation and protein synthesis and degradation to assure high-efficiency turnover (Viscomi and D’Amelio, 2012). Protein degradation plays an important role in the course of synaptic plasticity, but the involved molecular mechanisms are unclear (Haynes et al., 2015). Therefore, Autophagy is a quality control mechanism of organelles and proteins in neurons, which plays a crucial role in their physiology and pathology (Viscomi and D’Amelio, 2012). In a word, there is a close relationship between autophagy and brain plasticity, and the related mechanisms are summarized in this review paper (as Table 1 and Figure 1 demonstrate).
mTOR complex 1 (mTORC1) was unveiled as a master regulator of autophagy since inhibition of mTORC1 was required to initiate the autophagy process.


So... weve hyperactived our mTORC1.... we cannot initiate the autophagy process... what does this mean???
1) Hyperactivation of mTORC1 by TSC1/2 deletion induces aberrant growth, proliferation, and differentiation of neurons and astrocytes, resulting in neuronal dysplasia, abnormal neuronal architecture, reactive astrogliosis, and seizures (27, 40,42).
Hyperactivation of mTORC1 disrupts cellular homeostasis in cerebellar Purkinje cells
Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolism. The importance of mTORC1 signaling in neuronal development and functions has been highlighted by its strong relationship with many neurological and neuropsychiatric diseases. Previous studies demonstrated that hyperactivation of mTORC1 in forebrain recapitulates tuberous sclerosis and neurodegeneration. In the mouse cerebellum, Purkinje cell-specific knockout of Tsc1/2 has been implicated in autistic-like behaviors. However, since TSC1/2 activity does not always correlate with clinical manifestations as evident in some cases of tuberous sclerosis, the intriguing possibility is raised that phenotypes observed in Tsc1/2 knockout mice cannot be attributable solely to mTORC1 hyperactivation. Here we generated transgenic mice in which mTORC1 signaling is directly hyperactivated in Purkinje cells. The transgenic mice exhibited impaired synapse elimination of climbing fibers and motor discoordination without affecting social behaviors. Furthermore, mTORC1 hyperactivation induced prominent apoptosis of Purkinje cells, accompanied with dysregulated cellular homeostasis including cell enlargement, increased mitochondrial respiratory activity, and activation of pseudohypoxic response. These findings suggest the different contributions between hyperactivated mTORC1 and Tsc1/2 knockout in social behaviors, and reveal the perturbations of cellular homeostasis by hyperactivated mTORC1 as possible underlying mechanisms of neuronal dysfunctions and death in tuberous sclerosis and neurodegenerative diseases.
neurogenesis, dendrite formation, and synaptic integration:
Effects of mTOR activation during neurogenesis. Neural stem cells (blue) undergo proliferation and either give rise to more stem cells (self-renewal) or daughter cells (green, differentiation). Activation of mTORC2 promotes neural stem cells (NSC) cell cycle entry through Akt. Hyperactivation of mTORC1 results in diminished self-renewal, favoring differentiation and lineage expansion. Daughter cells then migrate (red) from proliferation zones to their terminal positions. Activation of mTORC1 results in aberrant migration of daughter cells. Upon reaching their terminal positions, newly born neurons (gray) extend neurites and properly form dendritic arbors. Cells with high levels of mTORC1 activity can severely alter dendrite formation and synaptic integration. Upward pointing arrows indicate increased activity of designated genes or proteins. Downward pointing arrows indicate decreased activity or knockdown of designated genes or proteins. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983636/
Induction of cellular stresses by mTORC1 hyperactivation
We explored the molecular mechanisms underlying Purkinje cell death by mTORC1 hyperactivation in PC-mTOR Tg mice. Activation of mTORC1 enhances the mitochondrial biogenesis by forming a complex with PGC1α and YY124. We observed mitochondrial morphology in Purkinje cells by using the electron microscopy (Fig. 6a–f). As expected, the remarkably enlarged mitochondria were often found in PC-mTOR Tg mice in both cell bodies (Fig. 6a and b) and dendrites (Fig. 6e and f) compared to control mice. Despite their abnormal morphology, the internal lamellar structure of cristae was almost preserved even in enlarged mitochondria in PC-mTOR Tg mice (Fig. 6c and d). To test the mitochondrial respiratory activity, the cytochrome c oxidase activity was visualized in the cerebellar slices. Mitochondrial activity was detected in both molecular and Purkinje cell layers of control mice (Fig. 6g and h). Although similar staining patterns were also observed in PC-mTOR Tg mice, the cell bodies of Purkinje cells were stained more densely than control mice. Thus, despite their abnormal morphology, the mitochondrial respiratory function was not impaired but rather enhanced in Purkinje cells of PC-mTOR Tg mice.
To assess the pre- and postsynaptic function of each mTOR complex in glutamatergic synaptic transmission, we inactivated mTORC1 signaling by conditionally deleting Raptor, or mTORC2 signaling by conditionally deleting Rictor, postmitotically in primary neuron cultures from mouse hippocampus. We then performed morphological and whole-cell patch-clamp analysis of synaptic and membrane properties of glutamatergic neurons. Our results showed that both mTOR complexes were necessary to support normal neuron growth and evoked excitatory synaptic transmission. Despite these similarities, the effects of mTORC1 on evoked EPSCs (eEPSCs) were postsynaptic, via reductions in synapse number, whereas mTORC2 regulated the presynaptic Ca2+ dependence of evoked SV release. Furthermore, although the mechanism through which mTORC1 inactivation decreased eEPSCs was postsynaptic, it also increased spontaneous SV release and SV pool replenishment, which are thought to be presynaptic processes. Overall, each mTOR complex affected distinct modes of SV release: mTORC1 inactivation enhanced modes with low rates of SV fusion, such as spontaneous release, and mTORC2 inactivation impaired modes with high rates of SV fusion, such as action potential-evoked release. Thus, via differential activation of these two complexes, the mTOR pathway is ideally poised to respond to external cues and make fine adjustments to glutamatergic synaptic transmission to maintain normal neural network function.
Previous studies showed that mTOR inhibition by rapamycin treatment reduces the number of AMPA receptors at the synapse (Wang et al., 2006), the number of synapses (Weston et al., 2012), and the number of SVs per synapse (Hernandez et al., 2012). Accordingly, mTOR hyperactivation increases mEPSC amplitude (Xiong et al., 2012), AMPA receptor number, and spine density (Tang et al., 2014Williams et al., 2015), and these effects are blocked by rapamycin. Thus, integrating our findings on specific mTORC1 inactivation with these previous findings, several lines of evidence now indicate that mTORC1 acts via a postsynaptic mechanism to bidirectionally regulate evoked glutamatergic synaptic strength.


GABA: We have FUCKED gaba (but you knew that didn't you)
The following is evidence of the reduced gaba hypothesis that is thrown around a lot. basically, disrupting the autophagy from mTOR (such as that which would occure through hyperactive MTORC1) causes a reduction in the surface expression of GABA A receptors ( thats the ones benzos increase activity on). The following is two studies, one is a good tldr and the other is much mor indepth.


Autophagy links MTOR and GABA signaling in the brain
The disruption of MTOR-regulated macroautophagy/autophagy was previously shown to cause autistic-like abnormalities; however, the underlying molecular defects remained largely unresolved. In a recent study, we demonstrated that autophagy deficiency induced by conditional Atg7 deletion in either forebrain GABAergic inhibitory or excitatory neurons leads to a similar set of autistic-like behavioral abnormalities even when induced following the peak period of synaptic pruning during postnatal neurodevelopment. Our proteomic analysis and molecular dissection further revealed a mechanism in which the GABAA receptor trafficking function of GABARAP (gamma-aminobutyric acid receptor associated protein) family proteins was compromised as they became sequestered by SQSTM1/p62-positive aggregates formed due to autophagy deficiency. Our discovery of autophagy as a link between MTOR and GABA signaling may have implications not limited to neurodevelopmental and neuropsychiatric disorders, but could potentially be involved in other human pathologies such as cancer and diabetes in which both pathways are implicated.


GABARAPs dysfunction by autophagy deficiency in adolescent brain impairs GABAA receptor trafficking and social behavior


Excessive p62 accumulation in autophagy-deficient and mTOR-hyperactivated neurons results in reduced GABAA receptor surface expression due to mislocalized GABARAPs
We next asked whether the observed disruption of GABAA receptor trafficking is specific to autophagy-deficient conditions or is due to a general increase in p62 levels. First, by immunofluorescence, we found that p62 overexpression in WT neurons led to the formation of p62+ aggregates that also sequestered GABARAPs (Fig. 5A and fig. S7, A to C). Second, we observed a significant reduction in surface GABAA receptors in WT neurons with p62 overexpression (Fig. 5B). Third, if the reduced surface expression of GABAA receptors was caused by sequestration of GABARAPs by p62, a reduction of p62 in Atg7 cKO neurons would be expected to restore such deficits. To test this hypothesis, we performed surface receptor biotinylation experiments on Atg7 cKO and control neurons with or without Sqstm1 (p62) knockdown. Consistent with our prediction, Sqstm1 knockdown in Atg7 cKO neurons reversed the reduction of surface GABAA receptors to control levels (Fig. 5C). Together, this series of experiments suggests that the pathologic accumulation of p62 in Atg7 cKO neurons sequesters GABARAPs and disrupts the normal functions of GABARAPs, resulting in a reduction of surface GABAA receptor levels.
Astrocytes: here we go
Astrocyte activation has been implicated in the pathogenesis of several neurological conditions, such as neurodegenerative diseases, infections, trauma, and ischemia. Reactive astrocytes are capable of producing a variety of pro-inflammatory mediators, including interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), neurotrophic factors [1], as well as potentially neurotoxic compounds, like nitric oxide (NO). NO, one of the smallest known bioactive products of mammalian cells, is biosynthesized by three distinct isoforms of NO synthase (NOS): the constitutively expressed neuronal (n)NOS and endothelial (e)NOS, and the inducible (i)NOS [2]. The expression of iNOS can be induced in different cell types and tissues by exposure to immunological and inflammatory stimuli [3]. In vitro, primary astrocyte cultures express iNOS in response to cytokines such as IL-1β [4], interferon γ (IFNγ), TNFα and/or the bacterial endotoxin, lipopolysaccharide (LPS) [5, 6]. Once induced, iNOS leads to continuous NO production, which is terminated by enzyme degradation, depletion of substrates, or cell death [7]. iNOS activity generates large amounts of NO (within the μM range) that can have antimicrobial, anti-atherogenic, or apoptotic actions [8]. However, aberrant iNOS induction exerts detrimental effects and seems to be involved in the pathophysiology of several human diseases [9, 10].
soo... this is where it gets bad.


