Constitutive downregulation protein kinase C epsilon in hSOD1G93A astrocytes influences mGluR5 signaling and the regulation of glutamate uptake.

Accumulating evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a non-cell-autonomous process and that impaired glutamate clearance by astrocytes, leading to excitotoxicity, could participate in progression of the disease. In astrocytes derived from an animal model of ALS (hSOD1G93A rats), activation of type 5 metabotropic glutamate receptor (mGluR5) fails to increase glutamate uptake, impeding a putative dynamic neuroprotective mechanism involving astrocytes. Using astrocyte cultures from hSOD1G93A rats, we have demonstrated that the typical Ca2+ oscillations associated with mGluR5 activation were reduced, and that the majority of cells responded with a sustained elevation of intracellular Ca2+ concentration. Since the expression of protein kinase C epsilon isoform (PKCɛ) has been found to be considerably reduced in astrocytes from hSOD1G93A rats, the consequences of manipulating its activity and expression on mGluR5 signaling and on the regulation of glutamate uptake have been examined. Increasing PKCɛ expression was found to restore Ca2+ oscillations induced by mGluR5 activation in hSOD1G93A -expressing astrocytes. This was also associated with an increase in glutamate uptake capacity in response to mGluR5 activation. Conversely, reducing PKCɛ expression in astrocytes from wild-type animals with specific PKCɛ-shRNAs was found to alter the mGluR5 associated oscillatory signaling profile, and consistently reduced the regulation of the glutamate uptake-mediated by mGluR5 activation. These results suggest that PKCɛ is required to generate Ca2+ oscillations following mGluR5 activation, which support the regulation of astrocytic glutamate uptake. Reduced expression of astrocytic PKCɛ could impair this neuroprotective process and participate in the progression of ALS.

PKC epsilon-dependent calcium oscillations associated with metabotropic glutamate receptor 5 prevent agonist-mediated receptor desensitization in astrocytes.

A critical role has been assigned to protein kinase C (PKC)ε in the control of intracellular calcium oscillations triggered upon activation of type 5 metabotropic glutamate receptor (mGluR5) in cultured astrocytes. Nevertheless, the physiological significance of this particular signalling profile in the response of astrocytes to glutamate remains largely unknown. Considering that kinases are frequently involved in the regulation of G protein-coupled receptors, we have examined a putative link between the nature of the calcium signals and the response regulation upon repeated exposures of astrocytes to the agonist (S)-3,5-dihydroxyphenylglycine. We show that upon repeated mGluR5 activations, a robust desensitization was observed in astrocytes grown in culture conditions favouring the peak-plateau-type response. At variance, in cell cultures where calcium oscillations were predominating, the response was fully preserved even during repeated challenges with the agonist. Pharmacological inhibition of PKCε or genetic suppression of this isoform using shRNA was found to convert an oscillatory calcium profile to a sustained calcium mobilization and this latter profile was subject to desensitization upon repetitive mGluR5 activation. Our results suggest a yet undocumented scheme in which the activity of PKCε contributes to preserve the receptor sensitivity upon repeated or sustained activations. Cover Image for this issue: doi: 10.1111/jnc.13797.

Inflammation-associated regulation of RGS in astrocytes and putative implication in neuropathic pain.

BACKGROUND: Regulators of G-protein signaling (RGS) are major physiological modulators of G-protein-coupled receptors (GPCR) signaling. Several GPCRs expressed in both neurons and astrocytes participate in the central control of pain processing, and the reduced efficacy of analgesics in neuropathic pain conditions may rely on alterations in RGS function. The expression and the regulation of RGS in astrocytes is poorly documented, and we herein hypothesized that neuroinflammation which is commonly observed in neuropathic pain could influence RGS expression in astrocytes. METHODS: In a validated model of neuropathic pain, the spared nerve injury (SNI), the regulation of RGS2, RGS3, RGS4, and RGS7 messenger RNA (mRNA) was examined up to 3 weeks after the lesion. Changes in the expression of the same RGS were also studied in cultured astrocytes exposed to defined activation protocols or to inflammatory cytokines. RESULTS: We evidenced a differential regulation of these RGS in the lumbar spinal cord of animals undergoing SNI. In particular, RGS3 appeared upregulated at early stages after the lesion whereas expression of RGS2 and RGS4 was decreased at later stages. Decrease in RGS7 expression was already observed after 3 days and outlasted until 21 days after the lesion. In cultured astrocytes, we observed that changes in the culture conditions distinctly influenced the constitutive expression of these RGS. Also, brief exposures (4 to 8 h) to either interleukin-1ß, interleukin-6, or tumor necrosis factor α caused rapid changes in the mRNA levels of the RGS, which however did not strictly recapitulate the regulations observed in the spinal cord of lesioned animals. Longer exposure (48 h) to inflammatory cytokines barely influenced RGS expression, confirming the rapid but transient regulation of these cell signaling modulators. CONCLUSION: Changes in the environment of astrocytes mimicking the inflammation observed in the model of neuropathic pain can affect RGS expression. Considering the role of astrocytes in the onset and progression of neuropathic pain, we propose that the inflammation-mediated modulation of RGS in astrocytes constitutes an adaptive mechanism in a context of neuroinflammation and may participate in the regulation of nociception.

