Neuronal Overexpression Research Articles

The influence of pro-longevity gene Gclc overexpression on the age-dependent changes in Drosophila transcriptome and biological functions.

BackgroundTranscriptional changes that contribute to the organism’s longevity and prevent the age-dependent decline of biological functions are not well understood. Here, we overexpressed pro-longevity gene encoding glutamate-cysteine ligase catalytic subunit (Gclc) and analyzed age-dependent changes in transcriptome that associated with the longevity, stress resistance, locomotor activity, circadian rhythmicity, and fertility.ResultsHere we reproduced the life extension effect of neuronal overexpression of the Gclc gene and investigated its influence on the age-depended dynamics of transcriptome and biological functions such as fecundity, spontaneous locomotor activity and circadian rhythmicity, as well as on the resistance to oxidative, proteotoxic and osmotic stresses. It was shown that Gclc overexpression reduces locomotor activity in the young and middle ages compared to control flies. Gclc overexpression slowed down the age-dependent decline of locomotor activity and circadian rhythmicity, and resistance to stress treatments. Gclc level demonstrated associations with the expression of genes involved in a variety of cellular processes including Jak-STAT, MAPK, FOXO, Notch, mTOR, TGF-beta signaling pathways, translation, protein processing in endoplasmic reticulum, proteasomal degradation, glycolysis, oxidative phosphorylation, apoptosis, regulation of circadian rhythms, differentiation of neurons, synaptic plasticity and transmission.ConclusionsOur study revealed that Gclc overexpression induces transcriptional changes associated with the lifespan extension and uncovered pathways that may be associated with the age-dependent decline of biological functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3356-0) contains supplementary material, which is available to authorized users.

Overexpression of STIM1 in neurons in mouse brain improves contextual learning and impairs long-term depression

STIM1 is an endoplasmic reticulum calcium sensor that is involved in several processes in neurons, including store-operated calcium entry. STIM1 also inhibits voltage-gated calcium channels, such as Cav1.2 and Cav3.1, and is thus considered a multifunctional protein. The aim of this work was to investigate the ways in which transgenic neuronal overexpression of STIM1 in FVB/NJ mice affects animal behavior and the electrophysiological properties of neurons in acute hippocampal slices. We overexpressed STIM1 from the Thy1.2 promoter and verified neuronal expression by quantitative reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry. Mature primary hippocampal cultures expressed STIM1 but exhibited no changes in calcium homeostasis. Basal synaptic transmission efficiency and short-term plasticity were comparable in slices that were isolated from transgenic mice, similarly as the magnitude of long-term potentiation. However, long-term depression that was induced by the glutamate receptor 1/5 agonist (S)-3,5-dihydroxyphenylglycine was impaired in STIM1 slices. Interestingly, transgenic mice exhibited a decrease in anxiety-like behavior and improvements in contextual learning. In summary, our data indicate that STIM1 overexpression in neurons in the brain perturbs metabotropic glutamate receptor signaling, leading to impairments in long-term depression and alterations in animal behavior. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

162 - GSNOR Impaired Cognitive Function in Aging Through De-Nitrosation of CamKIIalpha

As a result of the acceleration of population aging, an increasing number of people suffer from age related cognitive impairment. However, the mechanisms that underlie this process are unclear. Here we found that S-nitrosoglutathione reductase (GSNOR), the key enzyme metabolizing the intracellular nitric oxide (NO) and S-nitrosation, significantly increased in the hippocampus of both aging human and mice. Neuronal specific overexpression of GSNOR leads to cognitive impairment, long-term potentiation (LTP) defect and lower dendrite spine density. While knock out GSNOR rescued the age related cognitive impairment. We then performed quantitative proteomic analysis of protein S-nitrosation and found S-nitrosation of CamKIIα was significantly decreased in the hippocampus of aging mice and GSNOR transgenic mice. In consistent with the change of CamKIIα S-nitrosation, the accumulation of CamKIIα in hippocampal Synaptosomal and its downstream signaling p-GLUR1 and CREB/c-fos were also significantly decreased, which can all be rescued in GSNOR knockout mice. The cognitive impairment in GSNOR transgenic mice can be rescued by up-regulation of the NO signaling pathway or CamKIIα/CREB signaling pathway. In summary, our research demonstrated that GSNOR is a new potential target for treatment of the age related cognitive impairment. NO signaling deficiency may be the main cause to induce age-related cognitive impairment. In addition, we explored the physiological function of S-nitrosation of CamKIIα for the first time.