This study has revealed that the inactivation of mTORC1 in postmitotic neurons causes moderate reactive astrogliosis. The loss of neural mTORC1 activity may induce astrogliosis by reducing the neuronal secretion of FGF-2, thereby inhibiting FGF receptor signaling in astrocytes, which is required to maintain their nonreactive state (36) (Fig. 7). Although our present data could not identify the exact role of FGF-2 in this process, and the underlying mechanism needs to be further investigated, our findings have uncovered a novel mechanism for the regulation of astrocytes by dysfunctional neurons and have established a potential important link between mTORC1 signaling and CNS pathologies.
The function of mTORC1 in neurons and astrocytes has been extensively studied in conditional knock-out mice (26, 37,39). Hyperactivation of mTORC1 by TSC1/2 deletion induces aberrant growth, proliferation, and differentiation of neurons and astrocytes, resulting in neuronal dysplasia, abnormal neuronal architecture, reactive astrogliosis, and seizures (27, 40,42). Inactivation of mTORC1 in neuronal progenitors impairs the growth and proliferation of neurons and astrocytes, resulting in a smaller brain and in death shortly after birth (25, 43).
So we see that mTORC1 Hyperactivation results in an increase in cytokines. One of those is il-1b.... and its nefarious in the brain.
1. Increase in mTORC1
2. increase in IL-1b proinflammatory cytokine
3. decrease in cb1 recebtor binding
4. decreases CB1R's (gabaA) synapse binding in the striatum
5. causes behavioral manifestations closely resembling anxious-depressive symptoms in humans, including anhedonia, reduced exploratory behaviors, social withdrawal, fatigue, and sleep disturbances
6. This entire process requires "intact function of the transient receptor potential vanilloid 1 (TRPV1)" to work.
IL-1beta, but not IL-10 or tumour necrosis factor (TNF)-alpha, down-regulated the surface expression and Ser831 phosphorylation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1. Agents that block IL-1beta receptor activity abolished these effects. In contrast, no change in the surface expression of the N-methyl-d-aspartate (NMDA) receptor subunit NR1 was observed.
The inhibition of NMDA receptor activity or depletion of extracellular calcium blocked IL-1beta effects on GluR1 phosphorylation and surface expression. NMDA-mediated calcium influx was also regulated by IL-1beta.
These findings suggest that IL-1beta selectively regulates AMPA receptor phosphorylation and surface expression through extracellular calcium and an unknown mechanism involving NMDA receptor activity.
(PS Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory)
Blood brain barrier:
On the other hand, interleukin (IL)-1β significantly induces the production of MMP 1, 3, 10, and 13 via a mechanism that is independent of Ca2+
In summary, the results presented here are the first to reveal the function of MMP3 in the BBB and suggest that it has an essential role in the brain microvasculature that differs from its function in other vessels. We have shown that MMP3 increases BBB permeability by upregulating the ERK signaling pathway, which subsequently reduces TJ and AJ protein abundance in BMVECs. Oxidative stress often leads to impairment of BBB. Since the BBB is the primary regulator of exchange between the peripheral blood and the brain, our observations likely have important implications for treating neuroinflammatory conditions and other CNS disorders involving the endothelial MMP3 pathway.
TNF-a: this is a bit scary
TNF released by microglia has an important role in regulating synaptic plasticity [110]. Specifically, it controls a process called synaptic scaling, i.e., the adjustment of synaptic strength in response to prolonged changes in the electrical activity of neurons [110,111]. Indeed, a reduction of glutamate transmission increases microglial TNF release, which promotes the expression of AMPA glutamate receptors in neurons. Conversely, increased extracellular glutamate concentration inhibits TNF release from microglia, additional glutamate receptor expression, and declines neuronal activity [111–113]. The increase of AMPA receptor GluR1 subunit expression does not occur at mRNA level, but this is controlled by TNF at post-transcriptional level [114]. Subsequent studies revealed that TNF facilitates the trafficking and membrane insertion of AMPA receptors at the neuron surface, which are crucial for the homeostatic synaptic plasticity. Specifically, hippocampal neurons exposed to TNF increase surface expression of GluR1 subunit through modulation of NF-κB and acid sphingomyelinase pathways [115]. TNF not only controls homeostatic synaptic activity, but also induces neurotoxicity via autocrine/paracrine loops involving other endogenous mediators. First, TNF activates TNFR1 on microglia, amplifying its production and release [95]. Second, microglia-derived TNF activates TNFR1 expressed on astrocytes, allowing glutamate release from the glial cells. This, in turn, activates its specific receptors, including the metabotropic mGluR2 receptor on microglia, potentiating microglial TNF production and affecting synaptic transmission [110]. ATP, released by microglia concurrently with TNF, contributes to TNF-mediated neuronal damage by inducing a prolonged activation of microglial P2X7 receptor and release of both IL-1β and TNF inflammatory cytokines. In addition, both microglial TNF and ATP trigger adjacent astrocytes to release additional ATP, that amplifies microglia response and promotes astroglial release of glutamate, aggravating neuronal dysfunction [110]. Moreover, TNF mediates neuronal death by increasing extracellular levels of the excitotoxic transmitter glutamate and excessive AMPA receptor activation via downregulation of the astrocytic glutamate transporter EAAT2/GLT1 [116]. The effects of TNF on N-methyl-D-aspartate receptors (NMDARs) trafficking are less characterized. However, it has been demonstrated that, in hippocampal neurons, TNF increases the expression of the NR1 subunit of NMDAR and its specific clustering into lipid rafts [117]. Accordingly, treatment of human neuronal cultures with competitive (2-APV) and noncompetitive (MK-801) NMDA receptor antagonists reduced the glutamate neurotoxicity induced by TNF [118].
" In MS, glutamate-related excitotoxicity, caused by excessive activation of these receptors (leading to a Ca2+ overload), is responsible for neuronal and oligodendrocyte death [2, 16, 17]. In addition, microglia, the resident macrophages of the CNS, become activated by increased glutamate concentration. Activated microglia proliferate, secrete cytokines, chemokines, nitric oxide and ROS, and may become phagocytic; outcomes all of which cause further injury to the ailing CNS [9]. Oligodendrocytes have been found to be particularly susceptible to glutamate excitotoxicity, via the AMPA/kainate receptors. AMPA/kainate antagonists have been shown to increase oligodendrocyte survival as well as reducing axonal damage [16, 17]. "
--this is important to note because if we have abysmal gaba and high glutamate from the mTORC1 and from astrocyte issues then excitotoxic events are easily understandable. Here you can see how these events could lead to an out-of-control inflammatory response.


What do we do???? BEATS ME REALLY but heres some options
reduce mtor?
Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration
mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here, we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels, particularly when oxidative phosphorylation is impaired, such as in neurons treated with mitochondrial inhibitors, or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly, in mitochondrially defective neurons, but not neuroprogenitor cells, ribosomal S6 and S6 kinase phosphorylation increased over time, despite activation of AMPK, which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis, a major energy-consuming process, may account for its ATP-saving effect. We propose that a mild reduction in protein synthesis may have the potential to treat mitochondria-related neurodegeneration.
Autophagy Dysfunction and mTOR Hyperactivation Is Involved in Surgery: Induced Behavioral Deficits in Aged C57BL/6J Mice
Autophagy is crucial for cell survival, development, division, and homeostasis. The mammalian target of rapamycin (mTOR), which is the foremost negative controller of autophagy, plays a key role in many endogenous processes. The present study investigated whether rapamycin can ameliorate surgery—induced cognitive deficits by inhibiting mTOR and activating autophagy in the hippocampus. Both adult and aged C57BL/6J mice received an intraperitoneal injection of rapamycin (10 mg/kg/day) for 5 days per week for one and a half months. Mice were then subjected to partial hepatectomy under general anesthesia. Behavioral performance was assessed on postoperative days 3, 7, and 14. Hippocampal autophagy-related (Atg)-5, phosphorylated mTOR, and phosphorylated p70S6K were examined at each time point. Brain derived neurotrophic factor (BDNF), synaptophysin, and tau hyperphosphorylation (T396) in the hippocampus were also examined. Surgical trauma and anesthesia exacerbated spatial learning and memory impairment in aged mice on postoperative days 3 and 7. Following partial hepatectomy, the levels of phosphorylated mTOR, phosphorylated 70S6K, and phosphorylated tau were all increased in the hippocampus. A corresponding decline in BDNF and synaptophysin were observed. Rapamycin treatment restored autophagy function, attenuated phosphorylation of tau protein, and increased BDNF and synaptophysin expression in the hippocampus of surgical mice. Furthermore, surgery and anesthesia induced spatial learning and memory impairments were also reversed by rapamycin treatment. Autophagy impairments and mTOR hyperactivation were detected along with surgery—induced behavioral deficits. Inhibiting the mTOR signaling pathway with rapamycin successfully ameliorated surgery-related cognitive impairments by sustaining autophagic degradation, inhibiting tau hyperphosphorylation, and increasing synaptophysin and BDNF expression. https://link.springer.com/article/10.1007/s11064-019-02918-x


