Neurexin 2 p.G849D variant, implicated in Parkinson's disease, increases reactive oxygen species, and reduces cell viability and mitochondrial membrane potential in SH-SY5Y cells.

Parkinson's disease (PD) is a neurodegenerative movement disorder, affecting 1-2% of the human population over 65. A previous study by our group identified a p.G849D variant in neurexin 2α (NRXN2) co-segregating with PD, prompting validation of its role using experimental methods. This novel variant had been found in a South African family with autosomal dominant PD. NRXN2α is an essential synaptic maintenance protein with multiple functional roles at the synaptic cleft. The aim of the present study was to investigate the potential role of the translated protein NRXN2α and the observed mutant in PD by performing functional studies in an in vitro model. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells to assess the effect of the mutant on cell viability and apoptosis [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay; ApoTox-Glo™ Triplex Assay)], mitochondrial membrane potential (MMP; MitoProbe™ JC-1 Assay), mitochondrial network analysis (MitoTracker®) and reactive oxygen species (ROS; ROS-Glo™ H2O2 Assay). Cells transfected with the mutant NRXN2α plasmid showed decreased cell viability and MMP. They also exhibited increased ROS production. However, these cells showed no changes in mitochondrial fragmentation. Our findings led us to speculate that the p.G849D variant may be involved in a toxic feedback loop leading to neuronal death in PD. Mitochondrial dysfunction and synaptic dysfunction have been linked to PD. Therefore, findings from this exploratory study are in line with previous studies connecting these two processes and warrants further investigation into the role of this variant in other cellular and animal models.

Sildenafil, a Type-5 Phosphodiesterase Inhibitor, Fails to Reverse Myeloid-Derived Suppressor Cell-Mediated T Cell Suppression in Cells Isolated From Tuberculosis Patients.

Successful TB treatment is hampered by increasing resistance to the two most effective first-line anti-TB drugs, namely isoniazid and rifampicin, thus innovative therapies focused on host processes, termed host-directed therapies (HDTs), are promising novel approaches for increasing treatment efficacy without inducing drug resistance. We assessed the ability of Sildenafil, a type-5 phosphodiesterase inhibitor, as a repurposed compound, to serve as HDT target, by counteracting the suppressive effects of myeloid-derived suppressor cells (MDSC) obtained from active TB cases on T-cell responsiveness. We confirm that MDSC suppress non-specific T-cell activation. We also show that Sildenafil treatment fails to reverse the MDSC-mediated suppression of T-cell functions measured here, namely activation and proliferation. The impact of Sildenafil treatment on improved immunity, using the concentration tested here, is likely to be minimal, but further identification and development of MDSC-targeting TB host-directed therapies are warranted.

Advances in fluorescence microscopy can reveal important new aspects of tissue regeneration.

Advances in fluorescence microscopy can greatly facilitate research in regenerative health. Specifically, live cell imaging is a powerful tool to understand the underlying mechanisms of tissue regeneration, which is characterised by a dynamic interplay at cellular and molecular level. Recent advances in microscopy have aimed to overcome some of the most challenging limitations, such as slow acquisition speed, the resolution limit of light, low signal to noise ratio in thick samples, as well as photobleaching and phototoxicity. In applications such as lightsheet fluorescence microscopy and intra-vital multi-photon microscopy, improved deep tissue imaging have been achieved, and super-resolution technologies have shown to improve optical resolution far beyond the diffraction limit of light for better visualisation at the cellular and molecular level. By combining certain techniques, researchers can now image live samples at much higher resolution for a prolonged time. Advances in analytical technologies will enable researchers to gain an even better understanding of the processes involved to ultimately translate stem cell research into therapeutic interventions.

Model for Studying the Effects of Chronic Metabolic Disease on Endogenous Bone Marrow Stem Cell Populations.

Disease-associated impairment/dysfunction of stem cell populations is prominent in chronic metabolic and inflammatory diseases, such as type 2 diabetes mellitus (DM) where the multifunctional properties (viability, proliferation, paracrine secretion, multilineage differentiation) of bone marrow resident mesenchymal stem cells (MSCs) can be affected. The growth and viability impairments make it difficult to study the underlying molecular mechanisms related to the dysfunction of these cells in vitro. We have consequently optimized the isolation and culture conditions for impaired/dysfunctional bone marrow MSCs from B6.Cg-Lepob/J obese prediabetic mice. The method described here permits ex vivo investigations into disease-associated functional impairments and the dysregulated molecular mechanisms in these primary MSCs through direct comparisons with their healthy wild-type C57BL6/J control mouse counterparts.

CD4+ T cells in HIV infection show increased levels of expression of a receptor for vasoactive intestinal peptide, VPAC2.

Immune activation is a strong predictor of disease outcome in HIV infection and promotes the loss of CD4+ T cells. The neuropeptide, vasoactive intestinal peptide (VIP), has immune-modulating properties with specific receptors identified on lymphocytes; VPAC1 and VPAC2. Studies have shown that VIP limits immune activation and apoptosis in T cells by decreasing the expression of the apoptosis signaling molecule Fas ligand (FasL). VIP receptor surface expression has not been investigated by flow cytometry in the context of HIV infection and may represent a novel target for immune-modulating therapy. Eighty-seven untreated HIV-infected individuals with CD4 counts >200 and 57 uninfected controls were recruited from a primary health clinic in Cape Town, South Africa. Flow cytometry was used to determine levels of expression of VPAC1 and VPAC2, as well as FasL on CD4+ T cells, and these results were correlated with the immune activation phenotype %CD38+CD8+ T cells. VPAC2 expression was significantly increased in the HIV group (mean %VPAC2+CD4+ cells 19.25 vs. control 12.56; p ≤ 0.0001), but no difference in VPAC1 expression was observed. VPAC2 correlated positively with FasL (r = 0.310; p = 0.001), and there was a significant inverse correlation between FasL and the CD4 count (r = -0.211; p = 0.013) and a direct correlation with %CD38+CD8+ T cells (r = 0.39; p ≤ 0.0001). Thus, higher levels of immune activation correlated with higher levels of the death-signaling FasL and lower CD4 counts. VPAC2 may provide a novel target for the selective limitation of CD4+ T-cell death in HIV infection.

Short-term antiretroviral therapy fails to reduce the expanded activated CCR5-expressing CD4+ T lymphocyte population or to restore the depleted naive population in chronically HIV-infected individuals with active pulmonary tuberculosis.

The effective role of antiretroviral (ARV) therapy in the regulation of CD4 T cell subset distribution, coreceptor expression, and activation status in individuals with chronic HIV also presenting with active pulmonary TB is not clearly understood. A cross-sectional analysis was performed on a total of 137 South African individuals. CCR5, CXCR4, and CD38 expression of CD4 T cell subsets in HIV-infected individuals with and without active pulmonary tuberculosis (TB) disease, pre- and post-ARV therapy, were determined by flow cytometry. In treatment-naive patients, CD4 T cells showed elevated surface expression of CCR5 and CD38 in TB/HIV coinfection as compared to HIV infection alone despite the overall percentage of CD4 T cells expressing CCR5 being reduced. Total CD38+ CD4 T cells were not significantly increased in either group; however, mean CD38 fluorescence was significantly higher in the context of TB infection. HIV/TB-coinfected individuals also displayed an increased percentage of activated (CD38+) CCR5+ CD4 T cells as compared to HIV patients alone. The naive CD4 T cell subset was depleted similarly in both HIV and HIV/TB groups. Only the HIV treatment group and not the TB-coinfected treatment group showed significantly decreased activated CCR5+ CD4 T cells, an increased percentage of naive T cells, and a decreased percentage of antigen-experienced T cells. This study highlighted an association of TB disease with immune activation, particularly of the CCR5+ CD4 T cell subset in HIV infection and the differential impact of ARV treatment. Further studies are needed to understand how TB coinfection confounds normal responses to ARV.