Progranulin promotes peripheral nerve regeneration and reinnervation: role of notch signaling.

BackgroundPeripheral nerve injury is a frequent cause of lasting motor deficits and chronic pain. Although peripheral nerves are capable of regrowth they often fail to re-innervate target tissues.ResultsUsing newly generated transgenic mice with inducible neuronal progranulin overexpression we show that progranulin accelerates axonal regrowth, restoration of neuromuscular synapses and recovery of sensory and motor functions after injury of the sciatic nerve. Oppositely, progranulin deficient mice have long-lasting deficits in motor function tests after nerve injury due to enhanced losses of motor neurons and stronger microglia activation in the ventral horn of the spinal cord. Deep proteome and gene ontology (GO) enrichment analysis revealed that the proteins upregulated in progranulin overexpressing mice were involved in ‘regulation of transcription’ and ‘response to insulin’ (GO terms). Transcription factor prediction pointed to activation of Notch signaling and indeed, co-immunoprecipitation studies revealed that progranulin bound to the extracellular domain of Notch receptors, and this was functionally associated with higher expression of Notch target genes in the dorsal root ganglia of transgenic mice with neuronal progranulin overexpression. Functionally, these transgenic mice recovered normal gait and running, which was not achieved by controls and was stronger impaired in progranulin deficient mice.ConclusionWe infer that progranulin activates Notch signaling pathways, enhancing thereby the regenerative capacity of partially injured neurons, which leads to improved motor function recovery.Graphical abstract Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0132-1) contains supplementary material, which is available to authorized users.

Increased Expression of Caspase-12 After Experimental Subarachnoid Hemorrhage.

Convincing evidences have proved that apoptosis plays a vital role in the pathogenesis of early and delayed brain injury following subarachnoid hemorrhage (SAH). Recently, a novel caspase-12-mediated apoptotic pathway has been reported to be induced by excess endoplasmic reticulum (ER) stress. Extensive protein damage occurs after SAH, which may trigger ER stress-associated apoptotic pathway. Thus, we hypothesized that caspase-12, as the major molecular marker of this novel apoptotic pathway, may be activated and involved in the pathogenesis of apoptotic injury after SAH. This study sought to investigate the changes of caspase-12 expressions in both in vitro and in vivo SAH models. Western blot analysis found significantly increased protein expressions of both pro- and active forms of caspase-12 after SAH. Quantitative real-time PCR and immunohistochemistry assays confirmed elevated caspase-12 level after SAH in vivo. Further, double immunofluorescence staining revealed obvious caspase-12 over-expression in both cortical neurons and astrocytes. Moreover, immunofluorescent co-staining in vivo demonstrated that neural cells with high immunoreactivity of caspase-12 also expressed caspase-3, and dual-immunofluorescent staining for caspase-12 and TUNEL in vitro showed that TUNEL-positive cells were more likely to exhibit higher caspase-12 immunoreactivity, indicating a potential contribution of caspase-12 activation to apoptosis in SAH. Collectively, our results showed significant upregulation of caspase-12 expression after experimental SAH. These findings also offer important implications for further investigations of the therapeutic potential of caspase-12 associated apoptosis in SAH.

Tyrosine kinase inhibition reverses TDP-43 effects on synaptic protein expression, astrocytic function and amino acid dis-homeostasis.