Neuronal mTORC1 Is Required for Maintaining the Nonreactive State of Astrocytes
In summary, this study has demonstrated that the inactivation of mTORC1 in postmitotic neurons induces reactive astrogliosis, possibly by inhibiting FGF-2 secretion. mTORC1 activity in postmitotic neurons is required for maintaining astrocytes in a nonreactive state. Astrogliosis is likely to be regulated by various signaling pathways and various cell types in different nuclei on the CNS. In our studies, neuronal mTORC1 activity regulates astrocyte activation, possibly via multiple potential signals directly or indirectly. Further investigation is required to define the pathological consequences of astrogliosis induced by the loss of neuronal mTORC1 and its association with CNS disease. Manipulating mTORC1 in neurons or FGF-2 signaling in astrocytes may represent a novel therapeutic mechanism for treating CNS disorders and improving functional recovery in neuropathological conditions.
Medications: IM NOT A DOCTOR and honestly some of these are pretty extreme. Don't take these without research and consulting a medical professional.
The point of these medications:
  1. reduce damage caused by reaction. this includes microglial and astrocyte dysfunction and resulting inflammatory markers
  2. treat reaction at source
  3. Repair damage from the reaction
curative action: DRD3 (dopamine receptor D3) but not DRD2 activates autophagy through MTORC1 inhibition preserving protein synthesis
The results revealed that pramipexole induces autophagy through MTOR inhibition and a DRD3-dependent but DRD2-independent mechanism. DRD3 activated AMPK followed by inhibitory phosphorylation of RPTOR, MTORC1 and RPS6KB1 inhibition and ULK1 activation. Interestingly, despite RPS6KB1 inhibition, the activity of RPS6 was maintained through activation of the MAPK1/3-RPS6KA pathway, and the activity of MTORC1 kinase target EIF4EBP1 along with protein synthesis and cell viability, were also preserved. This pattern of autophagy through MTORC1 inhibition without suppression of protein synthesis, contrasts with that of direct allosteric and catalytic MTOR inhibitors and opens up new opportunities for G protein-coupled receptor ligands as autophagy inducers in the treatment of neurodegenerative and psychiatric diseases.
Palliative: this will help with the astrocyte insanity/out of control inflammatory cycle
Experimental autoimmune encephalomyelitis (EAE) is the most used animal model of multiple sclerosis (MS) for the development of new therapies. Dopamine receptors can modulate EAE and MS development, thus highlighting the potential use of dopaminergic agonists in the treatment of MS, which has been poorly explored. Herein, we hypothesized that pramipexole (PPX), a dopamine D2/D3 receptor-preferring agonist commonly used to treat Parkinson's disease (PD), would be a suitable therapeutic drug for EAE. Thus, we report the effects and the underlying mechanisms of action of PPX in the prevention of EAE. PPX (0.1 and 1 mg/kg) was administered intraperitoneally (i.p.) from day 0 to 40 post-immunization (p.i.). Our results showed that PPX 1 mg/kg prevented EAE development, abolishing EAE signs by blocking neuroinflammatory response, demyelination, and astroglial activation in spinal cord. Moreover, PPX inhibited the production of inflammatory cytokines, such as IL-17, IL-1β, and TNF-α in peripheral lymphoid tissue. PPX was also able to restore basal levels of a number of EAE-induced effects in spinal cord and striatum, such as reactive oxygen species, glutathione peroxidase, parkin, and α-synuclein (α-syn). Thus, our findings highlight the usefulness of PPX in preventing EAE-induced motor symptoms, possibly by modulating immune cell responses, such as those found in MS and other T helper cell-mediated inflammatory diseases.
The results indicated that TPM and LEV alleviated behavioral deficits and reduced amyloid plaques in APPswe/PS1dE9 transgenic mice. TPM and LEV increased Aβ clearance and up‐regulated Aβ transport and autophagic degradation. TPM and LEV inhibited Aβ generation and suppressed γ‐secretase activity. TPM and LEV inhibited GSK‐3β activation and increased the activation of AMPK/Akt activation. Further, TPM and LEV inhibited histone deacetylase activity in vivo.
  • activation of the ampk pathway reduces mtorc1
Baclofen: possible brain healing
GABA receptors play an important role in ischemic brain injury. Studies have indicated that autophagy is closely related to neurodegenerative diseases. However, during chronic cerebral hypoperfusion, the changes of autophagy in the hippocampal CA1 area, the correlation between GABA receptors and autophagy and their influences on hippocampal neuronal apoptosis have not been well established. Here, we found that chronic cerebral hypoperfusion resulted in rat hippocampal atrophy, neuronal apoptosis, enhancement and redistribution of autophagy, down-regulation of Bcl-2/Bax ratio, elevation of cleaved caspase-3 levels, reduction of surface expression of GABAA receptor α1 subunit and an increase in surface and mitochondrial expression of connexin 43 (CX43) and CX36. Chronic administration of GABAB receptors agonist baclofen significantly alleviated neuronal damage. Meanwhile, baclofen could up-regulate the ratio of Bcl-2/Bax and increase the activation of Akt, GSK-3β and ERK which suppressed cytodestructive autophagy. The study also provided evidence that baclofen could attenuate the decrease in surface expression of GABAA receptor α1 subunit and down-regulate surface and mitochondrial expression of CX43 and CX36, which might enhance protective autophagy. The current findings suggested that, under chronic cerebral hypoperfusion, the effects of GABAB receptors activation on autophagy regulation could reverse neuronal damage.


WARNING: baclofen interacts with ampa receptors causing an initial increase and then what seems like long term decrease. since there could be a loss of ampa function (whether through cell death or otherwise) this could worsen certain symptoms. Mainly low empathy/issues with anhedonia. Ampa receptors play a very complex role in synaptic plasticity, mood, and behavior.
I took baclofen in very high doses (100mg per day) a few years ago. It restored my cognition, made me sociopathic (not permanent), hypomanic (god i wish permanent but no), and gave me anhedonia that took 2 years to cease. But hey it actually did work to heal my brain which is neato ** I think i just overdid it. PAWS set in. Homotaurine may help with this should anyone wish to try baclofen for restoring their cognition.
Rapamycin: the ultimate mtor inhibitor, see basically all the studies.
The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes
In our previous studies, we observed that although rapamycin reduced iNOS expression mRNA and activity in microglial cells, and was without effect on astrocyte iNOS activity [21], it caused a rapid significant increase in iNOS mRNA levels in astrocytes induced by two different proinflammatory stimuli. Later time points were not examined; neither was the basis for this contrasting result examined. In the present paper we tested the hypothesis that while at early times rapamycin increases iNOS mRNA, at later times it modifies iNOS mRNA stability. Our results using primary rat astrocytes are consistent with this hypothesis, and suggest that inhibition of mTOR kinase activity in glial cells results in anti-inflammatory actions. Together with the marked anti-inflammatory effects observed in microglial cells [21], these data further provide pre-clinical evidence for a possible clinical use of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies.
but.... is it that simple??
Probably not. Inhibiting mtorc1 across the board surely has issues involved with it. These are very complex mechanisms at work... ive come across some stuff saying there are serious pros and cons for using drugs like rapamycin.
Some good reads:
tldr: Our brains are going fucking nuts. We have out of control inflammation, autophagy problems, gaba is gonnneeeee, glutamate is out of control... the list is almost endless really. There is so much more not mentioned in this post. this is like a giant tree-- you can follow the branches out very far. But it makes sense-- especially with the weird onset times of hppd. Some of this needs time to get bad enough that you notice it.
And if the mtor stays dysregulated you wont heal. For whatever reason, in some people it stays stuck. If we can reverse this issue it should provide at least a fair fight for healing. The sooner its dealt with the better as the more time spent in the "active" state of hppd the more damage is being done.
So why cant you see this on an MRI? thats a whole nother post. Youd be surprised what you cant see on an mri though lol.
One thing that is important to note--- if there is excitotoxicity of glutamate receptors in this process, reducing the thing that is boosting them, even if it is causing the pathology, may make a person feel WORSE initially. Or, likely, there are other things going on here too--- like how hallucinogens open up susceptibility to viral reactivation in the brain.... for another day.
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 In these five patients with positive respond, four of them were given therapy based on LEV, which may indicate LEV as a preferential choice for patients with DEPDC5 variants. Considering the fact that the loss-of-function variants in DEPDC5 will lead to over-activation of the mTOR pathway, the mTOR inhibitor, such as sirolimus or everolimus, may be a complementary treatment for DEDPC5related epilepsy.”



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  • 6 months later...

The following is a big dump of info I need to sort into a coherent train of though outside of just my brain lol...:



Mast cell









Interleukin1-b (and more...)



Pre Frontal Cortex PFC

retinal ganglion cell (RGC)




Little is known about the signals downstream of PI3K which regulate mast cell homeostasis and function following FcepsilonRI aggregation and Kit ligation. In this study, we investigated the role of the mammalian target of rapamycin complex 1 (mTORC1) pathway in these responses. In human and mouse mast cells, stimulation via FcepsilonRI or Kit resulted in a marked PI3K-dependent activation of the mTORC1 pathway, as revealed by the wortmannin-sensitive sequential phosphorylation of tuberin, mTOR, p70S6 kinase (p70S6K), and 4E-BP1. In contrast, in human tumor mast cells, the mTORC1 pathway was constitutively activated and this was associated with markedly elevated levels of mTORC1 pathway components. Rapamycin, a specific inhibitor of mTORC1, selectively and completely blocked the FcepsilonRI- and Kit-induced mTORC1-dependent p70S6K phosphorylation and partially blocked the 4E-BP1 phosphorylation. In parallel, although rapamycin had no effect on FcepsilonRI-mediated degranulation or Kit-mediated cell adhesion, it inhibited cytokine production, and kit-mediated chemotaxis and cell survival. Furthermore, Rapamycin also blocked the constitutive activation of the mTORC1 pathway and inhibited cell survival of tumor mast cells. These data provide evidence that mTORC1 is a point of divergency for the PI3K-regulated downstream events of FcepsilonRI and Kit for the selective regulation of mast cell functions. Specifically, the mTORC1 pathway may play a critical role in normal and dysregulated control of mast cell homeostasis.




What Are Microglia?

Microglial cells are the antigen presenting cells of the CNS and are key mediators of neuroinflammation (Kettenmann et al., 2011). They have a role in surveillance and phagocytosis of cellular debris (Sierra et al., 2013) and maintenance of brain function and are thought to regulate many processes including neurogenesis, synaptic plasticity and synaptic pruning (Aloisi, 2001; Tremblay et al., 2010; Ji et al., 2013; Schafer and Stevens, 2013; Zhan et al., 2014; Wu et al., 2015; Bar and Barak, 2019). They are derived from primitive myeloid progenitor cells and migrate into the CNS during embryogenesis, appearing before E8 in mice and 4.5–5 weeks in humans (Lichanska and Hume, 2000; Ginhoux et al., 2010), and increase in numbers rapidly from E16 onward in mice (Swinnen et al., 2013) and are functionally heterogeneous (Smolders et al., 2019; Mendes and Majewska, 2021). Microglial progenitors infiltrate the CNS before the vasculature is maturely formed, either migrating though the ventricular walls or through the meninges (Ginhoux et al., 2010; Swinnen et al., 2013; Reemst et al., 2016). Migration and distribution may also be further regulated by direct neuronal-microglial interactions though the chemokine CX3CL1, otherwise known as Fractalkine, and its corresponding receptor expressed on the microglia (Paolicelli et al., 2011). They then disperse in non-uniform manner, comprising 0.5–16.6% of the cell population depending on the region of the adult brain (Lawson et al., 1992) and differentiate to help regulate neurodevelopment, monitoring and maintaining synapses in the healthy, uninjured brain (Aloisi, 2001; Bar and Barak, 2019). We would direct readers to some excellent review articles on the role of microglia on neurodevelopment (Cowan and Petri, 2018; Coomey et al., 2020; Thion and Garel, 2020).


