The maintenance of genomic integrity is essential for normal cellular functions. However, it is difficult to maintain over a lifetime in postmitotic cells such as neurons, in which DNA damage increases with age and is exacerbated by multiple neurological disorders, including Alzheimer’s disease (AD). Here we used immunohistochemical staining to detect DNA double strand breaks (DSBs), the most severe form of DNA damage, in postmortem brain tissues from patients with mild cognitive impairment (MCI) or AD and from cognitively unimpaired controls. Immunostaining for γH2AX—a post-translational histone modification that is widely used as a marker of DSBs—revealed increased proportions of γH2AX-labeled neurons and astrocytes in the hippocampus and frontal cortex of MCI and AD patients, as compared to age-matched controls. In contrast to the focal pattern associated with DSBs, some neurons and glia in humans and mice showed diffuse pan-nuclear patterns of γH2AX immunoreactivity. In mouse brains and primary neuronal cultures, such pan-nuclear γH2AX labeling could be elicited by increasing neuronal activity. To assess whether pan-nuclear γH2AX represents DSBs, we used a recently developed technology, DNA damage in situ ligation followed by proximity ligation assay, to detect close associations between γH2AX sites and free DSB ends. This assay revealed no evidence of DSBs in neurons or astrocytes with prominent pan-nuclear γH2AX labeling. These findings suggest that focal, but not pan-nuclear, increases in γH2AX immunoreactivity are associated with DSBs in brain tissue and that these distinct patterns of γH2AX formation may have different causes and consequences. We conclude that AD is associated with an accumulation of DSBs in vulnerable neuronal and glial cell populations from early stages onward. Because of the severe adverse effects this type of DNA damage can have on gene expression, chromatin stability and cellular functions, DSBs could be an important causal driver of neurodegeneration and cognitive decline in this disease. Electronic supplementary material The online version of this article (10.1186/s40478-019-0723-5) contains supplementary material, which is available to authorized users.

SUMMARY The pathogenic mechanisms of frontotemporal dementia (FTD) remain poorly understood. Here we generated multiple induced pluripotent stem cell (iPSC) lines from a control subject, a patient with sporadic FTD, and an FTD patient with a novel GRN mutation (PGRN S116X). In neurons and microglia differentiated from PGRN S116X iPSCs, the levels of intracellular and secreted progranulin were reduced, establishing patient-specific cellular models of progranulin haploinsufficiency. Through a systematic screen of inducers of cellular stress, we found that PGRN S116X neurons, but not sporadic FTD neurons, exhibited increased sensitivity to staurosporine and other kinase inhibitors. Moreover, the serine/threonine kinase S6K2, a component of the PI3K and MAPK pathways, was specifically downregulated in PGRN S116X neurons. Both increased sensitivity to kinase inhibitors and reduced S6K2 were rescued by progranulin expression. Our findings identify cell-autonomous, reversible defects in patient neurons with progranulin deficiency and provide a new model for studying progranulin-dependent pathogenic mechanisms and testing potential therapies.

3-dimensional chromosomal conformations regulate transcription by moving enhancers and regulatory elements into spatial proximity with target genes. Here, we describe activity-regulated long-range loopings bypassing up to 0.5 megabase of linear genome to modulate NMDA glutamate receptor GRIN2B expression in human and mouse prefrontal cortex. Distal intronic and 3’ intergenic loop formations competed with repressor elements to access promoter-proximal sequences, and facilitated expression via a ‘cargo’ of AP-1 and NRF-1 transcription factors and TALE-based transcriptional activators. Neuronal deletion or overexpression of Kmt2a/Mll1 H3K4- and Kmt1e/Setdb1 H3K9-methyltransferase was associated with higher order chromatin changes at distal regulatory Grin2b sequences and impairments in working memory. Genetic polymorphisms and isogenic deletions of loop-bound sequences conferred liability for cognitive performance and decreased GRIN2B expression. Dynamic regulation of chromosomal conformations emerges as a novel layer for transcriptional mechanisms impacting neuronal signaling and cognition.

Calcium signaling is important for differentiation-dependent gene expression, but is also involved in other cellular functions. Therefore mechanisms must exist to distinguish calcium signaling relevant to differentiation. Calcineurin is a calcium-regulated phosphatase that is required for myogenic gene expression and skeletal muscle differentiation. Here, we demonstrate that inhibition of calcineurin blocks chromatin remodeling and that the Brg1 ATPase of the SWI/SNF chromatin remodeling enzyme, which is required for the activation of myogenic gene expression, is a calcineurin substrate. Furthermore, we identify the calcium-regulated classical protein kinase C beta (PKCβ) as a repressor of myogenesis and as the enzyme that opposes calcineurin function. Replacement of endogenous Brg1 with a phosphomimetic mutant in primary myoblasts inhibits myogenesis, while replacement with a non-phosphorylatable mutant allows myogenesis despite inhibition of calcineurin signaling, demonstrating the functionality of calcineurin/PKC modified residues. Thus the Brg1 chromatin remodeling enzyme integrates two antagonistic calcium-dependent signaling pathways that control myogenic differentiation.

