Adenosine signalling to astrocytes coordinates brain metabolism and function.

Brain computation performed by billions of nerve cells relies on a sufficient and uninterrupted nutrient and oxygen supply1,2. Astrocytes, the ubiquitous glial neighbours of neurons, govern brain glucose uptake and metabolism3,4, but the exact mechanisms of metabolic coupling between neurons and astrocytes that ensure on-demand support of neuronal energy needs are not fully understood5,6. Here we show, using experimental in vitro and in vivo animal models, that neuronal activity-dependent metabolic activation of astrocytes is mediated by neuromodulator adenosine acting on astrocytic A2B receptors. Stimulation of A2B receptors recruits the canonical cyclic adenosine 3',5'-monophosphate-protein kinase A signalling pathway, leading to rapid activation of astrocyte glucose metabolism and the release of lactate, which supplements the extracellular pool of readily available energy substrates. Experimental mouse models involving conditional deletion of the gene encoding A2B receptors in astrocytes showed that adenosine-mediated metabolic signalling is essential for maintaining synaptic function, especially under conditions of high energy demand or reduced energy supply. Knockdown of A2B receptor expression in astrocytes led to a major reprogramming of brain energy metabolism, prevented synaptic plasticity in the hippocampus, severely impaired recognition memory and disrupted sleep. These data identify the adenosine A2B receptor as an astrocytic sensor of neuronal activity and show that cAMP signalling in astrocytes tunes brain energy metabolism to support its fundamental functions such as sleep and memory.

Memantine suppresses the excitotoxicity but fails to rescue the ataxic phenotype in SCA1 model mice.

Spinocerebellar ataxia type 1 (SCA1) is a debilitating neurodegenerative disorder of the cerebellum and brainstem. Memantine has been proposed as a potential treatment for SCA1. It blocks N-methyl-D-aspartate (NMDA) receptors on neurons, reduces excitotoxicity and decreases neurodegeneration in Alzheimer models. However, in cerebellar neurodegenerative diseases, the potential value of memantine is still unclear. We investigated the effects of memantine on motor performance and synaptic transmission in the cerebellum in a mouse model where mutant ataxin 1 is specifically targeted to glia. Lentiviral vectors (LVV) were used to express mutant ataxin 1 selectively in Bergmann glia (BG). In mice transduced with the mutant ataxin 1, chronic treatment with memantine improved motor activity during initial tests, presumably due to preserved BG and Purkinje cell (PC) morphology and numbers. However, mice were unable to improve their rota rod scores during next days of training. Memantine also compromised improvement in the rota rod scores in control mice upon repetitive training. These effects may be due to the effects of memantine on plasticity (LTD suppression) and NMDA receptor modulation. Some effects of chronically administered memantine persisted even after its wash-out from brain slices. Chronic memantine reduced morphological signs of neurodegeneration in the cerebellum of SCA1 model mice. This resulted in an apparent initial reduction of ataxic phenotype, but memantine also affected cerebellar plasticity and ultimately compromised motor learning. We speculate that that clinical application of memantine in SCA1 might be hampered by its ability to suppress NMDA-dependent plasticity in cerebellar cortex.

Contributions of carotid bodies, retrotrapezoid nucleus neurons and preBötzinger complex astrocytes to the CO2 -sensitive drive for breathing.