Microglia and Neuroinflammation

Increased microglial-mediated neuroinflammation has been seen in numerous NDDs, including ASD (Morgan et al., 2010; Tetreault et al., 2012; Suzuki et al., 2013; Gupta et al., 2014; Lee et al., 2017), schizophrenia (Garey, 2010; Sellgren et al., 2019; Chini et al., 2020), ADHD (Anand et al., 2017), and TS (Lennington et al., 2016). Microglia in the developing brain are also sensitive to external perturbations such as maternal infections (Smolders et al., 2015; Bernstein et al., 2016; Rosin et al., 2021a; Rosin et al., 2021b). Microglia are thought to initiate an immune response to protect the brain, but altered microglial activity has also been implicated in disorders of neurodevelopment and neurodegeneration through an upregulation of neuroinflammation (Kettenmann et al., 2011; Salter and Stevens, 2017). The precise mechanisms how a microglial bias toward pro- or anti-inflammatory cytokine production can affect neurodevelopment and pathological states remains incompletely understood. As further outlined below histamine has been shown to trigger both anti-inflammatory and pro-inflammatory responses from the microglia (Biber et al., 2007; Zhang et al., 2020). An appreciation of the role of the microglia in neuroinflammation and neurodevelopment and their regulation by histamine is therefore an exciting prospect into understanding the pathophysiology of a range of neurodevelopmental disorders.


Histamine’s Regulation of Microglia

Microglia have been shown to express all four subsets of histamine receptor (Dong et al., 2014a; Haas and Panula, 2016; Zhang et al., 2020), which can differentially affect their behavior. For example,Frick et al. (2016) undertook one of the first in vivo studies on the role of histamine in microglial activation. The group used immunohistochemistry to assess the effect of either histamine deficiency (Hdc KO mouse model) or histamine stimulation in wild type mice on microglia. Histamine was shown to regulate microglia via the H4 receptor. Hdc KO mice have a normal number of microglia but with reduced ramifications, reduced insulin-like growth factor-1 (IGF-1) expression and reduced expression of H4 receptor that may indicate impairment in histaminergic regulation of microglia. Similar findings occurred by selective removal of histaminergic neurons in the TMN of the hypothalamus. IGF-1 expressing microglia are induced by cytokines released from T helper 2 cells, which may be neuroprotective, promoting neurogenesis. Furthermore, the pro-inflammatory microglial response to challenge with lipopolysaccharide (LPS) was greater in Hdc KO mice. This may indicate that a genetic predisposition such as histamine deficiency may increase the brain’s vulnerability to pro-inflammatory insults in neuropsychiatric disorders such as TS. Indeed exogenous histamine was able to reduce LPS induced inflammation in the hippocampus (Saraiva et al., 2019).

Interestingly, in vitro studies have shown both pro- and anti-inflammatory effects of histamine on microglial function (Biber et al., 2007). For example, histamine can reduce pro-inflammatory cytokine production such as IL-1β in response to mediators such as LPS as well modulate overall microglial motility (Ferreira et al., 2012). This may indicate an anti-inflammatory function of histamine on the microglia. However, in contrast to this, microglial secretion of the pro-inflammatory cytokines TNF-α and IL-6 is triggered by histaminergic stimulation of the H1 and H4 receptors (Dong et al., 2014a; Zhu et al., 2014). Specifically, Zhang et al. (2020) found that histamine could induce microglial activation and the production of the pro-inflammatory cytokines TNF-α and IL-1β that was partially negated with H1 and H4 receptor antagonists and stimulated with H1 and H4 receptor agonists. On the other hand, H2 and H3 receptor antagonists led to significant increases in TNF-α and IL-1β, and H2 and H3 receptor agonists significantly increased the release of the anti-inflammatory cytokine interleukin-10 (IL-10). This is further supported by both Chen et al. (2020) who found that H2 and H3 receptor agonism inhibited laparotomy- or LPS-induced microglial activation, pro-inflammatory cytokine production and cognitive decline and by Fang et al. (2020) whereby H4 receptor antagonism reduced microglial activation and TNF-α release in a rat model of Parkinson’s disease. Along with histamine’s ability to induce microglial activation and the subsequent release of both anti- and pro-inflammatory factors, it can also promote phagocytosis via H1 receptor activation and the production of reactive oxygen species (Rocha et al., 2016) and prostaglandin E2 (Lenz et al., 2018).

Overall, these findings highlight the pleiotropic nature of the microglia in mediating their immune response and suggest potential roles for histamine in regulating microglial-mediated inflammation. What remains poorly studied though, is which sources of histamine within the CNS may contribute to both microglia-mediated neuroinflammation and altered neurodevelopment.

What Are Astrocytes?

Astrocytes are the most numerous cell type found within the CNS, forming complex networks with neuronal and non-neuronal cells alike. They are dynamic cells that have a wide range of functions and are fundamental for brain homeostasis (Nedergaard et al., 2003; Escartin et al., 2021). During early neurodevelopment, astrocytes have a trophic effect, facilitating the generation and migration of neuronal cells, facilitating synaptogenesis and the creation and maintenance of neuronal circuits (Ricci et al., 2009). One astrocyte can communicate with multiple neurons, with most of these structures being tripartite in nature; structural units formed of pre- and postsynaptic components of two neurons and an astrocyte (Araque et al., 1999; Halassa et al., 2007; Cavaccini et al., 2020). They can sense and respond to changes in the local microenvironment (e.g., local neurotransmitters) to control neuronal signaling (Wahis et al., 2021) and protect neurons from oxidative damage and neuronal injury. They are also important in energy metabolism (Brown and Ransom, 2007; Parra-Abarca et al., 2019), ionic homeostasis (Olsen et al., 2015), blood flow regulation (Howarth, 2014) and the formation of the blood brain barrier and can release gliotransmitters such as adenosine triphosphate (ATP), glutamate and D-serine (Parpura et al., 1994; Zhang et al., 2003; Volterra and Meldolesi, 2005; Hamilton and Attwell, 2010).

Astrocytes and Neuroinflammation

There is a growing evidence to suggest that astrocytes can modulate the immune system within the CNS and are important in regulating neuroinflammation (Rothhammer and Quintana, 2015). We have already discussed that neuroinflammatory processes can have both protective or detrimental effects on the developing brain. So too can astrocytic activation (Cekanaviciute and Buckwalter, 2016). Astrocyte activation can lead to the release of trophic factors such neurotrophin-3 (NT-3), glial cell line-derived neurotrophic factor (GDNF) and BDNF (Jurič et al., 2011; Thomsen et al., 2017). These growth-promoting molecules promote neuronal survival and GDNF has also been shown to inhibit microglial activation (Ossola et al., 2011; Rocha et al., 2012) resulting in a dampening down of neuroinflammation. Conversely, astrocytic activation can also lead to pro-inflammatory cytokine release, alongside increased concentrations of chemokines and reactive oxygen species and microglial activation (Sochocka et al., 2017) resulting in increased excitotoxicity, apoptosis and neurodegeneration. The mechanisms underlying the induction of a specific neuroinflammatory process remains poorly understood. Recent cutting-edge approaches have started to describe key target molecules important for the interactions between astrocytes and microglia in these neuroinflammatory processes (Clark et al., 2021). It is hoped that improved understanding of astrocyte-microglia cross-talk may then reveal new potential therapeutic targets for modulation that could be relevant in an array of neurodevelopmental disorders.

Histamine’s Regulation of Astrocytes

The H1, H2, and H3 receptors are expressed on astrocytes (Jurič et al., 2016) (see Figure 2), though H3 receptor expression may be restricted to certain brain regions and may vary depending on the species that is studied (Karpati et al., 2018). Our current understanding of how astrocytes can respond to histaminergic activity in the brain originated over three decades ago (Hosli and Hosli, 1984; Hosli et al., 1984) and continue to be investigated. For example, Karpati et al. (2018) employed the human astrocytoma cell line 1321N1 to better establish the underlying mechanism and found that histamine can interact with astrocytic histamine receptors resulting in glutamate release in an H1 receptor-dependent and concentration-dependent manner suggesting that histamine can form part of neuron-astrocyte communications.

There is less data available if and how histaminergic activity might influence astrocytic immunomodulation. Some studies have shown that histamine can act synergistically with pro-inflammatory cytokines such as IL-1 (Lipnik-Stangelj and Carman-Krzan, 2006) and IL-6 (Lipnik-Štangelj and Čarman-Kržan, 2005; Ales et al., 2008) to modulate astrocytic release of neurotrophins such as NGF. For example, Xu et al. (2018) investigated the role of histamine on astrocytic neuromodulation and neuroprotection. They found that histamine selectively upregulated the expression of H1, H2, and H3 receptors, stimulated the synthesis of astrocytic GDNF and inhibited the production of pro-inflammatory cytokines, TNF-α and IL-1β in a concentration-dependent manner. The increased production of neurotrophic factors likely highlights an important mechanistic role in CNS recovery from injury by promoting neuronal survival and synaptogenesis (Lipnik-Stangelj and Carman-Krzan, 2004; Jurič et al., 2011; Xu et al., 2018). We have already discussed that released GDNF can inhibit microglial activation in vivo and in vitro, thereby revealing a possible interaction between these glial cells in modifying (microglial-mediated) neuroinflammation (Rocha et al., 2012; Zhang et al., 2014). In addition see also recent findings suggestive of purinergic signaling from astrocytes to microglia upon histaminergic stimulation (Xia et al., 2021). Moreover, changes in astrocyte-neuronal crosstalk have been implicated in the development of mental disorders, including depression, ASD and schizophrenia (Roman et al., 2020). However, to our knowledge, there are no studies that have investigated the specific role of histamine in directly modulating astrocytic behavior contributing to neurodevelopmental disorders.

