Patient-derived iPSC modeling of rare neurodevelopmental disorders: Molecular pathophysiology and prospective therapies.

The pathological alterations that manifest during the early embryonic development due to inherited and acquired factors trigger various neurodevelopmental disorders (NDDs). Besides major NDDs, there are several rare NDDs, exhibiting specific characteristics and varying levels of severity triggered due to genetic and epigenetic anomalies. The rarity of subjects, paucity of neural tissues for detailed analysis, and the unavailability of disease-specific animal models have hampered detailed comprehension of rare NDDs, imposing heightened challenge to the medical and scientific community until a decade ago. The generation of functional neurons and glia through directed differentiation protocols for patient-derived iPSCs, CRISPR/Cas9 technology, and 3D brain organoid models have provided an excellent opportunity and vibrant resource for decoding the etiology of brain development for rare NDDs caused due to monogenic as well as polygenic disorders. The present review identifies cellular and molecular phenotypes demonstrated from patient-derived iPSCs and possible therapeutic opportunities identified for these disorders. New insights to reinforce the existing knowledge of the pathophysiology of these disorders and prospective therapeutic applications are discussed.

Hepatic expression of lipopolysaccharide-binding protein (Lbp) is induced by the gut microbiota through Myd88 and impairs glucose tolerance in mice independent of obesity.

OBJECTIVE: Gut-derived inflammatory factors can impair glucose homeostasis, but the underlying mechanisms are not fully understood. In this study, we investigated how hepatic gene expression is regulated by gut colonization status through myeloid differentiation primary response 88 (MYD88) and how one of the regulated genes, lipopolysaccharide-binding protein (Lbp), affects insulin signaling and systemic glucose homeostasis. METHODS: Liver transcriptomics analysis was conducted on four groups of mice fed a chow diet: conventionally raised (CONV-R) wild-type, germ-free (GF) wild-type, CONV-R Myd88 KO, and GF Myd88 KO. Primary hepatocytes were exposed to combinations of lipopolysaccharide (LPS), LBP, and the LBP-blocking peptide LBPK95A, and the effect on insulin signaling was determined. To assess how LBP affects glucose metabolism in vivo, two mouse models were applied: treatment with LBPK95A and hepatic knockdown of Lbp using CRISPR-CAS9. RESULTS: We showed that the colonization status regulates gene expression in the liver and that a subset of these genes, including Lbp, is regulated through MYD88. Furthermore, we demonstrated that LBP impairs insulin signaling in hepatocytes in the presence of low levels of LPS and that the effect of LBP is abolished by LBPK95A. We showed that both systemic pharmacological blocking of LBP by LBPK95A and CRISPR-CAS9-mediated downregulation of hepatic Lbp improve glucose homeostasis. CONCLUSIONS: Our results demonstrate that the gut microbiota regulates hepatic expression of Lbp through MYD88-dependent signaling. LBP potentiates LPS inhibition of insulin signaling in vitro and impairs systemic glucose homeostasis in vivo.

Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy.

BACKGROUND: Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT. METHODS: In vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method. RESULTS: Photofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins. CONCLUSIONS: Altogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy.