Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons in the brain and spinal cord. Accumulating ...evidence suggests that ALS is not solely a neuronal cell- or brain tissue-autonomous disease and that neuroinflammation plays a key role in disease progression. Furthermore, whereas both CD4 and CD8 T cells were observed in spinal cords of ALS patients and in mouse models of the disease, their role in the neuroinflammatory process, especially considering their functional changes with age, is not fully explored. In this study, we revealed the structure of the CD4 T-cell compartment during disease progression of early-onset SOD1
and late-onset SOD1
mouse models of ALS. We show age-related changes in the CD4 T-cell subset organization between these mutant SOD1 mouse models towards increased frequency of effector T cells in spleens of SOD1
mice and robust infiltration of CD4 T cells expressing activation markers and the checkpoint molecule PD1 into the spinal cord. The frequency of infiltrating CD4 T cells correlated with the frequency of infiltrating CD8 T cells which displayed a more exhausted phenotype. Moreover, RNA-Seq and immunohistochemistry analyses of spinal cords from SOD1
mice with early clinical symptoms demonstrated immunological trajectories reminiscent of a neurotoxic inflammatory response which involved proinflammatory T cells and antigen presentation related pathways. Overall, our findings suggest that age-related changes of the CD4 T cell landscape is indicative of a chronic inflammatory response, which aggravates the disease process and can be therapeutically targeted.
The SIRT6 deacetylase has been implicated in DNA repair, telomere maintenance, glucose and lipid metabolism and, importantly, it has critical roles in the brain ranging from its development to ...neurodegeneration. Here, we combined transcriptomics and metabolomics approaches to characterize the functions of SIRT6 in mouse brains. Our analysis reveals that SIRT6 is a central regulator of mitochondrial activity in the brain. SIRT6 deficiency in the brain leads to mitochondrial deficiency with a global downregulation of mitochondria-related genes and pronounced changes in metabolite content. We suggest that SIRT6 affects mitochondrial functions through its interaction with the transcription factor YY1 that, together, regulate mitochondrial gene expression. Moreover, SIRT6 target genes include SIRT3 and SIRT4, which are significantly downregulated in SIRT6-deficient brains. Our results demonstrate that the lack of SIRT6 leads to decreased mitochondrial gene expression and metabolomic changes of TCA cycle byproducts, including increased ROS production, reduced mitochondrial number, and impaired membrane potential that can be partially rescued by restoring SIRT3 and SIRT4 levels. Importantly, the changes we observed in SIRT6-deficient brains are also occurring in aging human brains and particularly in patients with Alzheimer's, Parkinson's, Huntington's, and Amyotrophic lateral sclerosis disease. Overall, our results suggest that the reduced levels of SIRT6 in the aging brain and neurodegeneration initiate mitochondrial dysfunction by altering gene expression, ROS production, and mitochondrial decay.
Microglia play a key role in innate immunity in Alzheimer disease (AD), but their role as antigen-presenting cells is as yet unclear. Here we found that amyloid β peptide (Aβ)-specific T helper 1 ...(Aβ-Th1 cells) T cells polarized to secrete interferon-γ and intracerebroventricularly (ICV) injected to the 5XFAD mouse model of AD induced the differentiation of major histocompatibility complex class II (MHCII)+ microglia with distinct morphology and enhanced plaque clearance capacity than MHCII- microglia. Notably, 5XFAD mice lacking MHCII exhibited an enhanced amyloid pathology in the brain along with exacerbated innate inflammation and reduced phagocytic capacity. Using a bone marrow chimera mouse model, we showed that infiltrating macrophages did not differentiate to MHCII+ cells following ICV injection of Aβ-Th1 cells and did not support T cell-mediated amyloid clearance. Overall, we demonstrate that CD4 T cells induce a P2ry12+ MHCII+ subset of microglia, which play a key role in T cell-mediated effector functions that abrogate AD-like pathology.
The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated ...with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq (the assay for transposase-accessible chromatin with high-throughput sequencing) and chromatin conformation by Hi-C (high-throughput chromosome conformation capture).
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•Isolation of nuclei from mouse cortical tissue•Immunolabeling of nuclei and sorting by FACS•Fixed nuclei protocol•Preparation of ATAC-seq and Hi-C libraries
The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq and chromatin conformation by Hi-C.
The blood-brain barrier acts as a major barrier for the entrance of most therapeutics into the brain, impeding treatment for neurological disorders. Intracerebroventricular (ICV) injection of T cells ...is a useful tool for cell therapy of neurological disorders including neurodegenerative and neuropsychiatric diseases and brain tumors. Here, we present an optimized ICV injection of T cells with improved injection efficiency at pathological sites within the brain parenchyma. We describe details of the surgical procedure and verification of injection via immunohistochemistry.
For complete details on the use and execution of this protocol, please refer to Fisher et al. (2014); Strominger et al., (2018); Mittal et al. (2019); Eremenko et al. (2019).