Mast Cells as a Non-neuronal Source of Histamine

Mast cells are immune cells derived from hematopoietic precursors, originating within the bone marrow from CD34+/CD117+ pluripotent progenitors (Gilfillan et al., 2011). They then mature within the microenvironment of various tissues, including the vascular endothelium and the brain, where they participate in both innate and adaptive immune responses, even in the absence of antigen presentation (Dong et al., 2014b). Mast cells in general express H1 and H4 receptors, which have been implicated in the pathophysiology of peripheral type 1 hypersensitivity reactions and increased histamine and cytokine generation, respectively (Thangam et al., 2018) and guide chemotaxis (Hofstra et al., 2003; Halova et al., 2012). However, their expression in brain mast cells has yet to be confirmed. Mast cells are located in perivascular regions within close vicinity of neurons, especially in the hypothalamus, the pineal and pituitary glands (Theoharides, 2017), velum interpositum below the hippocampus (Panula et al., 2014), the meninges (Reuter et al., 2001; Galli et al., 2005a) and are able to cross the normal blood brain barrier (Silverman et al., 2000). The ability to traverse the blood brain barrier may be accentuated further by disease states affecting its integrity which can intimately be linked to mast cell activation and contribute to neuroinflammation and neurotoxicity (Theoharides et al., 2012), including during periods of neurodevelopment. Approximate mast cell numbers in the developing rodent brain have recently been described and are mainly localized to the pia mater and the thalamus. Within the pia mater, mast cells are most numerous during early development, with approximately 3,500 seen at birth, peaking at approximately 5,000 at postnatal day 11. Numbers then decline to approximately 1,500 at P15, though the remaining mast cells become more concentrated in the pia that overlies the anterior thalamus. The total numbers of mast cell within the pia then reach adult levels of approximately 50 by P30. Within the thalamus, around 140 mast cells are seen at P8, which then steadily increases to reach adult values of 1,500 at P30 (Khalil et al., 2007; Panula et al., 2014).

Mast cells produce a range of mediators, some of which are preformed, whereas others are synthesized upon activation. These mediators include the biogenic amines histamine and serotonin, cytokines, specifically IL-1, IL-6, TNF-α, interferon-γ (IFN-γ), TGF-β, enzymes such as phospholipases, chymase, and mast cell proteases and tryptase, lipid mediators such as leukotrienes and prostaglandins, growth factors, nitric oxide, heparin, ATP and neuropeptides (Johnson and Krenger, 1992; Skaper et al., 2001; Dong et al., 2014b). Despite their small numbers they can affect numerous processes in the brain that have a potentially underestimated impact on neuroinflammation (see Figure 2). Preformed mediators may be released from secretory granules within seconds, followed by de novo formation of lipid mediators, cytokines and chemokines (Galli et al., 2005b; Nelissen et al., 2013; Silver and Curley, 2013). Mast cells are a heterogeneous cell type, with wide variation in mediator synthesis and release and a wide response in signaling pathways (Dong et al., 2014b) some of which seems to depend on histamine synthesis by mast cells itself (Ohtsu et al., 2001). Mast cells are an important source of histamine in the brain, with up to 50% of brain histamine levels in rodents attributable to the presence of mast cells (Yamatodani et al., 1982). This was established using high-performance liquid chromatography in mast cell deficient (KitW/Wv) mice compared to controls at 2–4 months after birth (Yamatodani et al., 1982). Such mice have reduced c-kit tyrosine kinase-dependent signaling, leading to impaired mast cell development and survival (Kitamura et al., 1978; Grimbaldeston et al., 2005). These mice are profoundly deficient in mast cells, with adult mice containing no detectable mast cells across numerous anatomical sites by 6–8 weeks of age (Kitamura et al., 1978).

Mast Cell Interactions With Microglia

Mast cells are a non-neuronal source of histamine that can be released upon degranulation. We have already discussed the role of histamine-mediated microglial activation and the release of the pro-inflammatory cytokines IL-6 and TNF-α in vitro via H1 and H4 receptors and MAPK and PI3K/AKT pathway activation (Dong et al., 2014a). Other implicated pathways include the complement 5a receptor and chemokine receptor 4/12 (CXCr4 and CXCL12) (Dong et al., 2014a) and the chemoattractant, C-C Motif Chemokine Ligand 5 (CCL5) (Hendriksen et al., 2017). We can therefore see an array of in vitro evidence for bidirectional interactions between mast cells and microglia in regulating neuroinflammation some of which are highlighted below (see also Figure 2).Dong et al. (2017) provided the first data on in vivo mast cell-microglial interactions, demonstrating that activation of brain mast cells by injecting the mast cell degranulator, C48/80 directly into the hypothalamus triggered microglial activation and the release of the pro-inflammatory cytokines, IL-6 and TNF-α. In turn, this was opposed by mast cell stabilization using sodium cromoglycate. Indeed, this resulted in a decrease in pro-inflammatory cytokines and reduced expression of the innate immune protein, toll-like receptor 4 (TLR4), and H1 and H4 receptors on the microglia. In turn, there was no effect on microglial activation in mast-cell deficient KitW–sh/W/–sh mice. Similar to the KitW/Wv mice discussed previously, these mice have reduced c-kit tyrosine kinase-dependent signaling, leading to impaired mast cell development and survival (Kitamura et al., 1978; Grimbaldeston et al., 2005). However, the specific mutation used is thought to lead to fewer developmental abnormalities that the KitW/Wv model while still retaining the desired mast cell deficiency (Yamazaki et al., 1994; Grimbaldeston et al., 2005). The findings by Dong et al. (2017) are important not only in confirming an interaction between mast cells and microglia in vivo, but also in highlighting the importance of mast cell degranulation for this interaction. Given that mast cell activation may be the first responder to injury (Jin et al., 2009b), and not the microglia, inhibition of mast cell activation may inhibit the pro-inflammatory cascade and therefore protect against neuroinflammation. What remains poorly understood is the contribution and role of histamine, if any, in this interaction. However, as the altered microglial expression of the H1 and H4 receptors depends on the activation state of mast cells (Dong et al., 2017) this may be suggestive that mast cell sources of histamine, not just neuronal sources, are crucial in the initiation of neuroinflammation.

Mast Cell Interactions With Astrocytes and Neurons

As well as mast cell-microglial interactions, there is some emerging evidence that mast cells may have direct interactions with CNS neurons and astrocytes as outlined below. Indeed, mast cells tend to co-localize with neurons (Skaper et al., 2012; Silver and Curley, 2013) or even to strongly adhere to neurons (Hagiyama et al., 2011). Neuronal release of neuropeptides such as NGF, neurotensin and substance P have been shown to bind directly bind to mast cells, altering their activation state (Kulka et al., 2008). Conversely, mast cells may also communicate with neurons via transgranulation, whereby mast cell granules can be inserted into adjacent neurons that alters neuronal responsiveness to its microenvironment (Wilhelm et al., 2005) (see Figure 3). Kempuraj et al. (2019) investigated such interactions in a mouse model of Parkinson’s disease. They found that mouse mast cell protease-6 and 7 induced the release of interleukin 33 (IL-33) from astrocytes and a mixed culture of glia and neuronal cells. This suggested that mast cells might interact with astrocytes and neurons to accelerate neuroinflammation and neurodegeneration. Kim et al. (2010) investigated the signaling pathways of activated mast cells and their interaction with astrocytes in experimental allergic encephalomyelitis. This was used as a model for the chronic demyelinating disease, multiple sclerosis. Co-culturing of mast cells with astrocytes led to increased release of histamine, leukotrienes and pro-inflammatory cytokines. It does so via enhanced expression of CD40L on mast cells, which is the natural ligand for CD40 expressed on astrocytes. This CD40-CD40L may therefore be important in chronic disease associated with neuroinflammation. Lenz et al. (2018) investigated the role of mast cells, and specifically histamine released from mast cell degranulation on neuronal development in the preoptic area of the hypothalamus. This is a crucial brain region in determining sexual behavior. Mast cell activation with the estrogen steroid hormone, estradiol, was found to stimulate microglial activation, subsequent prostaglandin release which was associated with increased dendritic spine density and the dendritic spine protein, spinophilin, as well as more masculinized sexual behavior. A small number of mast cells therefore had a profound effect on overall brain development and resultant behavior. To our knowledge, there are no further studies that have investigated the effect of mast cell activation and non-neuronal histamine directly on the CNS in vivo. However, bi-directional communication was recently demonstrated between mast cells and neurons in the skin (Zhang et al., 2021), which may demonstrate a role in the mediation of epidermal and dermal inflammation. Mast cell sources of histamine have also been implicated in the pathophysiology of neuropathic pain (Rosa and Fantozzi, 2013).




2. Overview and activation of MCs

Although the role of MCs is overlooked compared with microglia, MCs remain an important factor in the immune signaling pathway (29). MCs, the effector cells of the innate immune system, are derived from hematopoietic stem cells and multifunctional antigen-presenting cells and have a pivotal role in immunoglobulin type E (IgE)-associated allergic and inflammation-associated diseases (35). Despite their low numbers in most organs, MCs are present in both healthy and disease states. MCs are the first line of defense against invading pathogens and are distributed in almost all organs and vascularized tissues (36). Blood MCs express CD34 and contain cytoplasmic granules filled with heparin and histamine, the latter of which is released after binding to IgE. Unlike other myeloid-derived cells, tissue MCs have a hematopoietic developmental lineage (37,38). During MC development, immature lineage progenitors enter the circulation and are recruited to peripheral tissues by endothelial cells, regulating the appearance of granules with proteases (37,38). Human MCs may be classified into mucosal and connective tissue types according to the type of proteases present in their cytoplasmic granules; the mucosal type contains tryptase, whereas the connective tissue type contains both tryptase and chymase (39). MCs act as first responders and environmental ‘sensors’ to interact with other cellular elements involved in physiological and immune responses, promoting the neuroinflammation process (40). MCs are present in various areas of the brain and meninges. Although less distributed in the brain, they are generally found in the subthalamic nucleus, choroid plexus and the parenchyma of the hypothalamic region (41). The pathogenic roles of MCs were indicated to extend from allergic disease to autoimmune diseases and carcinogenesis (42-47).

The most common way through which MCs perform their function is degranulation. The activation of the inflammatory process results in a rapid release of MC granules into the interstitium. MC granules contain pre-formed and newly synthesized reactive chemicals known as MC mediators. These mediators include histamine, tryptase, chymase, interleukin families, tumor necrosis factor-α (TNF-α), serotonin, heparin, proteoglycans, vascular endothelial growth factor (VEGF), prostaglandins, leukotrienes, chemokines and growth factors, several of these are unique to MCs (42,48). Studies have indicated that MC degranulation may cause cognitive dysfunction (49). Large-scale MC degranulation may cause fatal anaphylaxis; however, most physiological functions of MCs, including regulation of inflammatory processes, occur without complete degranulation (50). MCs are phenotypically and functionally heterogeneous. The pathways and results of MC activation are multifaceted. In addition to IgE, MCs may also be activated through a number of other stimuli, including trauma, other immunoglobulins, complements, toll-like receptors (TLRs), neuropeptides, cytokines, chemokines and other inflammatory products, causing mast cell activation and leading to the selective release of mediators and/or stimulating T-cell proliferation, differentiation and migration (51,52). A characteristic of MC physiology that has been overlooked is that MCs are able to secrete mediators via differential or selective release without significant degranulation. This process may be regulated by the action of distinct protein kinases on a unique phosphoprotein (53). MCs undergo changes in the core of the electron-dense granules but without overt degranulation, a process that has been termed as activation, intragranular activation or piecemeal degranulation (54). MCs are essential for the pathogenesis of numerous inflammatory diseases, but this effect may only be achieved if MCs release selective mediators without degranulation, which may otherwise cause allergic reactions (52). Under normal circumstances, the brain does not express IgE receptor (FcεRI), since the brain does not display any allergic reactions and IgE does not cross the blood-brain barrier (BBB) under normal conditions (55).