Current models of respiratory CO2 chemosensitivity are centred around the function of a specific population of neurons residing in the medullary retrotrapezoid nucleus (RTN). However, there is significant evidence suggesting that chemosensitive neurons exist in other brainstem areas, including the rhythm-generating region of the medulla oblongata - the preBötzinger complex (preBötC). There is also evidence that astrocytes, non-neuronal brain cells, contribute to central CO2 chemosensitivity. In this study, we reevaluated the relative contributions of the RTN neurons, the preBötC astrocytes, and the carotid body chemoreceptors in mediating the respiratory responses to CO2 in experimental animals (adult laboratory rats). To block astroglial signalling via exocytotic release of transmitters, preBötC astrocytes were targeted to express the tetanus toxin light chain (TeLC). Bilateral expression of TeLC in preBötC astrocytes was associated with ∼20% and ∼30% reduction of the respiratory response to CO2 in conscious and anaesthetized animals, respectively. Carotid body denervation reduced the CO2 respiratory response by ∼25%. Bilateral inhibition of RTN neurons transduced to express Gi-coupled designer receptors exclusively activated by designer drug (DREADDGi ) by application of clozapine-N-oxide reduced the CO2 response by ∼20% and ∼40% in conscious and anaesthetized rats, respectively. Combined blockade of astroglial signalling in the preBötC, inhibition of RTN neurons and carotid body denervation reduced the CO2 -induced respiratory response by ∼70%. These data further support the hypothesis that the CO2 -sensitive drive to breathe requires inputs from the peripheral chemoreceptors and several central chemoreceptor sites. At the preBötC level, astrocytes modulate the activity of the respiratory network in response to CO2 , either by relaying chemosensory information (i.e. they act as CO2  sensors) or by enhancing the preBötC network excitability to chemosensory inputs. KEY POINTS: This study reevaluated the roles played by the carotid bodies, neurons of the retrotrapezoid nucleus (RTN) and astrocytes of the preBötC in mediating the CO2 -sensitive drive to breathe. The data obtained show that disruption of preBötC astroglial signalling, blockade of inputs from the peripheral chemoreceptors or inhibition of RTN neurons similarly reduce the respiratory response to hypercapnia. These data provide further support for the hypothesis that the CO2 -sensitive drive to breathe is mediated by the inputs from the peripheral chemoreceptors and several central chemoreceptor sites.

Indirect Negative Effect of Mutant Ataxin-1 on Short- and Long-Term Synaptic Plasticity in Mouse Models of Spinocerebellar Ataxia Type 1.

Spinocerebellar ataxia type 1 (SCA1) is an intractable progressive neurodegenerative disease that leads to a range of movement and motor defects and is eventually lethal. Purkinje cells (PC) are typically the first to show signs of degeneration. SCA1 is caused by an expansion of the polyglutamine tract in the ATXN1 gene and the subsequent buildup of mutant Ataxin-1 protein. In addition to its toxicity, mutant Ataxin-1 protein interferes with gene expression and signal transduction in cells. Recently, it is evident that ATXN1 is not only expressed in neurons but also in glia, however, it is unclear the extent to which either contributes to the overall pathology of SCA1. There are various ways to model SCA1 in mice. Here, functional deficits at cerebellar synapses were investigated in two mouse models of SCA1 in which mutant ATXN1 is either nonspecifically expressed in all cell types of the cerebellum (SCA1 knock-in (KI)), or specifically in Bergmann glia with lentiviral vectors expressing mutant ATXN1 under the control of the astrocyte-specific GFAP promoter. We report impairment of motor performance in both SCA1 models. In both cases, prominent signs of astrocytosis were found using immunohistochemistry. Electrophysiological experiments revealed alteration of presynaptic plasticity at synapses between parallel fibers and PCs, and climbing fibers and PCs in SCA1 KI mice, which is not observed in animals expressing mutant ATXN1 solely in Bergmann glia. In contrast, short- and long-term synaptic plasticity was affected in both SCA1 KI mice and glia-targeted SCA1 mice. Thus, non-neuronal mechanisms may underlie some aspects of SCA1 pathology in the cerebellum. By combining the outcomes of our current work with our previous data from the B05 SCA1 model, we further our understanding of the mechanisms of SCA1.

Memantine Disrupts Motor Coordination through Anxiety-like Behavior in CD1 Mice.

