The identification of a Distinct Astrocyte Subtype that Diminishes in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the presence of two hallmark pathologies: the accumulation of Amyloid beta (Aß) and tau proteins in the brain. There is a growing body of evidence suggesting that astrocytes, a type of glial cell in the brain, play crucial roles in clearing Aß and binding to tau proteins. However, due to the heterogeneity of astrocytes, the specific roles of different astrocyte subpopulations in response to Aß and tau remain unclear. To enhance the understanding of astrocyte subpopulations in AD, we investigated astrocyte lineage cells based on single-nuclei transcriptomic data obtained from both human and mouse samples. We characterized the diversity of astrocytes and identified global and subpopulation-specific transcriptomic changes between control and AD samples. Our findings revealed the existence of a specific astrocyte subpopulation marked by low levels of GFAP and the presence of AQP4 and CD63 expression, which showed functional enrichment in Aß clearance and tau protein binding, and diminished in AD. We verified this type of astrocytes in mouse models and in AD patient brain samples. Furthermore, our research also unveiled significant alterations of the ligand-receptor interactions between astrocytes and other cell types. These changes underscore the complex interplay between astrocytes and neighboring cells in the context of AD. Overall, our work gives insights into astrocyte heterogeneity in the context of AD and reveals a distinct astrocyte subpopulation that holds potential for therapeutic interventions in AD. Targeting specific astrocyte subpopulations may offer new avenues for the development of novel treatments for AD.

Glial progenitor heterogeneity and key regulators revealed by single-cell RNA sequencing provide insight to regeneration in spinal cord injury.

Recent studies have revealed the heterogeneous nature of astrocytes; however, how diverse constituents of astrocyte-lineage cells are regulated in adult spinal cord after injury and contribute to regeneration remains elusive. We perform single-cell RNA sequencing of GFAP-expressing cells from sub-chronic spinal cord injury models and identify and compare with the subpopulations in acute-stage data. We find subpopulations with distinct functional enrichment and their identities defined by subpopulation-specific transcription factors and regulons. Immunohistochemistry, RNAscope experiments, and quantification by stereology verify the molecular signature, location, and morphology of potential resident neural progenitors or neural stem cells in the adult spinal cord before and after injury and uncover the populations of the intermediate cells enriched in neuronal genes that could potentially transition into other subpopulations. This study has expanded the knowledge of the heterogeneity and cell state transition of glial progenitors in adult spinal cord before and after injury.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3ß, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

Multi-target inhibition ability of neohesperidin dictates its neuroprotective activity: Implication in Alzheimer's disease therapeutics.

The polygenic nature of Alzheimer's disease (AD) and cross-talk between several signaling cascades make it harder to decode the disease pathogenesis. ß-secretase (BACE1) works upstream in the amyloidogenic processing of amyloid precursor protein (APP) to generate Aß that rapidly aggregates to form fibrils, the most abundant component of plaques observed in AD brains. Here, we report dual inhibition of BACE1 and Aß aggregation by neohesperidin, a flavonoid glycoconjugate, using multi-spectroscopic approaches, force microscopy, molecular modeling, and validated the potency in SH-SY5Y neuroblastoma cell lines. Steady-state and time-resolved fluorescence reveal that neohesperidin binds close to the catalytic aspartate dyad. This binding conformationally restricts the protein in closed form which possibly precludes APP recognition and thereby inhibits BACE1 activity. Neohesperidin also dose-dependently inhibits the amyloid fibril formation, as evident from ANS, ThT assay, and AFM. Neohesperidin ameliorates aggregated Aß25-35 induced ROS generation and mitochondrial dysfunction in the SH-SY5Y cell line. As a result, the amyloid induced apoptosis is significantly prohibited and normal neuronal morphology is rescued. These findings suggest neohesperidin as an inhibitor of the pathogenic conversion of Aß to fibrillar amyloid assembly. Neohesperidin thus emerges as a non-toxic multi-potent scaffold for the development of AD therapeutics.

Iron chelator Deferoxamine protects human neuroblastoma cell line SH-SY5Y from 6-Hydroxydopamine-induced apoptosis and autophagy dysfunction.