The ways in which the mediators are secreted depend on the given stimuli and microenvironmental conditions. For instance, serotonin may be selectively released without histamine or arachidonic acid metabolites (56). The combination of TLR4 and mast cells does not cause degranulation but results in the secretion of inflammation-associated mediators. TLR4 binds to the co-receptors CD14 and MD-2 expressed by MCs. Subsequently, activation by myeloid differentiation primary response protein MyD88 innate immune signal transduction adaptor results in activation of interleukin (IL) receptor-associated kinase family members and pyruvate dehydrogenase kinase isoform 1, mitogen-activated protein kinases (MAPKs) p38 and JNK and to phospholipase A2(57). TLR4 also binds to lipopolysaccharides (LPS) and induces TNF-α release without degranulation (58). LPS induces secretion of IL-5, IL-10 and IL-13 but not granulocyte-macrophage colony-stimulating factor, IL-1 or leukotriene C4 (LTC4) (58). The selective release of IL-6 occurs in the MC response to LPS, provided the presence of the PI3K inhibitor wortmannin or stem cell factors (59). Corticotropin-releasing hormone (CRH) was demonstrated to stimulate the selective release of VEGF without degranulation and histamine or tryptase release from the human leukemic mast cell line HMC-1 and human umbilical cord blood-derived mast cells (60). Neurotensin (NT) induces expression of CRH receptor (CRHR)-1 on MCs and NT and CRH are released under stress via NT-CRH crosstalk (61). IL-1 stimulates human MCs to selectively release IL-6 without degranulation, via a unique process utilizing 40-80 nm vesicles unrelated to the length of secretory granules (800-1,000 nm) (62). IL-33 may serve as a potent activator of MCs and was reported to promote MC survival, maturation, migration and adhesion, and to selectively produce a variety of pro-inflammatory cytokines, including IL-4, IL-5, IL-6, IL-8 and IL-13 and chemokines including macrophage inflammatory protein-1α and monocyte chemoattractant protein 1 (MCP-1) (63,64). IL-33 enhances the role of the pro-inflammatory peptide substance P in stimulating human MCs to secrete high levels of VEGF and TNF via the interaction of neurokinin 1 and ST2 receptors without concomitant secretion of tryptase (65). In the presence of stem cell factor, IL-33 may also induce TNF production in MCs via a MAPK-activated protein kinases 2 and 3, ERK1/2- and PI3K-dependent pathways (66). Understanding the selective release of mediators may explain how MCs participate in numerous biological processes and how they are capable of exerting both immunostimulatory and immunosuppressive effects.

3. MC-glia crosstalk

Microglia and MCs are the two most important cell types mediating and regulating neuroinflammation in the brain. There is a close association between MCs and glial cells. MCs are generally clustered near the glia in neuroinflammatory conditions to recruit and activate other inflammatory cells, where neuroinflammation already occurs in the brain. The contribution of MCs and glia to neuroinflammation is strongly influenced by the likelihood of their crosstalk and pathological exacerbation (29). MCs may interact with microglia and astrocytes via the complement system, proteases, TLRs and chemokines. MCs may participate in the migration and activation of glia, thereby affecting the release of inflammatory mediators. The expression of ligand-receptor pairings may be upregulated under inflammatory conditions, facilitating chemotactic actions through contact between MC and glia (27). For instance, C5a, the chemoattractant anaphylatoxin peptide and its receptor CD88 are upregulated in the glia of inflammatory CNS tissues (67-69). Complementary expression of the C5a receptor on activated MCs produces an intense chemoattractant signal to the C5a peptide and intense crosstalk between C5a and TLR4, which also has a role in neuroinflammation (67-69). TLRs are a major class of pattern recognition receptors involved in innate immunity. TLRs are associated with groups of pathogens recognized by innate immune system cells, including microglia and MCs, and act as a bridge between non-specific and specific immunity (70). Upregulation of C-C motif chemokine 5 (CCL5; also known as RANTES) by MC activation leads to a pro-inflammatory response in microglia, releasing IL-6 and CCL5, which in turn promotes chemokine expression in MC (71). IL-33 is an activator of MCs and IL-33 release from astrocytes may activate brain MCs and microglia (72). The binding of IL-33 to MC receptors leads to the secretion of IL-6, IL-13 and MCP-1 to regulate microglia activity. Furthermore, IL-33 may be stimulated from microglia pre-activated with pathogen-associated molecular patterns via TLRs (73,74). Together, MC protease and matrix metalloproteinase (MMP) activate p38, ERK1/2, MAPKs and transcription factors including NF-κB in astrocytes, microglia and MCs (75). IL-6 and TNF-α released from microglia upregulate protease-activated receptor 2 (PAR2) expression in MCs, causing MC activation and TNF-α release (76). MC tryptase may induce the release of pro-inflammatory mediators such as TNF-α, IL-6 and reactive oxygen species (ROS) via the PAR2/MAPK/NF-κB signaling pathway and activation of PAR2 receptors on MCs, which then contributes to the development of microglia-mediated inflammation in the brain (77). IL-6 induces IL-13 release from MCs, affecting the expression of TLR2/TLR4. Furthermore, TNF-α upregulates PAR2 expression in MCs and enhances PAR2-mediated MC activation and degranulation (78-80). C-X-C chemokine receptor type 4 (CXCR4; also known as stromal cell-derived factor 1) is an MC chemotaxin and studies have indicated that CXCR4 is upregulated in hypoxia and ischemia, promoting the migration and activation of microglia (81). In addition to microglia, astrocytes sharing a perivascular localization with MCs maintain the viability of MCs. Astrocytes express histamine receptors and release cytokines/chemokines through Rho-family GTPases/Ca2+-dependent protein kinase C isoforms, MAPK, NF-κB and signal transducer and activator of transcription 1 (82-84). These trigger MC degranulation and enhance CD40L and CD40 surface expression, leading to further inflammation (82-84). Both microglia and astrocytes express histamine receptor H1 (HRH1), HRH2 and HRH3 and MCs may affect the activity of microglia and astrocytes through these receptors (85,86). An in vitro study has indicated that MC proteases may induce demyelination and apoptosis of oligodendrocytes, while myelin promotes MC degranulation (87). Several experiments have confirmed the relationship between MCs and glia. Co-culture of microglia and HMC-1 cells revealed that activated HMC-1 cells stimulate the activation of microglia and subsequent production of pro-inflammatory factors TNF-α and IL-6(88). MC degranulator compound 48/80 induces microglia activation and inflammatory cytokine production, triggering an acute brain inflammatory response. However, the MC stabilizer cromolyn inhibits this effect, reduces inflammatory cytokines and inhibits the MAPK, AKT and NF-κB signaling pathways. Furthermore, cromolyn inhibits HRH1, HRH4, protease activity, PAR2 and TLR4 in microglia (49,89). Incubation of astrocytes and neurons with 1-methyl-4-phenylpyridinium, glia maturation factor (GMF), mouse MC protease-6 (MMCP-6) and MMCP-7 increased PAR-2 expression, suggesting contact between MCs and astrocytes (90).

4. MC-neuron interactions

The connection between MCs and neurons mainly occurs through peripheral interactions. A number of studies have revealed the association between MCs and neurons in CNS neuroinflammation. In the brain, the co-localization of MCs and neurons provides a basis for neuroimmunological interactions. Cell adhesion molecule-1 (CADM1), expressed by mature hippocampal neurons, may have an important role in the development of MC neuron interactions (91). In the CNS, MC-derived products may enter adjacent neurons to insert their granular contents, a process known as granulation. In this way, MCs change the internal environment of neurons, presenting a novel form of neuroimmunological interaction (92). In addition, MCs express a series of neurotransmitter receptors, which may be directly activated, enhanced [neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2] or inhibited (acetylcholine receptor) (93,94). Furthermore, it was reported that activated MCs enhanced excitotoxic damage to 60% when co-cultured with hippocampal neurons. In N-methyl-D-aspartate receptor-mediated synaptic neurotransmission, MC-derived histamine directly increases the death of hippocampal neurons (95). Tryptase released by MCs may directly activate proteinase-activated receptors on neurons and MC-derived TNF-α has a vital role in neuronal development, cell survival, synaptic plasticity and ionic homeostasis in the CNS (96). These MC-neuron interactions are thought to be involved in the pathogenesis of numerous neuroinflammatory diseases.

5. MCs and the HPA axis

The association between chronic stress and neuroinflammation has been confirmed by numerous studies. MCs have a vital role in the mechanism of brain damage caused by chronic stress on the brain. A variety of psychological and physiological stresses may lead to changes in the expression, distribution and activity of MCs in the CNS. Stress and pro-inflammatory cytokines activate the HPA axis, thus leading to an increase in CRH and arginine vasopressin release from the paraventricular nucleus of the hypothalamus. HPA axis activation also enhances the expression of CRH receptors, vascular permeability and MC activation (97). CRH released from MCs activates MCs and glia in the CNS in an autocrine and paracrine manner in the context of stress and neuroinflammation (98). In turn, activation of CNS MCs activates the HPA axis. MCs are located near CRH-positive neurons in the median eminence and are closely linked to corticotropin-releasing factor receptors, which may be activated by CRH (99). This may be closely associated with the meningeal vasodilation and increased secretion of cytokines during meningeal inflammation in migraines (46). Cao et al (100) indicated that intravesical stress, CRH, MC activation and VEGFs have a crucial role in the stress-induced deterioration of inflammation, which may provide insight into the mechanism of brain stress. MC activation and CRH release increase BBB permeability, leading to further brain damage and contributing to chronic neuroinflammation in the brain (60,101). Microglia express CRH receptors and activation of microglia by CRH leads to the release of harmful inflammatory mediators in psychiatric diseases, such as AD and pain (102,103). Human MCs synthesize and secrete CRH and express functional CRH receptors (CRHR1 and CRHR2) (104). CRHR1-mediated activation of microglia induces microglia proliferation, TNF-α release and activation of MAPK. CRHR1 also mediates stress-induced MC degranulation (105). CRH release from activated MCs may also activate glial cells in neurodegenerative diseases such as AD (103,106). Stressful conditions, including trauma or hypoxia, also activate peripheral MCs, which in turn activate CRH and substance P pathways, leading to BBB leakage and glial activation, causing further neuroinflammation and neurodegeneration (107). CRH concentrations are higher in brain regions prone to developing a pathology of AD (108). Elevated cortisol levels and HPA axis dysfunction are implicated in chronic stress, which releases amyloid beta (Aβ) that causes and/or worsens AD (109). CRHR1 antagonists have been indicated to decrease stress-mediated oxidative damage, prevent cognitive damage and loss of dendritic cells and reduce Aβ deposition in the brain (110). These results confirm the correlation between CRH and AD. Other neuropeptides, including NT, may work with CRH to enhance MC activation and release of excessive inflammatory mediators under stress (61). CRH may enhance VEGF release from human MCs and induce FcεRI expression in MCs, and this effect may be blocked by the natural flavone luteolin (111). CRH is also implicated in the pathogenesis of PD. Emotional chronic stress, which is closely associated with CRH, enhances glial activation and aggravates neuronal death through inflammation in the substantia nigra of the brain of patients with PD (107). Furthermore, observations in animal models of PD indicate that stress-induced striatal damage may subsequently worsen motor symptoms (112).