Memantine is an FDA approved drug for the treatment of Alzheimer's disease. It reduces neurodegeneration in the hippocampus and cerebral cortex through the inhibition of extrasynaptic NMDA receptors in patients and mouse models. Potentially, it could prevent neurodegeneration in other brain areas and caused by other diseases. We previously used memantine to prevent functional damage and to retain morphology of cerebellar neurons and Bergmann glia in an optogenetic mouse model of spinocerebellar ataxia type-1 (SCA1). However, before suggesting wider use of memantine in clinics, its side effects must be carefully evaluated. Blockers of NMDA receptors are controversial in terms of their effects on anxiety. Here, we investigated the effects of chronic application of memantine over 9 weeks to CD1 mice and examined rotarod performance and anxiety-related behaviors. Memantine-treated mice exhibited an inability to adapt to anxiety-causing conditions which strongly affected their rotarod performance. A tail suspension test revealed increased signs of behavioral despair. These data provide further insights into the potential deleterious effects of memantine which may result from the lack of adaptation to novel, stressful conditions. This effect of memantine may affect the results of tests used to assess motor performance and should be considered during clinical trials of memantine in patients.

Lactate-Mediated Signaling in the Brain-An Update.

Lactate is a universal metabolite produced and released by all cells in the body. Traditionally it was viewed as energy currency that is generated from pyruvate at the end of the glycolytic pathway and sent into the extracellular space for other cells to take up and consume. In the brain, such a mechanism was postulated to operate between astrocytes and neurons many years ago. Later, the discovery of lactate receptors opened yet another chapter in the quest to understand lactate actions. Other ideas, such as modulation of NMDA receptors were also proposed. Up to this day, we still do not have a consensus view on the relevance of any of these mechanisms to brain functions or their contribution to human or animal physiology. While the field develops new ideas, in this brief review we analyze some recently published studies in order to focus on some unresolved controversies and highlight the limitations that need to be addressed in future work. Clearly, only by using similar and overlapping methods, cross-referencing experiments, and perhaps collaborative efforts, we can finally understand what the role of lactate in the brain is and why this ubiquitous molecule is so important.

Feasibility of Photodynamic Therapy for Glioblastoma with the Mitochondria-Targeted Photosensitizer Tetramethylrhodamine Methyl Ester (TMRM).

One of the most challenging problems in the treatment of glioblastoma (GBM) is the highly infiltrative nature of the disease. Infiltrating cells that are non-resectable are left behind after debulking surgeries and become a source of regrowth and recurrence. To prevent tumor recurrence and increase patient survival, it is necessary to cleanse the adjacent tissue from GBM infiltrates. This requires an innovative local approach. One such approach is that of photodynamic therapy (PDT) which uses specific light-sensitizing agents called photosensitizers. Here, we show that tetramethylrhodamine methyl ester (TMRM), which has been used to asses mitochondrial potential, can be used as a photosensitizer to target GBM cells. Primary patient-derived GBM cell lines were used, including those specifically isolated from the infiltrative edge. PDT with TMRM using low-intensity green light induced mitochondrial damage, an irreversible drop in mitochondrial membrane potential and led to GBM cell death. Moreover, delayed photoactivation after TMRM loading selectively killed GBM cells but not cultured rat astrocytes. The efficacy of TMRM-PDT in certain GBM cell lines may be potentiated by adenylate cyclase activator NKH477. Together, these findings identify TMRM as a prototypical mitochondrially targeted photosensitizer with beneficial features which may be suitable for preclinical and clinical translation.

Expression of Microbial Enzymes in Mammalian Astrocytes to Modulate Lactate Release.