The trans-activating response of DNA/RNA-binding protein (TDP)-43 pathology is associated with many neurodegenerative diseases via unknown mechanisms. Here, we use a transgenic mouse model over-expressing human wild-type neuronal TDP-43 to study the effects of TDP-43 pathology on glutamate metabolism and synaptic function. We found that neuronal TDP-43 over-expression affects synaptic protein expression, including Synapsin I, and alters surrounding astrocytic function. TDP-43 over-expression is associated with an increase in glutamate and γ-amino butyric acid and reduction of glutamine and aspartate levels, indicating impairment of presynaptic terminal. TDP-43 also decreases tricarboxylic acid cycle metabolism and induces oxidative stress via lactate accumulation. Neuronal TDP-43 does not alter microglia activity or significantly changes systemic and brain inflammatory markers compared to control. We previously demonstrated that brain-penetrant tyrosine kinase inhibitors (TKIs), nilotinib and bosutinib, reduce TDP-43-induced cell death in transgenic mice. Here, we show that TKIs reverse the effects of TDP-43 on synaptic proteins, increase astrocytic function and restore glutamate and neurotransmitter balance in TDP-43 mice. Nilotinib, but not bosutinib, reverses mitochondrial impairment and oxidative metabolism. Taken together, these data suggest that TKIs can attenuate TDP-43 toxicity and improve synaptic and astrocytic function, independent of microglial or other inflammatory effects. In conclusion, our data demonstrate novel mechanisms of the effects of neuronal TDP-43 over-expression on synaptic protein expression and alteration of astrocytic function.

Transglutaminase 2 overexpression induces depressive-like behavior and impaired TrkB signaling in mice.

Serotonin (5-HT) and brain derived neurotrophic factor (BDNF) are two signaling molecules that play important regulatory roles in the development and plasticity of neural circuits that are known to be altered in depression. However, the mechanism by which 5-HT regulates BDNF signaling is unknown. In the present study, we found that 5-HT treatment increases BDNF receptor, TrkB (tropomyosin related kinase B) levels in mouse primary cortical neurons via a Rac1 (RAS-related C3 botulinum toxin substrate 1)-dependent mechanism. Significant increases in the levels of transglutaminase 2 (TG2, which is implicated in transamidation of 5-HT to Rac1) are observed in the mouse prefrontal cortex (PFC) following chronic exposure to stress. We also found that TG2 levels are increased in the postmortem PFC of depressed suicide subjects relative to matched controls. Moreover, in mice, neuronal overexpression of TG2 resulted in the atrophy of neurons and reduced levels of TrkB in the PFC as well as a depressive-like phenotype. Overexpression of TG2 in mouse cortical neurons reduced TrkB levels as a result of impaired endocytosis of TrkB. TG2 inhibition by either a viral particle or pharmacological approach attenuated behavioral deficits caused by chronic unpredictable stress. Moreover, the overexpression of TrkB in the mouse PFC ameliorated the depressive-like phenotype of TG2 overexpressed mice. Taken together, these postmortem and preclinical findings identify TG2 as a critical mediator of the altered TrkB expression and depressive-like behaviors associated with chronic exposure to stress and suggest that TG2 may represent a novel therapeutic target in depression.

Progranulin overexpression in sensory neurons attenuates neuropathic pain in mice: Role of autophagy