6. MCs and the BBB

The BBB is composed of functional cerebral blood vessels, which create a stable CNS environment and protect brain parenchymal cells from harmful substances in the immune cells and blood. The BBB consists of tightly connected endothelial junctions and several intact transmembrane proteins, including claudin and occludin, that ensure its integrity. The basal lamina, which is part of the extracellular matrix, connects the endothelial cells of the BBB to adjacent cell layers (113). BBB destruction involves the accumulation of multiple vascular and neurotoxic molecules within the brain parenchyma, decreased cerebral blood flow and hypoxia (114). MCs are present in the dura mater and meninges, as well as on the cerebral side of the BBB, and MCs are in contact with the distal ends of the astrocytes (115). MCs may cross the BBB and blood-spinal cord barrier when the barrier is damaged by CNS pathologies. Inflammatory factors released by MC activation, including histamine, tryptase, chymotrypsin and TNF-α, may regulate BBB permeability (116). Furthermore, TNF-α induces the expression of intercellular adhesion molecule 1 (ICAM-1) and allows leukocytes to enter the affected tissues in the brain (117). The me chanism by which MCs destroy the BBB and promote basal layer degradation may involve vascular activity and matrix degradation components of MCs. MCs affect the integrity of the BBB through MMPs, whose enzymatic activity may be regulated by tissue MMP inhibitors. These include histamine and protease chymase, trypsin and cathepsin G (118). Cathepsin G activates MMPs, which degrade most of the protein components of the neurovascular matrix (118). In cerebral ischemic disease, MC degranulation increases, and brain MCs affect the activation of acute microvascular gelatinases (MMP-2 and -9) by releasing proteases to affect BBB destruction. In addition, elevated levels of VEGF may cause BBB rupture, vascular leakage and edema, which in turn causes stroke (119,120). This process extravasates glutamate and albumin, activates astrocytes, alters K+ homeostasis in the brain parenchyma and leads to excessive neuronal excitation and inflammatory cell entry (119,120). In experimental autoimmune encephalomyelitis (EAE), activation of meningeal MCs leads to TNF-α production and early neutrophil recruitment (121). This promotes local BBB destruction, allowing initial immune cells to enter the CNS and aggravate neuroinflammation (121). An in vitro study revealed that TNF-α induces downregulation of tight junction proteins occludin, claudin-5 and vascular endothelial-cadherin via an increase in ROS, which leads to increased paracellular permeability (122). IL-6 participates in the effect of TNF-α on endothelial monolayers. TNF-α upregulates the expression of ICAM-1 and vascular cell adhesion molecule-1 on brain microvascular endothelial cells (123). ICAM-1 is involved in leukocyte adhesion to the endothelium and its upregulation and leukocyte-mediated BBB breakdown are one of the pathological mechanisms and characteristics of various brain inflammatory diseases, including MS (123). Brain MCs may induce post-operative cognitive dysfunction by destabilizing the BBB and acute stress may cause BBB breakdown by activating MCs (88,124). In addition to cerebral ischemia, BBB destruction has also been detected in dementia, motor neuron disease, MS, AD and other neuropsychiatric disorders (125-128). Substance P, which is released following traumatic brain injury or under stress, activates MCs and glia, releasing neuroinflammatory mediators and increasing BBB permeability (129). The release of CRH from MCs contributes to the subsequent release of various neuroinflammatory and neurotoxic mediators, leading to BBB rupture and glial cell activation, chronic neuroinflammation in the brain and causing autism (130). Cromoglycate, a MC-stabilizing agent, reversed BBB destruction, brain edema and neutrophil recruitment post-ischemia by inhibiting MC activation in a stroke model (131).





In summary, the results presented here are the first to reveal the function of MMP3 in the BBB and suggest that it has an essential role in the brain microvasculature that differs from its function in other vessels. We have shown that MMP3 increases BBB permeability by upregulating the ERK signaling pathway, which subsequently reduces TJ and AJ protein abundance in BMVECs. Oxidative stress often leads to impairment of BBB. Since the BBB is the primary regulator of exchange between the peripheral blood and the brain, our observations likely have important implications for treating neuroinflammatory conditions and other CNS disorders involving the endothelial MMP3 pathway.





4.1. Mast Cells: Guardians of Homeostasis in the Brain

Although they are often described in the context of pathology and disease, mast cells are likely important regulators of homeostasis, since mast cell mediators can have both beneficial and harmful effects depending on the context in which they are deployed. This may also be the case in the brain. For example, activated mast cells rapidly release a series of immunomodulatory molecules, such as histamine and TNF-α. In organotypic slice cultures and primary rat astrocyte-neuron co-cultures, exogenously added histamine was shown to protect hippocampal neurons against glutamate-induced excitotoxicity [63]. Neuroprotection was mediated by increased expression of astrocytic glutamate transporter-1 (GLT-1), probably due to reduction in extracellular glutamate levels. Mast cell-derived proteases and proteoglycans also might provide neuroprotection [64]. In a mouse model of ischemic injury, TNF-α was shown to promote the survival of hippocampal and striatal neurons, probably acting via its receptor (tumor necrosis factor receptor 2 (TNFR2)) [65]. The protective or detrimental effects of TNF-α might depend on the concentration and duration of release as well as receptor binding (TNFR1 vs TNFR2) [66]. Intracerebral mast cell secrete proteases, vasoactive molecules such as nitric oxide, lipid mediators, histamine, gonadotropin-releasing hormone and TNF-α which can increase BBB permeability by breaking down the tight junctions between brain endothelial cells [67]. Thus in pathological situations, mast cells appear to operate in a feed-forward mechanism; loss of BBB integrity might activate meningeal mast cells to recruit inflammatory cells to the CNS, leading to a vicious cycle of neuroinflammation [68]. These findings suggest that the beneficial or detrimental effects of mast cells to brain health might be dependent on several factors, including levels and time of cytokine release and the magnitude of the initial insult.


A growing number of recent studies have shown that under pathological conditions, microglia activates a defense program and transition from the ‘homeostatic state’ into the ‘disease-associated’ state and during disease progression might “lose control” and transition into a ‘neurodegenerative-disease state’ and become dysfunctional and destructive to the CNS [50,77]. Microglia from each disease-associated state express a unique transcriptional signature [78,79]. Whether microglia exacerbate or inhibit disease progression is still being actively debated, with both beneficial and detrimental roles ascribed to these cells in neurodegeneration [80].


Under pathological state, microglia continuously release excessive amounts of pathogenic pro-inflammatory mediators, excitatory amino acids (e.g., glutamate), complement activation products, proteolytic enzymes and RNOS, which, in turn, generates oxidative stress [84]. Exposure of in vitro neuronal-glia cultures to glutamate resulted in oxidative stress and neurotoxicity [85]. Microglia respond to misfolded β-amyloid by producing RNOS, notably superoxide anions via the NADPH oxidase. These reactive species can be inactivated by extracellular SOD to produce hydrogen peroxide, which can be further detoxified by the antioxidant defense systems, namely SOD, GPX, PRX, and HO [82]. Superoxide can also react rapidly with nitric oxide to produce peroxynitrite, a potent oxidizing and nitrating agent [86]. Interestingly, NADPH oxidase activity is increased in activated microglia in early stage AD patients as compared to age-matched controls [87].



Mast cells and microglia are found in close proximity to each other in the CNS, facilitating active communication. Mast cells likely use their surface receptors, adhesion molecules and ‘mast cell mediators’ to engage in a complex cross-talk with other brain-resident cells, which could be both unidirectional and bidirectional. Increasing evidence suggests that the initiation and propagation of neuroinflammation relies on the interactions between these cell types. Mast cells also express several costimulatory and inhibitory surface molecules that allows them to communicate with other immunocompetent cells, such as T-cells and B-cells, positioning them as a main bridge between innate and adaptive immunity [37,100].

Activated brain mast cells release histamine that can cause phenotypic changes and activation of microglial cells [101]. Exogenously added histamine triggered activation of cultured primary cortical microglia, murine N9 microglia and hippocampal organotypic slice cultures to secrete TNF-α and IL-6 and RNOS [102,103]. Although histamine stimulated microglial cell motility in control microglia, histamine inhibited microglial migration and IL-1β release in LPS-stimulated microglia, suggesting a dual role of histamine in modulating microglia-induced inflammatory responses [102,104]. Histamine exerts its functions by signaling through four types of G-protein coupled receptors, namely Histamine 1 receptor (H1R), H2R, H3R, and H4R, which are expressed on innate immune cells, neurons and endothelial cells [105]. In these cultures, the anti-inflammatory effects of histamine were mediated by activation of H4R which involved α5β1 integrin, p38 and protein kinase B (AKT) signaling to restrain exacerbated microglial responses in neuroinflammation [102]. All four types of histamine receptors are expressed by microglial cells and can modulate microglia-mediated neuroinflammation [104]. Rocha SM et. al., directly injected histamine into the substantia nigra of mice and studied its effects on microglial activity and dopaminergic neuron survival [106]. In accordance with other studies, histamine induced microglial activation and dopaminergic neuronal toxicity via H1R activation, probably through NADPH oxidase dependent oxidative stress signaling pathways and microglia phagocytosis. Altogether, these studies show that histamine per se triggers a pro-inflammatory response and under inflammatory conditions, histamine activates an anti-inflammatory response, dampens microglial-induced inflammation and is associated with neuroprotection.