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•Bilateral intracerebroventricular injection of T cells using an optimized needle gauge•Reduced damage and increased survival of the injected cells•Enhanced migration of the injected cells within the brain parenchyma
The blood-brain barrier acts as a major barrier for the entrance of most therapeutics into the brain, impeding treatment for neurological disorders. Intracerebroventricular (ICV) injection of T cells is a useful tool for cell therapy of neurological disorders including neurodegenerative and neuropsychiatric diseases and brain tumors. Here, we present an optimized ICV injection of T cells with improved injection efficiency at pathological sites within the brain parenchyma. We describe details of the surgical procedure and verification of injection via immunohistochemistry.
The headwaters of fluvial systems on the East European Plain between the boundaries of the Marine Isotope Stage 2 (MIS 2) and MIS 6 glaciations evolved during the last 150,000 years. At least three ...main events of high surface runoff caused intensive erosion: at the end of MIS 6, at the end of MIS 2 and in the Middle Holocene. Erosion developed in the territory with variable resistance of geological substrate, from hard-to-erode tills to weak sandy deposits. All erosional features in moraines formed in the pre-Holocene time. Even relatively large forms, such as balkas (small dry valleys), have not yet reached concave longitudinal profiles. A general tendency of their development was deepening. Short episodes of incision occurring during climatic events with increased water flow alternated with long periods of stabilization. Sand-covered areas are most favorable for linear erosion. The gullies formed in the Middle Holocene developed concave longitudinal profiles. The diversity of catchment areas, initial slope inclinations and sediment properties causing their resistance to erosion led to greater differences in the relief features and evolution of the upper reaches of the fluvial systems within the MIS 6 glaciation area compared to the more uniform landscape conditions in the extraglacial regions.
Transduction of primary T cells has become prominent with the introduction of chimeric antigen receptor T-cell therapy. Although there are many protocols for the transduction of human T cells, it ...remains a challenge to transduce murine T cells. We present an optimized protocol for the retroviral transduction of murine CD4 T cells, which overcomes major challenges including large-scale production and long-term culturing of transduced cells. The optimized protocol combines high transduction efficiency with a low rate of cell death.
For complete details on the use and execution of this protocol, please refer to Eremenko et al., 2019.
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•Detailed description of retroviral transduction of murine CD4 T cells•Large-scale production of murine CD4-transduced T cells•Enhanced proliferation and viability of murine CD4-transduced T cells with IL-2/IL-7
Transduction of primary T cells has become prominent with the introduction of chimeric antigen receptor T-cell therapy. Although there are many protocols for the transduction of human T cells, it remains a challenge to transduce murine T cells. We present an optimized protocol for the retroviral transduction of murine CD4 T cells, which overcomes major challenges including large-scale production and long-term culturing of transduced cells. The optimized protocol combines high transduction efficiency with a low rate of cell death.
Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we ...present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Age-associated changes in CD4 T-cell functionality have been linked to chronic inflammation and decreased immunity. However, a detailed characterization of CD4 T cell phenotypes that could explain ...these dysregulated functional properties is lacking. We used single-cell RNA sequencing and multidimensional protein analyses to profile thousands of CD4 T cells obtained from young and old mice. We found that the landscape of CD4 T cell subsets differs markedly between young and old mice, such that three cell subsets-exhausted, cytotoxic, and activated regulatory T cells (aT
)-appear rarely in young mice but gradually accumulate with age. Most unexpected were the extreme pro- and anti-inflammatory phenotypes of cytotoxic CD4 T cells and aT
, respectively. These findings provide a comprehensive view of the dynamic reorganization of the CD4 T cell milieu with age and illuminate dominant subsets associated with chronic inflammation and immunity decline, suggesting new therapeutic avenues for age-related diseases.
Sex-specific predilection in neurological diseases caused by mutations in autosomal genes is a phenomenon whose molecular basis is poorly understood. We studied females of consanguineous Bedouin ...kindred presenting with severe global developmental delay and epilepsy.
Linkage analysis, whole exome sequencing, generation of CRISPR/cas9 knock-in mice, mouse behaviour and molecular studies RESULTS: Linkage analysis and whole exome sequencing studies of the affected kindred delineated a ~5 Mbp disease-associated chromosome 2q35 locus, containing a novel homozygous frameshift truncating mutation in
, in line with recent studies depicting similar
putative loss-of-function human phenotypes with female preponderance. We generated knock-in mice with a truncating mutation adjacent to the human mutation in the mouse ortholog. Behaviour studies of homozygous
mice showed significant phenotype only in mutant females, with learning and memory deficits, hyperactivity and aberrant loss of fear of open spaces. Bone marrow and spleen of homozygous
mice showed depletion of lymphoid and haematopoietic cells, mostly in females. RT-PCR showed lower expression of
in brain compartments of female versus male wild-type mice. RNA-seq studies of hippocampus, hypothalamus, cortex and cerebellum of female wild-type versus homozygous
mice demonstrated differentially expressed genes. Notably, expression of
in the cortex and of
in the hippocampus was downregulated in homozygous
mice.
mutations have been associated with aberrant neurodevelopment and behaviour.
expression is regulated by sex hormones and
null-mutant mice present with haematopoietic, immune system and female-specific behaviour phenotypes.
mutation downregulates
and
causing a neurodevelopmental phenotype in humans and mice with female preponderance.
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