Astrocytes support and modulate neuronal activity through the release of L-lactate. The suggested roles of astrocytic lactate in the brain encompass an expanding range of vital functions, including central control of respiration and cardiovascular performance, learning, memory, executive behaviour and regulation of mood. Studying the effects of astrocytic lactate requires tools that limit the release of lactate selectively from astrocytes. Here, we report the validation in vitro of novel molecular constructs derived from enzymes originally found in bacteria, that when expressed in astrocytes, interfere with lactate handling. When lactate 2-monooxygenase derived from M. smegmatis was specifically expressed in astrocytes, it reduced intracellular lactate pools as well as lactate release upon stimulation. D-lactate dehydrogenase derived from L. bulgaricus diverts pyruvate towards D-lactate production and release by astrocytes, which may affect signalling properties of lactate in the brain. Together with lactate oxidase, which we have previously described, this set of transgenic tools can be employed to better understand astrocytic lactate release and its role in the regulation of neuronal activity in different behavioural contexts.

Chronic optogenetic stimulation of Bergman glia leads to dysfunction of EAAT1 and Purkinje cell death, mimicking the events caused by expression of pathogenic ataxin-1.

Bergmann glia (BG) are highly specialized radial astrocytes of the cerebellar cortex, which play a key role in the uptake of synaptic glutamate via the excitatory amino acid transporter EAAT1. Multiple lines of evidence suggest that in cerebellar neurodegenerative diseases reactive BG has a negative impact on neuronal function and survival through compromised EAAT activity. A family of such diseases are those caused by expansion of CAG repeats in genes of the ataxin family, resulting in spinocerebellar ataxias (SCA). We investigated the contribution of BG to the pathogenesis of cerebellar neurodegeneration in a model of SCA1, which was induced by expression of a polyglutamine mutant of ataxin-1 (ATXN1[Q85]) in BG specifically. We compared the outcomes with a novel model where we triggered excitotoxicity by a chronic optogenetic activation of BG with channelrhodopsin-2 (ChR2). In both cases we detected evidence of reduced glutamate uptake manifested by prolongation of excitatory postsynaptic currents in Purkinje cells which is consistent with documented reduction of expression and/or function of EAAT1. In both models we detected astroglyosis and Purkinje cells atrophy. Finally, the same pattern was detected in a knock-in mouse which expresses a polyglutamine mutant ataxin-1 ATXN1[Q154] in a non-cell-selective manner. Our results suggest that ATXN1[Q85] and ChR2-induced insult targeted to BG closely mimics SCA1 pathology, where excessive glutamate signaling appears to be a common feature likely being an important contributor to cerebellar neurodegeneration.

Reducing l-lactate release from hippocampal astrocytes by intracellular oxidation increases novelty induced activity in mice.

Astrocytes are in control of metabolic homeostasis in the brain and support and modulate neuronal function in various ways. Astrocyte-derived l-lactate (lactate) is thought to play a dual role as a metabolic and a signaling molecule in inter-cellular communication. The biological significance of lactate release from astrocytes is poorly understood, largely because the tools to manipulate lactate levels in vivo are limited. We therefore developed new viral vectors for astrocyte-specific expression of a mammalianized version of lactate oxidase (LOx) from Aerococcus viridans. LOx expression in astrocytes in vitro reduced their intracellular lactate levels as well as the release of lactate to the extracellular space. Selective expression of LOx in astrocytes of the dorsal hippocampus in mice resulted in increased locomotor activity in response to novel stimuli. Our findings suggest that a localized decreased intracellular lactate pool in hippocampal astrocytes could contribute to greater responsiveness to environmental novelty. We expect that use of this molecular tool to chronically limit astrocytic lactate release will significantly facilitate future studies into the roles and mechanisms of intercellular lactate communication in the brain.

Selective optogenetic stimulation of efferent fibers in the vagus nerve of a large mammal.