Similar to peripheral mast cells, brain mast cells are also known to secrete proteases, such as tryptase. Exposure of primary microglia to mast cell-derived tryptase stimulated microglia to subsequently secrete TNF-α and IL-6 and RNOS. These effects were mediated by protease-activated receptor-2 (PAR-2) signaling via activation of mitogen-activated protein (MAP) kinase (Erk and p38) and NF-kappa B (NF-kB) pathways [68]. Furthermore, PAR-2 activation induces the expression of ATP-sensitive ionotropic P2X4 receptors on microglia and exposure to ATP leads to secretion of BDNF, a potent trophic factor [107]. The presence of functional P2X4 receptors are also expressed by human mast cell lines [108]. Since mast cells play a pivotal role in neuroinflammation, it is important to determine the exact molecular mechanisms employed by activated mast cells and microglia and their role in disease progression.

Mast cell-derived pro-inflammatory cytokines such as CCL2, TNF-α and IL1β can also influence microglia activation. To recapitulate in vitro mast cell-glia-neuron crosstalk during neuroinflammation, mast cells were cocultured with mixed cultures of neuron and glia or enriched cultures of neurons or astroglia and challenged with MPP+ or GMF or mast cell proteases [109]. Mast cells cocultured with glia had increased production of CCL2 and IL-33, highlighting the importance of mast cell-glia coupling and their role in neuroinflammation [110]. Increased CCL2 levels have been demonstrated in AD patients which is associated with accelerated cognitive decline and AD progression [111]. CCL2 expression altered β-amyloid phagocytosis, supporting the notion that microglial phagocytosis could be regulated by mast cells [112]. Although there is no direct evidence of CCL2 expression in the brain of PD patients, increased serum levels of CCL2 have been reported [113]. Recently, two polymorphisms have been reported in the promoter region of CCL2 which are associated with an increased risk of PD [114]. The exact role of CCL2-CCR2 axis in regulating mast cells and microglia in neurodegenerative diseases is still not well understood.

Mast cell degranulation has been shown to activate microglia. Stereotaxic injection of a mast cell degranulation compound 48/80 (C48/80) and activator of the mas-related G protein-coupled receptor Mrgpr [115] in the hypothalamus of rats induced mast cell degranulation, production of pro-inflammatory cytokines and microglia activation [103]. These effects were mediated by activation of MAP kinase and AKT pathways and an increased protein expression of H1R, H4R, PAR-2 and TLR4 on microglial cells. Treatment with a mast cell stabilizer disodium cromoglycate (cromolyn), inhibited microglial activation and downstream signaling, suggesting mast cell involvement. Most importantly, C48/80 had no effect on microglial activation in mast cell-deficient Kitw-sh/w-sh mice. These data support the notion that stabilization of brain mast cells during neuroinflammation could be a new therapeutic strategy to restrain microglial hyperactivity. Tranilast has been used to inactivate the NLR family pyrin domain containing 3 (NLRP3) inflammasome, yet is also used as an anti-allergy medication as a “mast cell stabilizer” [116] suggesting that its effect in the brain may also modulate mast cell functions via the inflammasome.




TNF released by microglia has an important role in regulating synaptic plasticity [110]. Specifically, it controls a process called synaptic scaling, i.e., the adjustment of synaptic strength in response to prolonged changes in the electrical activity of neurons [110,111]. Indeed, a reduction of glutamate transmission increases microglial TNF release, which promotes the expression of AMPA glutamate receptors in neurons. Conversely, increased extracellular glutamate concentration inhibits TNF release from microglia, additional glutamate receptor expression, and declines neuronal activity [111–113]. The increase of AMPA receptor GluR1 subunit expression does not occur at mRNA level, but this is controlled by TNF at post-transcriptional level [114]. Subsequent studies revealed that TNF facilitates the trafficking and membrane insertion of AMPA receptors at the neuron surface, which are crucial for the homeostatic synaptic plasticity. Specifically, hippocampal neurons exposed to TNF increase surface expression of GluR1 subunit through modulation of NF-κB and acid sphingomyelinase pathways [115]. TNF not only controls homeostatic synaptic activity, but also induces neurotoxicity via autocrine/paracrine loops involving other endogenous mediators. First, TNF activates TNFR1 on microglia, amplifying its production and release [95]. Second, microglia-derived TNF activates TNFR1 expressed on astrocytes, allowing glutamate release from the glial cells. This, in turn, activates its specific receptors, including the metabotropic mGluR2 receptor on microglia, potentiating microglial TNF production and affecting synaptic transmission [110]. ATP, released by microglia concurrently with TNF, contributes to TNF-mediated neuronal damage by inducing a prolonged activation of microglial P2X7 receptor and release of both IL-1β and TNF inflammatory cytokines. In addition, both microglial TNF and ATP trigger adjacent astrocytes to release additional ATP, that amplifies microglia response and promotes astroglial release of glutamate, aggravating neuronal dysfunction [110]. Moreover, TNF mediates neuronal death by increasing extracellular levels of the excitotoxic transmitter glutamate and excessive AMPA receptor activation via downregulation of the astrocytic glutamate transporter EAAT2/GLT1 [116]. The effects of TNF on N-methyl-D-aspartate receptors (NMDARs) trafficking are less characterized. However, it has been demonstrated that, in hippocampal neurons, TNF increases the expression of the NR1 subunit of NMDAR and its specific clustering into lipid rafts [117]. Accordingly, treatment of human neuronal cultures with competitive (2-APV) and noncompetitive (MK-801) NMDA receptor antagonists reduced the glutamate neurotoxicity induced by TNF [118].




Interleukin-1β Causes Anxiety by Interacting with the Endocannabinoid System



1. increase in IL-1b proinflammatory cytokine

2 decrease in cb1 recebtor binding

3. decreases CB1R's (gabaA) synapse binding in striatum

4. causes behavioral manifestations closely resembling anxious-depressive symptoms in humans, including anhedonia, reduced exploratory behaviors, social withdrawal, fatigue, and sleep disturbances

5. This entire process requires "intact function of the transient receptor potential vanilloid 1 (TRPV1)" to work.



IL-1beta, but not IL-10 or tumour necrosis factor (TNF)-alpha, down-regulated the surface expression and Ser831 phosphorylation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1. Agents that block IL-1beta receptor activity abolished these effects. In contrast, no change in the surface expression of the N-methyl-d-aspartate (NMDA) receptor subunit NR1 was observed.

The inhibition of NMDA receptor activity or depletion of extracellular calcium blocked IL-1beta effects on GluR1 phosphorylation and surface expression. NMDA-mediated calcium influx was also regulated by IL-1beta.

These findings suggest that IL-1beta selectively regulates AMPA receptor phosphorylation and surface expression through extracellular calcium and an unknown mechanism involving NMDA receptor activity.

Interleukin-1 beta modulates AMPA receptor expression and phosphorylation in hippocampal neurons




Losing Connections, Losing Memory: AMPA Receptor Endocytosis as a Neurobiological Mechanism of Forgetting







Functional role of the endocannabinoid system and AMPA/kainate receptors in 5-HT2A receptor-mediated wet dog shakes.


AMPA/kainate receptors play a role in the mediation of 5-HT2A receptor activity, whereas the endocannabinoid system may act as a regulatory buffer system during periods of elevated activity, but not under basal conditions.





The prefrontal cortex (PFC) plays a key role in many high-level cognitive processes. It is densely innervated by serotonergic neurons originating from the dorsal and median raphe nuclei, which profoundly influence PFC activity. Among the 5-HT receptors abundantly expressed in PFC, 5-HT2A receptors located in dendrites of layer V pyramidal neurons control neuronal excitability and mediate the psychotropic effects of psychedelic hallucinogens, but their impact on glutamatergic transmission and synaptic plasticity remains poorly characterized. Here, we show that a 20-min exposure of mouse PFC slices to serotonin or the 5-HT2A receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) produces a long-lasting depression of evoked AMPA excitatory postsynaptic currents in layer V pyramidal neurons. DOI-elicited long-term depression (LTD) of synaptic transmission is absent in slices from 5-HT2A receptor-deficient mice, is rescued by viral expression of 5-HT2A receptor in pyramidal neurons and occludes electrically induced long-term depression. Furthermore, 5-HT2A receptor activation promotes phosphorylation of GluA2 AMPA receptor subunit at Ser880 and AMPA receptor internalization, indicating common mechanisms with electrically induced LTD. These findings provide one of the first examples of LTD gating under the control of a G protein-coupled receptor that might lead to imbalanced synaptic plasticity and memory impairment following a nonphysiological elevation of extracellular serotonin.


Sustained Activation of Postsynaptic 5-HT2A Receptors Gates Plasticity at Prefrontal Cortex Synapses


- ----


Seven days post-intervention, there was still significantly higher SV2A density in hippocampus (+9.24%) and PFC (+6.1%) whereas there were no longer any differences in 5-HT2AR density. Our findings suggest that psilocybin’s antidepressive actions are linked to increased persistent synaptogenesis and possibly also to an acute decrease in 5-HT2AR density.


A Single Dose of Psilocybin Increases Synaptic Density and Decreases 5-HT2A Receptor Density in the Pig Brain



THE EYE: Research more**

A retinal ganglion cell (RGC) is a type of neuron located near the inner surface (the ganglion cell layer) of the retina of the eye.

A retinal ganglion cell function and output can be influenced by retinal glia that include Müller cells that span the tissue (and provide critical ionic, metabolic and modulatory of support for neurons, [68]), protoplasmic astrocytes that line the inner limiting membrane (ILM) and (in vascularized retinas) control the permeability of the blood-retina barrier, and microglia, which represent the resident immune cells [69]. Whilst organization of retinal circuits represents a key determinant of visual information processing, the physiological state of every cell type is dynamically altered through activity-dependent and neurodegeneration-driven changes in calcium homeostasis, functional and structural connectivity across and between retinal laminae [67,69,70].





sleepy..... so much turkey... 

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question: in what scenario does a brains astrocytes become so over reactive they cannot shut off?

Chronic mast cell activation

mtorc1 inhibition (mtorc1 activation critical for astrocyte "deactivation" and to "turn off" mast cells)

cb1: antagonization: CB1 receptor blockade with rimonabant attenuated mTORC1 overactivation


How could this occur?

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