BACKGROUND: Intracellular iron involves in Fenton's reaction-mediated Hydroxyl radical (OH·) generation by reacting with the neurotoxic agent 6-Hydroxydopamine (6-OHDA) autoxidation derivative Hydrogen Peroxide (H2O2). Several studies have been conducted so far on the neuroprotective activities of the iron chelator Deferoxamine (DFO) but little or no clear evidence about the underlying cellular mechanism is available. METHODS: The present study was conducted on Human neuroblastoma cell line SH-SY5Y in the absence or presence of 6-OHDA or H2O2 and / or DFO. Following incubation, cell viability assay, intracellular reactive oxygen species (ROS) determination, flow cytometric quantification of apoptotic cells followed by nuclear staining, intracellular tracking of transfected fusion construct of microtubule-associated protein 1B-light chain with Green fluorescent protein - Red fluorescent protein (LC3B-GFP-RFP reporters) and immunocytochemistry of intracellular Cathepsin protein by confocal microscopy, were conducted. In addition, western blotting was carried out to detect expressions of apoptotic and autophagy related proteins. RESULTS: This study confirmed the neuroprotective potential of DFO by inhibiting 6-OHDA-mediated cell death and ROS generation. Reduced percentage of apoptotic cells and appearance of altered nuclei architecture followed by a reduced expression of cleaved PARP (Poly-ADP-ribose Polymerase) and cleaved Caspase-3 were observed upon DFO treatment against 6-OHDA, and as well as against H2O2 in SH-SY5Y cell lines. Besides, DFO induced the intracellular autophagolysosome formation (red puncta) rather than autophagosome (yellow puncta) only. Thereafter it was observed that DFO restored the expression of intracellular lysosomal protease Cathepsin and reduced the expression of the LC3-II. CONCLUSION: Taken together, this study clearly demonstrated that the anti-Fenton activity of DFO inhibited apoptosis and caused blockade in ALP or autophagy dysfunction in SH-SY5Y cell lines. These outcomes further suggest that DFO provides neuroprotection by inhibiting apoptosis and inducing the progression of Autophagy- lysosomal pathway (ALP).

Iron-Induced Apoptotic Cell Death and Autophagy Dysfunction in Human Neuroblastoma Cell Line SH-SY5Y.

Iron accumulation plays a major role in neuronal cell death which has severe effects on mental health like neurodegenerative disorders. The present work aims to explore the involvement of molecular pathways involved in iron-mediated neuronal cell death using Ferric Ammonium Citrate (FAC) as a source of iron to treat neuroblastoma SH-SY5Y cells. In this study, it was found that cytotoxicity induced by iron treatment is highly correlated with enhanced intracellular reactive oxygen species (ROS) generation and loss of mitochondrial integrity. Appearance of early and late apoptotic cells with altered nuclear morphology and increased expression of effector proteins, i.e., cleaved Caspase 3 and cleaved PARP (Poly-ADP-ribose Polymerase), clearly confirmed iron-induced apoptotic cell deaths. Furthermore, excess accumulation of acidic vesicles and microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3II/I expressions were observed. Simultaneously, ultrastructural studies of SH-SY5Y cells demonstrated the accumulation of a large number of autophagosomes, autophagic vacuolization, and swollen mitochondria which further confirmed the induction of autophagy concomitant with mitochondrial damage. Furthermore, increased incorporation of lysosome-specific dye, LysoTracker Deep Red, and the red fluorescence retention of LC3-GFP-RFP constructs indicates the incomplete autophagy or autophagy dysfunction due to altered lysosomal activity. Hence, the present work unveiled the interruption in autophagy progression caused by the plausible suppression of lysosomal activity due to iron treatment resulting in autophagic cell death in SH-SY5Y cell lines. In general, both apoptotic and autophagic pathways were prominent and each of the pathways played their prospective roles, in iron-mediated neuronal cell death.

Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid.

Cinnamic acid (C9H8O2), is a major constituent of the oriental Ayurvedic plant Cinnamomum cassia (Family: Lauraceae). This phenolic acid has been reported to possess various pharmacological properties of which its antioxidant activity is a prime one. Therefore it is rational to hypothesize that it may ameliorate myelosuppression and oxidative stress induced by cyclophosphamide, a widely used chemotherapeutic agent. Commercial cyclophosphamide, Endoxan, was administered intraperitoneally to Swiss albino mice (50mg/kg) pretreated with 15, 30 and 60mg/kg doses of cinnamic acid orally at alternate days for 15days. Cinnamic acid pre-treatment was found to reduce cyclophosphamide induced hypocellularity in the bone marrow and spleen. This recovery was also reflected in the peripheral blood count. Amelioration of hypocellularity could be correlated with the modulation of cell cycle phase distribution. Cinnamic acid pre-treatment reduced bone marrow and hepatic oxidative stress as evident by lipid peroxidation and activity assays of antioxidant enzymes such as superoxide dismutase, catalase and glutathione-S-transferase. The present study indicates that cinnamic acid pretreatment has protective influence on the myelosuppression and oxidative stress induced by cyclophosphamide. This investigation is an attempt and is the first of its kind to establish cinnamic acid as an agent whose consumption provides protection to normal cells from the toxic effects of a widely used anti-cancer drug.