Little is known about chromosomal loopings involving proximal promoter and distal enhancer elements regulating GABAergic gene expression, including changes in schizophrenia and other psychiatric conditions linked to altered inhibition. Here, we map in human chromosome 2q31 the 3D configuration of 200 kb of linear sequence encompassing the GAD1 GABA synthesis enzyme gene locus, and we describe a loop formation involving the GAD1 transcription start site and intergenic noncoding DNA elements facilitating reporter gene expression. The GAD1-TSS -50kbLoop was enriched with nucleosomes epigenetically decorated with the transcriptional mark, histone H3 trimethylated at lysine 4, and was weak or absent in skin fibroblasts and pluripotent stem cells compared with neuronal cultures differentiated from them. In the prefrontal cortex of subjects with schizophrenia, GAD1-TSS -50kbLoop was decreased compared with controls, in conjunction with downregulated GAD1 expression. We generated transgenic mice expressing Gad2 promoter-driven green fluorescent protein-conjugated histone H2B and confirmed that Gad1-TSS -55kbLoop , the murine homolog to GAD1-TSS -50kbLoop, is a chromosomal conformation specific for GABAergic neurons. In primary neuronal culture, Gad1-TSS -55kbLoop and Gad1 expression became upregulated when neuronal activity was increased. We conclude that 3D genome architectures, including chromosomal loopings for promoter-enhancer interactions involved in the regulation of GABAergic gene expression, are conserved between the rodent and primate brain, and subject to developmental and activity-dependent regulation, and disordered in some cases with schizophrenia. More broadly, the findings presented here draw a connection between noncoding DNA, spatial genome architecture, and neuronal plasticity in development and disease.

BackgroundTriglyceride and glucose (TyG) index and nonalcoholic fatty liver disease (NAFLD) both bave been related to insulin resistance (IR). The study aimed to investigate the longitudinal relationship between TyG index and NAFLD and to evaluate the ability of TyG, through comparing with the predictive value of other indexes, to identify individuals at risk for NAFLD.MethodsFour thousand and five hundred thirty nine subjects without NAFLD initially were followed up for 9 years. Cox regression models were used to analyze the risk factors of NAFLD.ResultsCox regression analyses indicated the TyG index was independently and positively associated with the risk of incident NAFLD. In receiver operating characteristic (ROC) curve analysis, the optimal cut-off level for TyG to predict incident NAFLD was 8.52 and the area under the ROC curve (AUC) was 0.76 (95% CI 0.74–0.77), which was larger than that of TG, ALT and FPG.ConclusionThis study demonstrated that the elevation of the TyG index might predict increase risk for incident NAFLD and it may be suitable as a diagnostic criterion for NAFLD.

The Shank genes (SHANK1, 2, 3) encode scaffold proteins highly enriched in postsynaptic densities where they regulate synaptic structure in spiny neurons. Mutations in human Shank genes are linked to autism spectrum disorder (ASD) and schizophrenia. Shank1 mutant mice exhibit intriguing cognitive phenotypes reminiscent of ASD individuals. However, the molecular mechanisms leading to the human pathophysiological phenotypes and mouse behaviors have not been elucidated. We show in this study that Shank1 protein is highly localized in Parvalbumin-expressing (PV+) fast-spiking inhibitory interneurons in hippocampus. Importantly, lack of Shank1 in hippocampal CA1 PV+ neurons reduced excitatory synaptic inputs and inhibitory synaptic outputs to pyramidal neurons. Furthermore, we demonstrate that hippocampal CA1 pyramidal neurons in Shank1 mutant mice exhibit a shift in the excitatory and inhibitory balance (E-I balance), a pathophysiological hallmark of ASD. The mutant mice also exhibit lower expression of gephyrin (a scaffold component of inhibitory synapses), supporting the dysregulation of E-I balance in hippocampus. These results suggest that Shank1 scaffold in PV+ interneurons regulates excitatory synaptic strength and participates in the maintenance of E-I balance in excitatory neurons.

Development of biocompatible nanomaterials with multiple functionalities for combination of radiotherapy and chemotherapy has attracted tremendous attention in cancer treatment. Herein, poly(ethylene glycol) (PEG) modified polydopamine (PDA) nanoparticles were successfully developed as a favorable biocompatible nanoplatform for co-loading antitumor drugs and radionuclides to achieve imaging-guided combined radio-chemotherapy. It is demonstrated that PEGylated PDA nanoparticles can effectively load two different drugs including sanguinarine (SAN) and metformin (MET), as well as radionuclides I in one system. The loaded SAN and MET could inhibit tumor growth via inducing cell apoptosis and relieving tumor hypoxia, while labeling PDA-PEG withI enables in vivo radionuclide imaging and radioisotope therapy. As revealed by the therapeutic efficacy both in cell and animal levels, the multifunctional PDA nanoparticles (I-PDA-PEG-SAN-MET) can effectively repress the growth of cancer cells in a synergistic manner without significant toxic side effects, exhibiting superior treatment outcome than the respective monotherapy. Therefore, this study provides a promising polymer-based platform to realize imaging-guided radioisotope/chemotherapy combination cancer treatment in future clinical application.