Peripheral or central nerve injury is a frequent cause of chronic pain and the mechanisms are not fully understood. Using newly generated transgenic mice we show that progranulin overexpression in sensory neurons attenuates neuropathic pain after sciatic nerve injury and accelerates nerve healing. A yeast-2-hybrid screen revealed putative interactions of progranulin with autophagy-related proteins, ATG12 and ATG4b. This was supported by colocalization and proteomic studies showing regulations of ATG13 and ATG4b and other members of the autophagy network, lysosomal proteins and proteins involved in endocytosis. The association of progranulin with the autophagic pathway was functionally confirmed in primary sensory neurons. Autophagy and survival were impaired in progranulin-deficient neurons and improved in progranulin overexpressing neurons. Nerve injury in vivo caused an accumulation of LC3b-EGFP positive bodies in neurons of the dorsal root ganglia and nerves suggesting an impairment of autophagic flux. Overexpression of progranulin in these neurons was associated with a reduction of the stress marker ATF3, fewer protein aggregates in the injured nerve and enhanced stump healing. At the behavioral level, further inhibition of the autophagic flux by hydroxychloroquine intensified cold and heat nociception after sciatic nerve injury and offset the pain protection provided by progranulin. We infer that progranulin may assist in removal of protein waste and thereby helps to resolve neuropathic pain after nerve injury.

A22 Genetic tools to validate and measure NRF2 in HD

Huntington’s disease (HD) is an autosomal dominant inherited disease leading to significant neurodegeneration. The neuropathological characterisation of the disease is the loss of medium spiny neurons in the striatum. A hallmark of HD is the intracellular aggregation of huntingtin protein. Multiple mechanisms for neuronal cell death have been proposed including oxidative stress, mitochondrial dysfunction and impaired proteostasis. The Nrf2 (NF-E2-related factor 2) pathway has the potential to modulate all of these pathologic processes. Nrf2 is a transcription factor that binds to the ARE (antioxidant response element) and controls a powerful array of endogenous cellular antioxidant systems. Genes regulated through Nrf2 activation code for proteins that are direct antioxidants, enzymes that inactivate ROS (reactive oxygen species), produce reductive co-factors such as glutathione, combat endoplasmic reticulum stress, and modulate protein degradation pathways which affect protein turnover and homeostasis. Thus, Nrf2 is an attractive target for therapeutic intervention for neurodegenerative diseases. Previously, our laboratory demonstrated that transgenic mice with astrocyte-specific overexpression of Nrf2 (GFAP-Nrf2 mice) reduces chemical-mediated neurotoxicity in PD and HD models, as well as genetically-induced motor neuron degeneration in models of amyotrophic lateral sclerosis and Parkinson’s disease. The GFAP-Nrf2 mice were crossed with R6/2 mice. Overexpression of Nrf2 in astrocytes did not extend lifespan of the R6/2 mice. There was however some protection by astrocytic Nrf2 activation in the early stages of pathogenesis based on the rotarod, open field testing, and multiplex mRNA analysis in the GFAP-Nrf2/R6/2 compared to the R6/2 mice. On going studies are evaluating Nrf2 overexpression in neurons using transgenic mice and AAV-mediated gene delivery to the brains of R6/2 mice. Funded by the CHDI Foundation.

Neuronal overexpression of human VAPB slows motor impairment and neuromuscular denervation in a mouse model of ALS.

Four mutations in the VAMP/synaptobrevin-associated protein B (VAPB) gene have been linked to amyotrophic lateral sclerosis (ALS) type 8. The mechanism by which VAPB mutations cause motor neuron disease is unclear, but studies of the most common P56S variant suggest both loss of function and dominant-negative sequestration of wild-type protein. Diminished levels of VAPB and its proteolytic cleavage fragment have also been reported in sporadic ALS cases, suggesting that VAPB loss of function may be a common mechanism of disease. Here, we tested whether neuronal overexpression of wild-type human VAPB would attenuate disease in a mouse model of familial ALS1. We used neonatal intraventricular viral injections to express VAPB or YFP throughout the brain and spinal cord of superoxide dismutase (SOD1) G93A transgenic mice. Lifelong elevation of neuronal VAPB slowed the decline of neurological impairment, delayed denervation of hindlimb muscles, and prolonged survival of spinal motor neurons. Collectively, these changes produced a slight but significant extension in lifespan, even in this highly aggressive model of disease. Our findings lend support for a protective role of VAPB in neuromuscular health.

