Probes for INS

Synthesis and regulation of catecholamine neurotransmitters in the central nervous system are implicated in the pathogenesis of a number of neuropsychiatric disorders. To identify factors that regulate the presynaptic synthesis of catecholamines, we tested the hypothesis that the rate-limiting enzyme of the catecholamine biosynthetic pathway, tyrosine hydroxylase (TH), is locally synthesized in axons and presynaptic nerve terminals of noradrenergic neurons. To isolate pure axonal mRNA and protein, rat superior cervical ganglion sympathetic neurons were cultured in compartmentalized Campenot chambers. qRT-PCR and RNA in situ hybridization analyses showed that TH mRNA is present in distal axons. Colocalization experiments with nerve terminal marker proteins suggested that both TH mRNA and protein localize in regions of the axon that resemble nerve terminals (i.e., synaptic boutons). Analysis of polysome-bound RNA showed that TH mRNA is present in polysomes isolated from distal axons. Metabolic labeling of axonally synthesized proteins labeled with the methionine analog, L-azidohomoalanine, showed that TH is locally synthesized in axons. Moreover, the local transfection and translation of exogenous TH mRNA into distal axons facilitated axonal dopamine synthesis. Finally, using chimeric td-Tomato-tagged constructs, we identified a sequence element within the TH 3'UTR that is required for the axonal localization of the reporter mRNA. Taken together, our results provide the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated. These findings raise the interesting possibility that the biosynthesis of the catecholamine neurotransmitters is locally regulated in the axon and/or presynaptic nerve terminal.

Severe influenza is often associated with disease manifestations outside the respiratory tract. Whilst pro-inflammatory cytokines can be detected in the lungs and blood of infected patients, the role of extra-respiratory organs in the production of pro-inflammatory cytokines is unknown. Here, we show that both pandemic H1N1 and highly pathogenic H5N1 virus induce expression of TNFα, IL-6 and IL-8 in the respiratory tract and central nervous system. In addition, H5N1 virus induced cytokines in the heart, pancreas, spleen, liver and jejunum. Together, these data suggest that extra-respiratory tissues contribute to systemic cytokine responses which may increase the severity of influenza.

The present study demonstrates HIV-1 Tat-mediated epigenetic downregulation of microglial miR-124 and its association with microglial activation. Exposure of mouse primary microglia isolated from newborn pups of either sex to HIV-1 Tat resulted in decreased expression of primary miR-124-1, primary miR-124-2 as well as the mature miR-124. In parallel, HIV-1 Tat exposure to mouse primary microglial cellsresulted in increased expression of DNA methylation enzymes, such as DNMT1, DNMT3A, and DNMT3B that were also accompanied by increased global DNA methylation. Bisulfite-converted genomic DNA sequencing in the HIV-1 Tat exposed mouse primary microglial cellsfurther confirmed increased DNA methylation of the primary miR-124-1 and primary miR-124-2 promoters. Bioinformatic analyses identified MECP2 as a novel 3'-UTR target of miR-124. This was further validated in mouse primary microglial cells wherein HIV-1 Tat-mediated downregulation of miR-124 resulted in increased expression of MECP2, leading in turn to further repression of miR-124 via the feedback loop. In addition to MECP2, miR-124 also modulated the levels of STAT3 through its binding to the 3'-UTR, leading to microglial activation. Luciferase assays and Ago2 immunoprecipitation determined the direct binding between miR-124 and 3'-UTR of both MECP2 and STAT3. Gene silencing of MECP2 and DNMT1 and overexpression of miR-124 blocked HIV-1 Tat-mediated downregulation of miR-124 and microglial activation. In vitro findings were also confirmed in the basal ganglia of SIV-infected rhesus macaques (both sexes). In summary, our findings demonstrate a novel mechanism of HIV-1 Tat-mediated activation of microglia via downregulation of miR-124, leading ultimately to increased MECP2 and STAT3 signaling.
SIGNIFICANCE STATEMENT
Despite the effectiveness of combination antiretroviral therapy in controlling viremia, the CNS continues to harbor viral reservoirs. The persistence of low-level virus replication leads to the accumulation of early viral proteins including HIV-1 Tat protein. Understanding the epigenetic/molecular mechanism(s) by which viral proteins such as HIV-1 Tat can activate microglia is thus of paramount importance. This study demonstrated HIV-1 Tat-mediated DNA methylation of the miR-124 promoter leads to its downregulation with a concomitant upregulation of the MECP2-STAT3-IL6 resulting in microglial activation. These findings reveal an unexplored epigenetic/molecular mechanism(s) underlying HIV-1 Tat-mediated microglial activation, thereby providing a potential target for the development of therapeutics aimed at ameliorating microglial activation and neuroinflammation in the context of HIV-1 infection.

A subset of midbrain dopamine (DA) neurons express vesicular glutamate transporter 2 (VgluT2), which facilitates synaptic vesicle loading of glutamate. Recent studies indicate that such expression can modulate DA-dependent reward behaviors, but little is known about functional consequences of DA neuron VgluT2 expression in neurodegenerative diseases like Parkinson's disease (PD). Here, we report that selective deletion of VgluT2 in DA neurons in conditional VgluT2-KO (VgluT2-cKO) mice abolished glutamate release from DA neurons, reduced their expression of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB), and exacerbated the pathological effects of exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, viral rescue of VgluT2 expression in DA neurons of VglutT2-cKO mice restored BDNF/TrkB expression and attenuated MPTP-induced DA neuron loss and locomotor impairment. Together, these findings indicate that VgluT2 expression in DA neurons is neuroprotective. Genetic or environmental factors causing reduced expression or function of VgluT2 in DA neurons may place some individuals at increased risk for DA neuron degeneration. Therefore, maintaining physiological expression and function of VgluT2 in DA neurons may represent a valid molecular target for the development of preventive therapeutic interventions for PD.

Parkinson's disease is characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). DA neurons in the ventral tegmental area are more resistant to this degeneration than those in the SNc, though the mechanisms for selective resistance or vulnerability remain poorly understood. A key to elucidating these processes may lie within the subset of DA neurons that corelease glutamate and express the vesicular glutamate transporter VGLUT2. Here, we addressed the potential relationship between VGLUT expression and DA neuronal vulnerability by overexpressing VGLUT in DA neurons of flies and mice. In Drosophila, VGLUT overexpression led to loss of select DA neuron populations. Similarly, expression of VGLUT2 specifically in murine SNc DA neurons led to neuronal loss and Parkinsonian behaviors. Other neuronal cell types showed no such sensitivity, suggesting that DA neurons are distinctively vulnerable to VGLUT2 expression. Additionally, most DA neurons expressed VGLUT2 during development, and coexpression of VGLUT2 with DA markers increased following injury in the adult. Finally, conditional deletion of VGLUT2 made DA neurons more susceptible to Parkinsonian neurotoxins. These data suggest that the balance of VGLUT2 expression is a crucial determinant of DA neuron survival. Ultimately, manipulation of this VGLUT2-dependent process may represent an avenue for therapeutic development.