Early ALS-type gait abnormalities in AMP-dependent protein kinase-deficient mice suggest a role for this metabolic sensor in early stages of the disease.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motoneurons. While the principal cause of the disease remains so far unknown, the onset and progression of the pathology are increasingly associated with alterations in the control of cell metabolism. On the basis of the well-known key roles of 5'-adenosine monophosphate-activated protein kinase (AMPK) in sensing and regulating the intracellular energy status, we hypothesized that mice with a genetic deletion of AMPK would develop locomotor abnormalities that bear similarity with those detected in the very early disease stage of mice carrying the ALS-associated mutated gene hSOD1(G93A). Using an automated gait analysis system (CatWalk), we here show that hSOD1(G93A) mice and age-matched mice lacking the neuronal and skeletal muscle predominant α2 catalytic subunit of AMPK showed an altered gait, clearly different from wild type control mice. Double mutant mice lacking AMPK α2 and carrying hSOD1(G93A) showed the same early gait abnormalities as hSOD1(G93A) mice over an age span of 8 to 16 weeks. Taken together, these data support the concept that altered AMPK function and associated bioenergetic abnormalities could constitute an important component in the early pathogenesis of ALS. Therapeutic interventions acting on metabolic pathways could prove beneficial on early locomotor deficits, which are sensitively detectable in rodent models using the CatWalk system.

Amyloid Precursor Protein (APP) Controls the Expression of the Transcriptional Activator Neuronal PAS Domain Protein 4 (NPAS4) and Synaptic GABA Release.

The amyloid precursor protein (APP) has been extensively studied as the precursor of the ß-amyloid (Aß) peptide, the major component of the senile plaques found in the brain of Alzheimer's disease (AD) patients. However, the function of APP per se in neuronal physiology remains to be fully elucidated. APP is expressed at high levels in the brain. It resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell-cell interaction and/or activation of intracellular signaling pathways. In this respect, the APP intracellular domain (AICD) was reported to act as a transcriptional regulator. Here, we used a transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and on maturation of primary cortical neurons. Surprisingly, the overall transcriptional changes were subtle, but a more detailed analysis pointed to genes clustered in neuronal-activity dependent pathways. In particular, we observed a decreased transcription of neuronal PAS domain protein 4 (NPAS4) in APP-/- neurons. NPAS4 is an inducible transcription factor (ITF) regulated by neuronal depolarization. The downregulation of NPAS4 co-occurs with an increased production of the inhibitory neurotransmitter GABA and a reduced expression of the GABAA receptors α1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABAA receptors activity, but long-term potentiation (LTP) measurement supported an increased GABA component in synaptic transmission of APP-/- mice. Together, NPAS4 appears to be a downstream target involved in APP-dependent regulation of inhibitory synaptic transmission.

The anti-inflammatory peptide stearyl-norleucine-VIP delays disease onset and extends survival in a rat model of inherited amyotrophic lateral sclerosis.

Vasoactive intestinal peptide (VIP) has potent immune modulatory actions that may influence the course of neurodegenerative disorders associated with chronic inflammation. Here, we show the therapeutic benefits of a modified peptide agonist stearyl-norleucine-VIP (SNV) in a transgenic rat model of amyotrophic lateral sclerosis (mutated superoxide dismutase 1, hSOD1(G93A)). When administered by systemic every-other-day intraperitoneal injections during a period of 80 days before disease, SNV delayed the onset of motor dysfunction by no less than three weeks, while survival was extended by nearly two months. SNV-treated rats showed reduced astro- and microgliosis in the lumbar ventral spinal cord and a significant degree of motor neuron preservation. Throughout the treatment, SNV promoted the expression of the anti-inflammatory cytokine interleukin-10 as well as neurotrophic factors commonly considered as beneficial in amyotrophic lateral sclerosis management (glial derived neuroptrophic factor, insulin like growth factor, brain derived neurotrophic factor). The peptide nearly totally suppressed the expression of tumor necrosis factor-α and repressed the production of the pro-inflammatory mediators interleukin-1ß, nitric oxide and of the transcription factor nuclear factor kappa B. Inhibition of tumor necrosis factor-α likely accounted for the observed down-regulation of nuclear factor kappa B that modulates the transcription of genes specifically involved in amyotrophic lateral sclerosis (sod1 and the glutamate transporter slc1a2). In line with this, levels of human superoxide dismutase 1 mRNA and protein were decreased by SNV treatment, while the expression and activity of the glutamate transporter-1 was promoted. Considering the large diversity of influences of this peptide on both clinical features of the disease and associated biochemical markers, we propose that SNV or related peptides may constitute promising candidates for amyotrophic lateral sclerosis treatment.

Immunomodulatory influence of bone marrow-derived mesenchymal stem cells on neuroinflammation in astrocyte cultures.

The therapeutic benefits associated with mesenchymal stem cells (MSCs) largely result from their immunomodulatory and neurotrophic properties. In this study, we evaluated the effects of MSCs on astrocyte cultures exposed to lipopolysaccharide. In response to this inflammatory trigger, astrocytes showed an increased expression of pro-inflammatory genes (IL-1ß, TNFα, IL-6), which was attenuated by pre-exposure to MSC conditioned medium. Furthermore, mediators released by MSCs increased cell proliferation and altered the regulation of intermediate filaments (GFAP, vimentin), pro-inflammatory enzymes (iNOS, COX-2) and receptors (TLR4, CD14, mGluR3, mGluR5). These data demonstrate that MSCs influence diverse cell types participating in the response to neuroinflammation.