The brain-tumor related protein podoplanin regulates synaptic plasticity and hippocampus-dependent learning and memory

Introduction: Podoplanin is a cell-surface glycoprotein constitutively expressed in the brain and implicated in human brain tumorigenesis. The intrinsic function of podoplanin in brain neurons remains however uncharacterized.Materials and methods: Using an established podoplanin-knockout mouse model and electrophysiological, biochemical, and behavioral approaches, we investigated the brain neuronal role of podoplanin.Results: Ex-vivo electrophysiology showed that podoplanin deletion impairs dentate gyrus synaptic strengthening. In vivo, podoplanin deletion selectively impaired hippocampus-dependent spatial learning and memory without affecting amygdala-dependent cued fear conditioning. In vitro, neuronal overexpression of podoplanin promoted synaptic activity and neuritic outgrowth whereas podoplanin-deficient neurons exhibited stunted outgrowth and lower levels of p-Ezrin, TrkA, and CREB in response to nerve growth factor (NGF). Surface Plasmon Resonance data further indicated a physical interaction between podoplanin and NGF.Discussion: This work proposes podoplanin as a novel component of the neuronal machinery underlying neuritogenesis, synaptic plasticity, and hippocampus-dependent memory functions. The existence of a relevant cross-talk between podoplanin and the NGF/TrkA signaling pathway is also for the first time proposed here, thus providing a novel molecular complex as a target for future multidisciplinary studies of the brain function in the physiology and the pathology.Key messagesPodoplanin, a protein linked to the promotion of human brain tumors, is required in vivo for proper hippocampus-dependent learning and memory functions.Deletion of podoplanin selectively impairs activity-dependent synaptic strengthening at the neurogenic dentate-gyrus and hampers neuritogenesis and phospho Ezrin, TrkA and CREB protein levels upon NGF stimulation.Surface plasmon resonance data indicates a physical interaction between podoplanin and NGF. On these grounds, a relevant cross-talk between podoplanin and NGF as well as a role for podoplanin in plasticity-related brain neuronal functions is here proposed.

Enhancing survival motor neuron expression extends lifespan and attenuates neurodegeneration in mutant TDP-43 mice.

Defects in the RNA-binding proteins survival motor neuron (SMN) and TAR DNA-binding protein 43 (TDP-43) cause progressive motor neuron degeneration in spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), respectively. While low levels of SMN protein in motor neurons result in SMA, recent studies implicate abnormal SMN levels and function in ALS pathogenesis. Here, we determine that SMN protein is upregulated early and progressively in spinal and cortical motor neurons of male transgenic mutant TDP-43A315T mice. Cytoplasmic SMN aggregates that contain TDP-43 and HuR were identified in motor neurons of TDP-43A315T mice, consistent with the incorporation of SMN into stress granules. To test the impact of augmenting SMN levels in TDP-43 proteinopathy, we demonstrate that neuronal overexpression of human SMN in TDP-43A315T mice delayed symptom onset and prolonged survival. SMN upregulation also countered motor neuron degeneration, attenuated activation of astrocytes and microglia and restored AMP kinase activation in spinal cords of TDP-43A315T mice. We also reveal that expression of another factor conferring motor neuron vulnerability, androgen receptor (AR), is reduced in spinal cords of male TDP-43A315T mice. These results establish that SMN overexpression in motor neurons slows disease onset and outcome by ameliorating pathological signs in this model of mutant TDP-43-mediated ALS. Further approaches to augment SMN levels using pharmacological or gene therapy agents may therefore be warranted in ALS. Our data also reinforce a novel potential link between ALS and spinal bulbar muscular atrophy (SBMA), another motor neurodegenerative disease mediated by reduced AR function in motor neurons.

The Influence of Differentially Expressed Tissue-Type Plasminogen Activator in Experimental Autoimmune Encephalomyelitis: Implications for Multiple Sclerosis.