BACKGROUND: Electrical stimulation applied to individual organs, peripheral nerves, or specific brain regions has been used to treat a range of medical conditions. In cardiovascular disease, autonomic dysfunction contributes to the disease progression and electrical stimulation of the vagus nerve has been pursued as a treatment for the purpose of restoring the autonomic balance. However, this approach lacks selectivity in activating function- and organ-specific vagal fibers and, despite promising results of many preclinical studies, has so far failed to translate into a clinical treatment of cardiovascular disease. OBJECTIVE: Here we report a successful application of optogenetics for selective stimulation of vagal efferent activity in a large animal model (sheep). METHODS AND RESULTS: Twelve weeks after viral transduction of a subset of vagal motoneurons, strong axonal membrane expression of the excitatory light-sensitive ion channel ChIEF was achieved in the efferent projections innervating thoracic organs and reaching beyond the level of the diaphragm. Blue laser or LED light (>10 mW mm-2; 1 ms pulses) applied to the cervical vagus triggered precisely timed, strong bursts of efferent activity with evoked action potentials propagating at speeds of ∼6 m s-1. CONCLUSIONS: These findings demonstrate that in species with a large, multi-fascicled vagus nerve, it is possible to stimulate a specific sub-population of efferent fibers using light at a site remote from the vector delivery, marking an important step towards eventual clinical use of optogenetic technology for autonomic neuromodulation.

Viral Vectors as Gene Therapy Agents for Treatment of Glioblastoma.

In this review, we scrutinize the idea of using viral vectors either as cytotoxic agents or gene delivery tools for treatment of glioblastoma multiforme (GBM) in light of the experience that our laboratory has accumulated over ~20 years when using similar vectors in experimental neuroscience. We review molecular strategies and current clinical trials and argue that approaches which are based on targeting a specific biochemical pathway or a characteristic mutation are inherently prone to failure because of the high genomic instability and clonal selection characteristics of GBM. For the same reasons, attempts to develop a viral system which selectively transduces only GBM cells are also unlikely to be universally successful. One of the common gene therapy approaches is to use cytotoxic viruses which replicate and cause preferential lysis of the GBM cells. This strategy, in addition to its reliance on the specific biochemical makeup of the GBM cells, bears a risk of necrotic cell death accompanied by release of large quantities of pro-inflammatory molecules. On the other hand, engaging the immune system in the anti-GBM response seems to be a potential avenue to explore further. We suggest that a plausible strategy is to focus on viral vectors which efficiently transduce brain cells via a non-selective, ubiquitous mechanism and which target (ideally irreversibly) processes that are critical only for dividing tumor cells and are dispensable for quiescent brain cells.

Astrocytes Modulate Baroreflex Sensitivity at the Level of the Nucleus of the Solitary Tract.

Maintenance of cardiorespiratory homeostasis depends on autonomic reflexes controlled by neuronal circuits of the brainstem. The neurophysiology and neuroanatomy of these reflex pathways are well understood, however, the mechanisms and functional significance of autonomic circuit modulation by glial cells remain largely unknown. In the experiments conducted in male laboratory rats we show that astrocytes of the nucleus of the solitary tract (NTS), the brain area that receives and integrates sensory information from the heart and blood vessels, respond to incoming afferent inputs with [Ca2+]i elevations. Astroglial [Ca2+]i responses are triggered by transmitters released by vagal afferents, glutamate acting at AMPA receptors and 5-HT acting at 5-HT2A receptors. In conscious freely behaving animals blockade of Ca2+-dependent vesicular release mechanisms in NTS astrocytes by virally driven expression of a dominant-negative SNARE protein (dnSNARE) increased baroreflex sensitivity by 70% (p < 0.001). This effect of compromised astroglial function was specific to the NTS as expression of dnSNARE in astrocytes of the ventrolateral brainstem had no effect. ATP is considered the principle gliotransmitter and is released by vesicular mechanisms blocked by dnSNARE expression. Consistent with this hypothesis, in anesthetized rats, pharmacological activation of P2Y1 purinoceptors in the NTS decreased baroreflex gain by 40% (p = 0.031), whereas blockade of P2Y1 receptors increased baroreflex gain by 57% (p = 0.018). These results suggest that glutamate and 5-HT, released by NTS afferent terminals, trigger Ca2+-dependent astroglial release of ATP to modulate baroreflex sensitivity via P2Y1 receptors. These data add to the growing body of evidence supporting an active role of astrocytes in brain information processing.SIGNIFICANCE STATEMENT Cardiorespiratory reflexes maintain autonomic balance and ensure cardiovascular health. Impaired baroreflex may contribute to the development of cardiovascular disease and serves as a robust predictor of cardiovascular and all-cause mortality. The data obtained in this study suggest that astrocytes are integral components of the brainstem mechanisms that process afferent information and modulate baroreflex sensitivity via the release of ATP. Any condition associated with higher levels of "ambient" ATP in the NTS would be expected to decrease baroreflex gain by the mechanism described here. As ATP is the primary signaling molecule of glial cells (astrocytes, microglia), responding to metabolic stress and inflammatory stimuli, our study suggests a plausible mechanism of how the central component of the baroreflex is affected in pathological conditions.

