Functional consequences of familial ALS-associated SOD1L84F in neuronal and muscle cells.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder characterized by progressive skeletal muscle denervation and loss of motor neurons that results in muscle atrophy and eventual death due to respiratory failure. Previously, we identified a novel SOD1L84F variation in a familial ALS case. In this study, we examined the functional consequences of SOD1L84F overexpression in the mouse motor neuron cell line (NSC-34). The cells expressing SOD1L84F showed increased oxidative stress and increased cell death. Interestingly, SOD1L84F destabilized the native dimer and formed high molecular weight SDS-resistant protein aggregates. Furthermore, SOD1L84F also decreased the percentage of differentiated cells and significantly reduced neurite length. A plethora of evidence suggested active involvement of skeletal muscle in disease initiation and progression. We observed differential processing of the mutant SOD1 and perturbations of cellular machinery in NSC-34 and muscle cell line C2C12. Unlike neuronal cells, mutant protein failed to accumulate in muscle cells probably due to the activated autophagy, as evidenced by increased LC3-II and reduced p62. Further, SOD1L84F altered mitochondrial dynamics only in NSC-34. In addition, microarray analysis also revealed huge variations in differentially expressed genes between NSC-34 and C2C12. Interestingly, SOD1L84F hampered the endogenous FUS autoregulatory mechanism in NSC-34 by downregulating retention of introns 6 and 7 resulting in a two-fold upregulation of FUS. No such changes were observed in C2C12. Our findings strongly suggest the differential processing and response towards the mutant SOD1 in neuronal and muscle cell lines.

Melanoma-associated repair-like Schwann cells suppress anti-tumor T-cells via 12/15-LOX/COX2-associated eicosanoid production.

Peripheral glia, specifically the Schwann cells (SCs), have been implicated in the formation of the tumor microenvironment (TME) and in cancer progression. However, in vivo and ex vivo analyses of how cancers reprogram SC functions in different organs of tumor-bearing mice are lacking. We generated Plp1-CreERT/tdTomato mice which harbor fluorescently labeled myelinated and non-myelin forming SCs. We show that this model enables the isolation of the SCs with high purity from the skin and multiple other organs. We used this model to study phenotypic and functional reprogramming of the SCs in the skin adjacent to melanoma tumors. Transcriptomic analyses of the peritumoral skin SCs versus skin SCs from tumor-free mice revealed that the former existed in a repair-like state typically activated during nerve and tissue injury. Peritumoral skin SCs also downregulated pro-inflammatory genes and pathways related to protective anti-tumor responses. In vivo and ex vivo functional assays confirmed immunosuppressive activities of the peritumoral skin SCs. Specifically, melanoma-reprogrammed SCs upregulated 12/15-lipoxygenase (12/15-LOX) and cyclooxygenase (COX)-2, and increased production of anti-inflammatory polyunsaturated fatty acid (PUFA) metabolites prostaglandin E2 (PGE2) and lipoxins A4/B4. Inhibition of 12/15-LOX or COX2 in SCs, or EP4 receptor on lymphocytes reversed SC-dependent suppression of anti-tumor T-cell activation. Therefore, SCs within the skin adjacent to melanoma tumors demonstrate functional switching to repair-like immunosuppressive cells with dysregulated lipid oxidation. Our study suggests the involvement of the melanoma-associated repair-like peritumoral SCs in the modulation of locoregional and systemic anti-tumor immune responses.

Interaction of novel proteins, centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth.