Tissue type plasminogen activator (t-PA) has been implicated in the development of multiple sclerosis (MS) and in rodent models of experimental autoimmune encephalomyelitis (EAE). We show that levels of t-PA mRNA and activity are increased ~4 fold in the spinal cords of wild-type mice that are mice subjected to EAE. This was also accompanied with a significant increase in the levels of pro-matrix metalloproteinase 9 (pro-MMP-9) and an influx of fibrinogen. We next compared EAE severity in wild-type mice, t-PA-/- mice and T4+ transgenic mice that selectively over-express (~14-fold) mouse t-PA in neurons of the central nervous system. Our results confirm that t-PA deficient mice have an earlier onset and more severe form of EAE. T4+ mice, despite expressing higher levels of endogenous t-PA, manifested a similar rate of onset and neurological severity of EAE. Levels of proMMP-9, and extravasated fibrinogen in spinal cord extracts were increased in mice following EAE onset regardless of the absence or over-expression of t-PA wild-type. Interestingly, MMP-2 levels also increased in spinal cord extracts of T4+ mice following EAE, but not in the other genotypes. Hence, while the absence of t-PA confers a more deleterious form of EAE, neuronal over-expression of t-PA does not overtly protect against this condition with regards to symptom onset or severity of EAE.

Hippocampal MicroRNA-124 Enhances Chronic Stress Resilience in Mice.

Chronic stress-induced aberrant gene expression in the brain and subsequent dysfunctional neuronal plasticity have been implicated in the etiology and pathophysiology of mood disorders. In this study, we examined whether altered expression of small, regulatory, noncoding microRNAs (miRNAs) contributes to the depression-like behaviors and aberrant neuronal plasticity associated with chronic stress. Mice exposed to chronic ultra-mild stress (CUMS) exhibited increased depression-like behaviors and reduced hippocampal expression of the brain-enriched miRNA-124 (miR-124). Aberrant behaviors and dysregulated miR-124 expression were blocked by chronic treatment with an antidepressant drug. The depression-like behaviors are likely not conferred directly by miR-124 downregulation because neither viral-mediated hippocampal overexpression nor intrahippocampal infusion of an miR-124 inhibitor affected depression-like behaviors in nonstressed mice. However, viral-mediated miR-124 overexpression in hippocampal neurons conferred behavioral resilience to CUMS, whereas inhibition of miR-124 led to greater behavioral susceptibility to a milder stress paradigm. Moreover, we identified histone deacetylase 4 (HDAC4), HDAC5, and glycogen synthase kinase 3β (GSK3β) as targets for miR-124 and found that intrahippocampal infusion of a selective HDAC4/5 inhibitor or GSK3 inhibitor had antidepressant-like actions on behavior. We propose that miR-124-mediated posttranscriptional controls of HDAC4/5 and GSK3β expressions in the hippocampus have pivotal roles in susceptibility/resilience to chronic stress. Depressive disorders are a major public health concern worldwide. Although a clear understanding of the etiology of depression is still lacking, chronic stress-elicited aberrant neuronal plasticity has been implicated in the pathophysiology of depression. We show that the hippocampal expression of microRNA-124 (miR-124), an endogenous small, noncoding RNA that represses gene expression posttranscriptionally, controls resilience/susceptibility to chronic stress-induced depression-like behaviors. These effects on depression-like behaviors may be mediated through regulation of the mRNA or protein expression levels of histone deacetylases HDAC4/5 and glycogen synthase kinase 3β, all highly conserved miR-124 targets. Moreover, miR-124 contributes to stress-induced dendritic hypotrophy and reduced spine density of dentate gyrus granule neurons. Modulation of hippocampal miR-124 pathways may have potential antidepressant effects.

ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aβ peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30mut) did not affect Aβ secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aβ42 secretion in neuron primary cultures, soluble Aβ42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aβ production, thereby contributing to Alzheimer's disease development.