Using Light for Therapy of Glioblastoma Multiforme (GBM).

: Glioblastoma multiforme (GBM) is the most malignant form of primary brain tumour with extremely poor prognosis. The current standard of care for newly diagnosed GBM includes maximal surgical resection followed by radiotherapy and adjuvant chemotherapy. The introduction of this protocol has improved overall survival, however recurrence is essentially inevitable. The key reason for that is that the surgical treatment fails to eradicate GBM cells completely, and adjacent parenchyma remains infiltrated by scattered GBM cells which become the source of recurrence. This stimulates interest to any supplementary methods which could help to destroy residual GBM cells and fight the infiltration. Photodynamic therapy (PDT) relies on photo-toxic effects induced by specific molecules (photosensitisers) upon absorption of photons from a light source. Such toxic effects are not specific to a particular molecular fingerprint of GBM, but rather depend on selective accumulation of the photosensitiser inside tumour cells or, perhaps their greater sensitivity to the effects, triggered by light. This gives hope that it might be possible to preferentially damage infiltrating GBM cells within the areas which cannot be surgically removed and further improve the chances of survival if an efficient photosensitiser and hardware for light delivery into the brain tissue are developed. So far, clinical trials with PDT were performed with one specific type of photosensitiser, protoporphyrin IX, which tends to accumulate in the cytoplasm of the GBM cells. In this review we discuss the idea that other types of molecules which build up in mitochondria could be explored as photosensitisers and used for PDT of these aggressive brain tumours.

Astrocytes monitor cerebral perfusion and control systemic circulation to maintain brain blood flow.

Astrocytes provide neurons with essential metabolic and structural support, modulate neuronal circuit activity and may also function as versatile surveyors of brain milieu, tuned to sense conditions of potential metabolic insufficiency. Here we show that astrocytes detect falling cerebral perfusion pressure and activate CNS autonomic sympathetic control circuits to increase systemic arterial blood pressure and heart rate with the purpose of maintaining brain blood flow and oxygen delivery. Studies conducted in experimental animals (laboratory rats) show that astrocytes respond to acute decreases in brain perfusion with elevations in intracellular [Ca2+]. Blockade of Ca2+-dependent signaling mechanisms in populations of astrocytes that reside alongside CNS sympathetic control circuits prevents compensatory increases in sympathetic nerve activity, heart rate and arterial blood pressure induced by reductions in cerebral perfusion. These data suggest that astrocytes function as intracranial baroreceptors and play an important role in homeostatic control of arterial blood pressure and brain blood flow.

Identification of neuron-type specific promoters in monkey genome and their functional validation in mice.