Centrins are cytoskeletal proteins associated with the centrosomes or basal bodies in the eukaryotes. We previously reported the involvement of Centrin 1-3 proteins in cell division in the protozoan parasites Leishmania donovani and Trypanosoma brucei. Centrin4 and 5, unique to such parasites, had never been characterized in Leishmania parasite. In the current study, we addressed the function of centrin4 (LdCen4) in Leishmania. By dominant-negative study, the episomal expression of C-terminal truncated LdCen4 in the parasite reduced the parasite growth. LdCen4 double allele gene deletion by either homologous recombination or CRISPR-Cas9 was not successful in L. donovani. However, CRISPR-Cas9-based deletion of the homologous gene was possible in L. mexicana, which attenuated the parasite growth in vitro, but not ex vivo in the macrophages. LdCen4 also interacts with endogenous and overexpressed LdPOC protein, a homolog of centrin reacting human POC (protein of centriole) in a calcium sensitive manner. LdCen4 and LdPOC binding has also been confirmed through in silico analysis by protein structural docking and validated by co-immunoprecipitation. By immunofluorescence studies, we found that both the proteins share a common localization at the basal bodies. Thus, for the first time, this article describes novel centrin4 and its binding protein in the protozoan parasites.

Tumor-Induced T Cell Polarization by Schwann Cells.

Nerve-cancer crosstalk resulting in either tumor neurogenesis or intratumoral neurodegeneration is critically controlled by Schwann cells, the principal glial cells of the peripheral nervous system. Though the direct stimulating effect of Schwann cells on malignant cell proliferation, motility, epithelial-mesenchymal transition, and the formation of metastases have been intensively investigated, the ability of Schwann cells to affect the effector and regulatory immune cells in the tumor environment is significantly less studied. Here, we demonstrated that tumor cells could stimulate Schwann cells to produce high levels of prostaglandin E, which could be blocked by COX-2 inhibitors. This effect was mediated by tumor-derived TGF-ß as neutralization of this cytokine in the tumor-conditioned medium completely blocked the inducible prostaglandin E production by Schwann cells. Similar protective effects were also induced by the Schwann cell pretreatment with TGF-ßR1/ALK4/5/7 and MAPK/ERK kinase inhibitors of the canonical and non-canonical TGF-ß signaling pathways, respectively. Furthermore, prostaglandin E derived from tumor-activated Schwann cells blocked the proliferation of CD3/CD28-activated T cells and upregulated the expression of CD73 and PD-1 on both CD4+ and CD8+ T cells, suggesting T cell polarization to the exhausted phenotype. This new pathway of tumor-induced T cell inhibition via the activation of neuroglial cells represents new evidence of the importance of nerve-cancer crosstalk in controlling tumor development and progression. A better understanding of the tumor-neuro-immune axis supports the development of efficient targets for harnessing this axis and improving the efficacy of cancer therapy.

Sensory Nerves Impede the Formation of Tertiary Lymphoid Structures and Development of Protective Antimelanoma Immune Responses.

Peripheral neurons comprise a critical component of the tumor microenvironment (TME). The role of the autonomic innervation in cancer has been firmly established. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Specifically, sensory ablation led to improved leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Cutaneous sensory nerves hindered the maturation of intratumoral high endothelial venules and limited the formation of mature tertiary lymphoid-like structures containing organized clusters of CD4+ T cells and B cells. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Importantly, CD8a depletion prevented denervation-dependent antitumor effects. Finally, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby defining a novel target for therapeutic intervention in the melanoma setting.

Keratinocyte death by ferroptosis initiates skin inflammation after UVB exposure.

The ultraviolet B radiation (UVB) causes skin inflammation, which contributes to the causality and the exacerbation of a number of cutaneous diseases. However, the mechanism of UVB-driven inflammation in the skin remains poorly understood. We show that ferroptosis, a non-apoptotic programmed cell death pathway that is promoted by an excessive phospholipid peroxidation, is activated in the epidermal keratinocytes after their exposure to UVB. The susceptibility of the keratinocytes to UVB-induced ferroptosis depends on the extent of pro-ferroptosis death signal generation and the dysregulation of the glutathione system. Inhibition of ferroptosis prevents the release of HMGB1 from the human epidermal keratinocytes, and blocks necroinflammation in the UVB-irradiated mouse skin. We show that while apoptosis and pyroptosis are also detectable in the keratinocytes after UVB exposure, ferroptosis plays a significant role in initiating UVB-induced inflammation in the skin. Our results have important implications for the prevention and the treatment of a broad range of skin diseases which are fostered by UVB-induced inflammation.