Targeted overexpression of tumor necrosis factor-α increases cyclin-dependent kinase 5 activity and TRPV1-dependent Ca2+ influx in trigeminal neurons.

We reported earlier that TNF-α, a proinflammatory cytokine implicated in many inflammatory disorders causing orofacial pain, increases the activity of Cdk5, a key kinase involved in brain development and function and recently found to be involved in pain signaling. To investigate a potential mechanism underlying inflammatory pain in trigeminal ganglia (TGs), we engineered a transgenic mouse model (TNF) that can conditionally overexpresses TNF-α upon genomic recombination by Cre recombinase. TNF mice were bred with Nav1.8-Cre mouse line that expresses the Cre recombinase in sensory neurons to obtain TNF-α:Nav1.8-Cre (TNF-α cTg) mice. Although TNF-α cTg mice appeared normal without any gross phenotype, they displayed a significant increase in TNF-α levels after activation of NFκB signaling in the TG. IL-6 and MCP-1 levels were also increased along with intense immunostaining for Iba1 and GFAP in TG, indicating the presence of infiltrating macrophages and the activation of satellite glial cells. TNF-α cTg mice displayed increased trigeminal Cdk5 activity, and this increase was associated with elevated levels of phospho-T407-TRPV1 and capsaicin-evocated Ca influx in cultured trigeminal neurons. Remarkably, this effect was prevented by roscovitine, an inhibitor of Cdk5, which suggests that TNF-α overexpression induced sensitization of the TRPV1 channel. Furthermore, TNF-α cTg mice displayed more aversive behavior to noxious thermal stimulation (45°C) of the face in an operant pain assessment device as compared with control mice. In summary, TNF-α overexpression in the sensory neurons of TNF-α cTg mice results in inflammatory sensitization and increased Cdk5 activity; therefore, this mouse model would be valuable for investigating the mechanism of TNF-α involved in orofacial pain.

Thrombospondin-4 divergently regulates voltage-gated Ca2+ channel subtypes in sensory neurons after nerve injury

Loss of high-voltage-activated (HVA) calcium current (ICa) and gain of low-voltage-activated (LVA) ICa after painful peripheral nerve injury cause elevated excitability in sensory neurons. Nerve injury is also accompanied by increased expression of the extracellular matrix glycoprotein thrombospondin-4 (TSP4), and interruption of TSP4 function can reverse or prevent behavioral hypersensitivity after injury. We therefore investigated TSP4 regulation of ICa in dorsal root ganglion (DRG) neurons. During depolarization adequate to activate HVA ICa, TSP4 decreases both N- and L-type ICa and the associated intracellular calcium transient. In contrast, TSP4 increases ICa and the intracellular calcium signal after low-voltage depolarization, which we confirmed is due to ICa through T-type channels. These effects are blocked by gabapentin, which ameliorates neuropathic pain by targeting the α2δ1 calcium subunit. Injury-induced changes of HVA and LVA ICa are attenuated in TSP4 knockout mice. In the neuropathic pain model of spinal nerve ligation, TSP4 application did not further regulate ICa of injured DRG neurons. Taken together, these findings suggest that elevated TSP4 after peripheral nerve injury may contribute to hypersensitivity of peripheral sensory systems by decreasing HVA and increasing LVA in DRG neurons by targeting the α2δ1 calcium subunit. Controlling TSP4 overexpression in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain.

α-Synuclein induced toxicity in brain stem serotonin neurons mediated by an AAV vector driven by the tryptophan hydroxylase promoter.

