Water-insoluble hexavalent chromium compounds are well-established human lung carcinogens. Lead chromate, a model insoluble Cr(VI) compound, induces DNA damage, chromosome aberrations, and dose-dependent cell death in human and Chinese hamster ovary (CHO) cells. The relationship between lead chromate-induced DNA damage and chromosome aberrations is unknown. Our study focus was on examining the role of XRCC1 in lead chromate-induced cytotoxicity and structural chromosomal aberrations in CHO cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant), and H9T3 (EM9 complemented with human XRCC1 gene). Cytotoxicity was significantly higher in EM9 cells when compared to AA8 and H9T3 cells, indicating that XRCC1 is important for protecting cells from lead chromate particles-induced cell death. The frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total amount of Cr(VI)-induced chromosome damage was exacerbated by XRCC1 deficiency, and the spectrum of damage changed dramatically. Chromatid and isochromatid lesions were the most prominent aberrations induced in all cell lines. XRCC1 was essential to reduce the formation of chromatid lesions but not for isochromatid lesions. In addition, XRCC1 deficiency resulted in a dramatic increase in the number of chromatid exchanges, indicating that XRCC1 is involved in protection from lead chromate-induced chromosome instability.

C57BL/6 (B6) mice develop peripheral neuropathy post-LP-BM5 infection, a murine model of HIV-1 infection, along with the up-regulation of select spinal cord cytokines. We investigated if calcitonin gene-related peptide (CGRP) contributed to the development of peripheral neuropathy by stimulating glial responses. An increased expression of lumbar spinal cord CGRP was observed in vivo, post-LP-BM5 infection. Consequently, in vitro CGRP co-treatments led to a microglial content-dependent attenuation of viral loads in spinal cord mixed glia infected with selected doses of LP-BM5. This inhibition was neither caused by the loss of glia nor induced via the direct inhibition of LP-BM5 by CGRP.

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Water-insoluble hexavalent chromium compounds are well-established human lung carcinogens. Lead chromate, a model insoluble Cr(VI) compound, induces DNA damage, chromosome aberrations, and dose-dependent cell death in human and Chinese hamster ovary (CHO) cells. The relationship between lead chromate-induced DNA damage and chromosome aberrations is unknown. Our study focus was on examining the role of XRCC1 in lead chromate-induced cytotoxicity and structural chromosomal aberrations in CHO cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant), and H9T3 (EM9 complemented with human XRCC1 gene). Cytotoxicity was significantly higher in EM9 cells when compared to AA8 and H9T3 cells, indicating that XRCC1 is important for protecting cells from lead chromate particles-induced cell death. The frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total amount of Cr(VI)-induced chromosome damage was exacerbated by XRCC1 deficiency, and the spectrum of damage changed dramatically. Chromatid and isochromatid lesions were the most prominent aberrations induced in all cell lines. XRCC1 was essential to reduce the formation of chromatid lesions, but not for isochromatid lesions. In addition, XRCC1 deficiency resulted in a dramatic increase in the number of chromatid exchanges, indicating that XRCC1 is involved in protection from lead chromate-induced chromosome instability.

Primary sensory Dorsal Root Ganglia (DRG) neurons are diverse, with distinct populations that respond to specific stimuli. Previously, we observed that functionally distinct populations of DRG neurons express mRNA transcript variants with different 3` untranslated regions (3`UTRs). 3`UTRs harbor binding sites for interaction with RNA-binding proteins (RBPs) critical for targeting mRNAs to subcellular domains, modulating transcript stability and regulating the rate of translation. In the current study we sought to determine if 3`UTR-binding proteins are restricted to specific DRG neuron populations. Analysis of publicly available single-cell RNA-Sequencing (scRNA-Seq) data generated from adult mice revealed that 17 3`UTR-binding RBPs were enriched in specific populations of DRG neurons. This included 4 members of the CUGBP Elav-Like Family (CELF). CELF2 and CELF4 were enriched in peptidergic, CELF6 in both peptidergic and nonpeptidergic and CELF3 in tyrosine hydroxylase-expressing neurons. CELF4 is a known regulator of neural excitability, likely through modulation of protein synthesis via binding to interaction sites within the 3`UTRs of mRNAs. Immunofluorescence studies showed 60% of CELF4+ neurons are small diameter C fibers and 33% medium diameter myelinated (likely Aδ) fibers. Co-expression analyses using transcriptomic data and quantitative immunofluorescence revealed that CELF4 is enriched in nociceptive neurons that express GFRA3, CGRP and the capsaicin receptor TRPV1. Finally, genes with CELF4 binding motifs expressed in CELF4+ neurons are significantly associated with gene ontology (GO) terms such as RNA-binding and translation. We propose that CELF4 may therefore control a novel regulon that coordinates the translation of mRNAs encoding components of the protein translation apparatus in nociceptors.

LP-BM5 retrovirus infection is an established murine model of HIV infection in humans due to that susceptible strains of mice (such as B6) develop immunodeficiency syndrome. Previously, we have shown that infected B6 mice developed peripheral neuropathy and CD40 knockout (KO) mice displayed less mechanical hypersensitivity (behavioral sign of peripheral neuropathy) post-LP-BM5 infection. The spinal cord viral RNA levels were significantly lower in CD40 KO mice compared to wild type (WT) B6 mice, which is correlated with decreased lumbar spinal cord cytokines, IL-1-beta, IL-6 and IFN-gamma. It is known that LP-BM5 can infect glial cells directly. We hypothesized that glial cells in CD40 KO mice were less susceptible to LP-BM5 infection. To test this hypothesis, primary spinal cord mixed glial cells from both WT and CD40 KO mice were infected with various doses of LP-BM5. Consistent with our in vivo findings, levels of viral RNA are significantly lower in mixed glial cells from CD40 KO mice than that from WT mice. Further, levels of cytokines, IL-1-beta, IL-6, TNF-alpha, IFN-gamma, and IL-12p40 were measured in the supernatants. LP-BM5 only induced minimal increase of these cytokines and there no significant differences between WT and CD40 KO mice were observed. Our data indicate that glial cells lacking CD40 are more resistant to LP-BM5 infection and this resistance does not seem to be due to the decreased production of proinflammatory cytokines by glial cells.

LP-BM5 is a murine retrovirus that induces peripheral neuropathy and AIDS like immune deficiency in B6 mice. Upon infection, primary sensory neurons in the spinal cord express elevated levels of calcitonin gene related protein (CGRP). Previously, we showed that CGRP induced reduction of LP-BM5 viral loads in primary mixed glial cells. The purpose of this current study was to investigate possible CGRP downstream targets that may contribute to its antiretroviral response. We hypothesized that CGRP decreases the viral load in glial cells by modifying the CD40 expression; increasing in CD40 signaling may promote production of cytokines and chemokines with antiretroviral properties. Using CD40 knockout mice we showed that CD40 is required for CGRP mediated antiretroviral response in mixed glia. In microglial cell line, CD40 expression was elevated after treatment with CGRP. Similar effect was seen in microglia from spinal cord derived mixed glial cultures. While LP-BM5 increased microglial CD40 expression gradually over time up to 7 days post-infection in primary mixed glia, CGRP caused an early elevation (24 hr post-infection) of microglial CD40 expression in microglia and stayed relatively steady levels 7 days after infection. CGRP also promoted chemokine CXCL1(KC) production in mixed glia. Our data suggest that activation of CD40 mediated signaling may result in increased production of chemokines that may interfere with retroviral replication. Future studies will be directed to specifically delineate CD40 downstream target involved in antiretroviral response in glial cells.