Viral gene delivery is one of the most versatile techniques for elucidating the mechanisms underlying brain dysfunction, such as neuropsychiatric disorders. Due to the complexity of the brain, expression of genetic tools, such as channelrhodopsin and calcium sensors, often has to be restricted to a specified cell type within a circuit implicated in these disorders. Only a handful of promoters targeting neuronal subtypes are currently used for viral gene delivery. Here, we isolated conserved promoter regions of several subtype-specific genes from the macaque genome and investigated their functionality in the mouse brain when used within lentiviral vectors (LVVs). Immunohistochemical analysis revealed that transgene expression induced by the promoter sequences for somatostatin (SST), cholecystokinin (CCK), parvalbumin (PV), serotonin transporter (SERT), vesicular acetylcholine transporter (vAChT), substance P (SP) and proenkephalin (PENK) was largely colocalized with specific markers for the targeted neuronal populations. Moreover, by combining these results with in silico predictions of transcription factor binding to the isolated sequences, we identified transcription factors possibly underlying cell-type specificity. These findings lay a foundation for the expansion of the current toolbox of promoters suitable for elucidating these neuronal phenotypes.

Manipulation of dorsal raphe serotonergic neurons modulates active coping to inescapable stress and anxiety-related behaviors in mice and rats.

Major depression and anxiety disorders are a social and economic burden worldwide. Serotonergic signaling has been implicated in the pathophysiology of these disorders and thus has been a crucial target for pharmacotherapy. However, the precise mechanisms underlying these disorders are still unclear. Here, we used species-optimized lentiviral vectors that were capable of efficient and specific transduction of serotonergic neurons in mice and rats for elucidation of serotonergic roles in anxiety-like behaviors and active coping behavior in both species. Immunohistochemical analyses revealed that lentiviral vectors with an upstream sequence of tryptophan hydroxylase 2 gene efficiently transduced serotonergic neurons with a specificity of approximately 95% in both mice and rats. Electrophysiological recordings showed that these lentiviral vectors induced sufficient expression of optogenetic tools for precise control of serotonergic neurons. Using these vectors, we demonstrate that acute activation of serotonergic neurons in the dorsal raphe nucleus increases active coping with inescapable stress in rats and mice in a time-locked manner, and that acute inhibition of these neurons increases anxiety-like behaviors specifically in rats. These findings further our understanding of the pathophysiological role of dorsal raphe serotonergic neurons in different species and the role of these neurons as therapeutic targets in major depression and anxiety disorders.

Signal transduction in astrocytes: Localization and release of inorganic polyphosphate.

Inorganic polyphosphate (polyP) is present in every cell and is highly conserved from primeval times. In the mammalian cells, polyP plays multiple roles including control of cell bioenergetics and signal transduction. In the brain, polyP mediates signaling between astrocytes via activation of purinergic receptors, however, the mechanisms of polyP release remain unknown. Here we report identification of polyP-containing vesicles in cortical astrocytes and the main triggers that evoke vesicular polyP release. In cultured astrocytes, polyP was localized predominantly within the intracellular vesicular compartments which express vesicular nucleotide transporter VNUT (putative ATP-containing vesicles), but not within the compartments expressing vesicular glutamate transporter 2 (VGLUT2). The number of lysosomes which contain polyP was dependent on the conditions of astrocytes. Release of polyP from a proportion of lysosomes could be induced by calcium ionophores. In contrast, polyP release from the VNUT-containing vesicles could be triggered by various physiological stimuli, such as pH changes, polyP induced polyP release and other stimuli which increase [Ca2+ ] i . These data suggest that astrocytes release polyP predominantly via exocytosis from the VNUT-containing vesicles. © 2018 Wiley Periodicals, Inc.

Glio- and neuro-protection by prosaposin is mediated by orphan G-protein coupled receptors GPR37L1 and GPR37.