We studied the impact of α-synuclein overexpression in brainstem serotonin neurons using a novel vector construct where the expression of human wildtype α-synuclein is driven by the tryptophan hydroxylase promoter, allowing expression of α-synuclein at elevated levels, and with high selectivity, in serotonergic neurons. α-Synuclein induced degenerative changes in axons and dendrites, displaying a distorted appearance, suggesting accumulation and aggregation of α-synuclein as a result of impaired axonal transport, accompanied by a 40% loss of terminals, as assessed in the hippocampus. Tissue levels of serotonin and its major metabolite 5-HIAA remained largely unaltered, and the performance of the α-synuclein overexpressing rats in tests of spatial learning (water maze), anxiety related behavior (elevated plus maze) and depressive-like behavior (forced swim test) was not different from control, suggesting that the impact of the developing axonal pathology on serotonin neurotransmission was relatively mild. Overexpression of α-synuclein in the raphe nuclei, combined with overexpression in basal forebrain cholinergic neurons, resulted in more pronounced axonal pathology and significant impairment in the elevated plus maze. We conclude that α-synuclein pathology in serotonergic or cholinergic neurons alone is not sufficient to impair non-motor behaviors, but that it is their simultaneous involvement that determines severity of such symptoms.

Relationship between ketamine-induced developmental neurotoxicity and NMDA receptor-mediated calcium influx in neural stem cell-derived neurons

Ketamine, a noncompetitive NMDA receptor antagonist, is used as a general anesthetic and recent data suggest that general anesthetics can cause neuronal damage when exposure occurs during early brain development. To elucidate the underlying mechanisms associated with ketamine-induced neurotoxicity, stem cell-derived models, such as rodent neural stem cells harvested from rat fetuses and/or neural stem cells derived from human induced pluripotent stem cells (iPSC) can be utilized. Prolonged exposure of rodent neural stem cells to clinically-relevant concentrations of ketamine resulted in elevated NMDA receptor levels as indicated by NR1subunit over-expression in neurons. This was associated with enhanced damage in neurons. In contrast, the viability and proliferation rate of undifferentiated neural stem cells were not significantly affected after ketamine exposure. Calcium imaging data indicated that 50μM NMDA did not cause a significant influx of calcium in typical undifferentiated neural stem cells; however, it did produce an immediate elevation of intracellular free Ca2+ [Ca2+]i in differentiated neurons derived from the same neural stem cells.This paper reviews the literature on this subject and previous findings suggest that prolonged exposure of developing neurons to ketamine produces an increase in NMDA receptor expression (compensatory up-regulation) which allows for a higher/toxic influx of calcium into neurons once ketamine is removed from the system, leading to neuronal cell death likely due to elevated reactive oxygen species generation. The absence of functional NMDA receptors in cultured neural stem cells likely explains why clinically-relevant concentrations of ketamine did not affect undifferentiated neural stem cell viability.

DEPTOR in POMC neurons affects liver metabolism but is dispensable for the regulation of energy balance.

We have recently demonstrated that specific overexpression of DEP-domain containing mTOR-interacting protein (DEPTOR) in the mediobasal hypothalamus (MBH) protects mice against high-fat diet-induced obesity, revealing DEPTOR as a significant contributor to energy balance regulation. On the basis of evidence that DEPTOR is expressed in the proopiomelanocortin (POMC) neurons of the MBH, the present study aimed to investigate whether these neurons mediate the metabolic effects of DEPTOR. Here, we report that specific DEPTOR overexpression in POMC neurons does not recapitulate any of the phenotypes observed when the protein was overexpressed in the MBH. Unlike the previous model, mice overexpressing DEPTOR only in POMC neurons 1) did not show differences in feeding behavior, 2) did not exhibit changes in locomotion activity and oxygen consumption, 3) did not show an improvement in systemic glucose metabolism, and 4) were not resistant to high-fat diet-induced obesity. These results support the idea that other neuronal populations are responsible for these phenotypes. Nonetheless, we observed a mild elevation in fasting blood glucose, insulin resistance, and alterations in liver glucose and lipid homeostasis in mice overexpressing DEPTOR in POMC neurons. Taken together, these results show that DEPTOR overexpression in POMC neurons does not affect energy balance regulation but could modulate metabolism through a brain-liver connection.