Discovery of neuroprotective pathways is one of the major priorities for neuroscience. Astrocytes are natural neuroprotectors and it is likely that brain resilience can be enhanced by mobilizing their protective potential. Among G-protein coupled receptors expressed by astrocytes, two highly related receptors, GPR37L1 and GPR37, are of particular interest. Previous studies suggested that these receptors are activated by a peptide Saposin C and its neuroactive fragments (prosaptide TX14(A)), which were demonstrated to be neuroprotective in various animal models by several groups. However, pairing of Saposin C or prosaptides with GPR37L1/GPR37 has been challenged and presently GPR37L1/GPR37 have regained their orphan status. Here, we demonstrate that in their natural habitat, astrocytes, these receptors mediate a range of effects of TX14(A), including protection from oxidative stress. The Saposin C/GPR37L1/GPR37 pathway is also involved in the neuroprotective effect of astrocytes on neurons subjected to oxidative stress. The action of TX14(A) is at least partially mediated by Gi-proteins and the cAMP-PKA axis. On the other hand, when recombinant GPR37L1 or GPR37 are expressed in HEK293 cells, they are not functional and do not respond to TX14(A), which explains unsuccessful attempts to confirm the ligand-receptor pairing. Therefore, this study identifies GPR37L1/GPR37 as the receptors for TX14(A), and, by extension of Saposin C, and paves the way for the development of neuroprotective therapeutics acting via these receptors.

Differences in autonomic innervation to the vertebrobasilar arteries in spontaneously hypertensive and Wistar rats.

KEY POINTS: Essential hypertension is associated with hyperactivity of the sympathetic nervous system and hypoperfusion of the brainstem area controlling arterial pressure. Sympathetic and parasympathetic innervation of vertebrobasilar arteries may regulate blood perfusion to the brainstem. We examined the autonomic innervation of these arteries in pre-hypertensive (PHSH) and hypertensive spontaneously hypertensive (SH) rats relative to age-matched Wistar rats. Our main findings were: (1) an unexpected decrease in noradrenergic sympathetic innervation in PHSH and SH compared to Wistar rats despite elevated sympathetic drive in PHSH rats; (2) a dramatic deficit in cholinergic and peptidergic parasympathetic innervation in PHSH and SH compared to Wistar rats; and (3) denervation of sympathetic fibres did not alter vertebrobasilar artery morphology or arterial pressure. Our results support a compromised vasodilatory capacity in PHSH and SH rats compared to Wistar rats, which may explain their hypoperfused brainstem. ABSTRACT: Neurogenic hypertension may result from brainstem hypoperfusion. We previously found remodelling (decreased lumen, increased wall thickness) in vertebrobasilar arteries of juvenile, pre-hypertensive spontaneously hypertensive (PHSH) and adult spontaneously hypertensive (SH) rats compared to age-matched normotensive rats. We tested the hypothesis that there would be a greater density of sympathetic to parasympathetic innervation of vertebrobasilar arteries in SH versus Wistar rats irrespective of the stage of development and that sympathetic denervation (ablation of the superior cervical ganglia bilaterally) would reverse the remodelling and lower blood pressure. Contrary to our hypothesis, immunohistochemistry revealed a decrease in the innervation density of noradrenergic sympathetic fibres in adult SH rats (P < 0.01) compared to Wistar rats. Unexpectedly, there was a 65% deficit in parasympathetic fibres, as assessed by both vesicular acetylcholine transporter (α-VAChT) and vasoactive intestinal peptide (α-VIP) immunofluorescence (P < 0.002) in PHSH rats compared to age-matched Wistar rats. Although the neural activity of the internal cervical sympathetic branch, which innervates the vertebrobasilar arteries, was higher in PHSH relative to Wistar rats, its denervation had no effect on the vertebrobasilar artery morphology or persistent effect on arterial pressure in SH rats. Our neuroanatomic and functional data do not support a role for sympathetic nerves in remodelling of the vertebrobasilar artery wall in PHSH or SH rats. The remodelling of vertebrobasilar arteries and the elevated activity in the internal cervical sympathetic nerve coupled with their reduced parasympathetic innervation suggests a compromised vasodilatory capacity in PHSH and SH rats that could explain their brainstem hypoperfusion.