Subhas Chandra Biswas

Parkinson′s disease (PD) is characterized by a progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta and degeneration of their projections in the striatum (STR) of the brain. Astrocytes are recognized as active contributors to PD pathogenesis, yet the role of calcineurin (CN), a Ca/calmodulin-dependent phosphatase which is well characterized in neurons, remains unexplored in astrocytes in the context of PD. Here, we show that CN protein expression is upregulated in the STR of MPTP-treated mice, coinciding with DA neurodegeneration and astrogliosis. In human astrocytoma cells and primary rat astrocytes, the neurotoxin Rotenone induced rapid intracellular Calcium elevation and CN activation. Unexpectedly, this was accompanied by a decrease in intracellular levels of miR-23a, an astrocyte-enriched microRNA previously linked to CN signalling in cardiomyocytes. We found that this decrease reflected active exosomal export: Rotenone increased both exosomal miR-23a levels and total exosome number, while blocking exosome biogenesis with GW4869 restored intracellular miR-23a levels. Functionally, both astrocyte-derived exosomes and miR-23a overexpression protected neuronal cells (SH-SY5Y, PC12) against Rotenone-, 6-OHDA-, and MPP+-induced toxicity. Mechanistically, miR-23a showed direct binding to the 3′UTR of the pro-apoptotic BH3-only protein NOXA, thereby suppressing its expression, subsequently attenuating caspase-3 activation. Finally, exosomal miR-23a levels were significantly elevated in plasma from PD patients compared with age-matched controls, supporting clinical relevance. Together, these findings identify a previously unrecognized astrocyte-to-neuron communication axis during PD pathogenesis, in which astrocytic CN activation accompanied by exosomal miR-23a release represents a neuroprotective stress-responsive pathway with potential biomarker and therapeutic relevance in PD.

Microgliosis is one of the early hallmarks of Alzheimer's disease (AD) that plays a crucial role in disease pathogenesis. Microglia play a defensive role by phagocytosing and clearing amyloid‐β (Aβ) aggregates. However, excessive uptake of Aβ leads to impairment of its clearing ability, which results in neuroinflammation and eventually neurodegeneration. Thus, enhancing microglial phagocytosis and reducing its pro‐inflammatory functions are promising strategies for AD therapy. However, harnessing microglial activation for long‐term benefits in controlling disease pathogenesis in AD is currently lacking. Our recent findings revealed that the astrocyte secreted cytokine Intercellular adhesion molecule 1 (ICAM‐1) improves memory and cognitive impairments in a 5xFAD mouse model of AD. Here, we investigated the involvement of microglia in ICAM‐1 function since its receptor, LFA‐1, is expressed in microglia. We found that ICAM‐1 blocks Aβ‐mediated microglial inflammatory activation by inhibiting the ERK‐STAT3 pathway, which is indispensable for microglial inflammation. Further, we found that ICAM‐1 potentiates microglial phagocytic ability to eliminate Aβ in primary culture. Additionally, ICAM‐1 reduced Aβ plaque load and associated microglial reactivation in the 5xFAD mouse hippocampus. This reduction in plaque‐associated microgliosis led to improved synaptic protein expressions which was reflected in significant cognitive improvement. Moreover, blocking the binding between ICAM‐1 with its receptor LFA‐1 partially reduced ICAM‐1‐mediated microglial modification. Collectively, these findings suggest that ICAM‐1 plays a pivotal role in modifying microglial phagocytic and inflammatory functions via the ERK‐STAT3 signalling pathway, thereby contributing to Aβ clearance and cognitive improvements.

Glial-secreted molecules influence neuronal function in Alzheimer’s disease (AD), but their mechanisms of action are partially understood. Anti-inflammatory cytokine tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is secreted by astrocytes early in response to amyloid-β and is suggested to have a neuroprotective function. We demonstrated that TIMP-1 levels are increased in 7-day-old 5xFAD versus wild-type mice but are drastically decreased from two months onwards. Administration of TIMP-1 protein in 5xFAD mice ameliorated AD-associated cognitive impairments. TIMP-1 regulated both neuronal apoptosis and autophagy by binding to CD63 receptors in an AD model. Synaptosomal and electrophysiological studies revealed that TIMP-1 reduces AD-related synaptic deficits, likely by promoting post-synaptic long-term potentiation in the hippocampus, independent of pre-synaptic activity. TIMP-1 induced brain-derived neurotrophic factor (BDNF) and BDNF-mediated post-synaptic signaling. These findings suggest that TIMP-1 functions as a multifunctional cytokine with protective and long-term benefits for neurons and may be a promising therapeutic candidate in AD.

Background: Astrocytes play an integral role in Alzheimer’s disease (AD) pathology, where they may act as a double-edged sword. The existing serum-supplemented in vitro astrocyte culture models are not suitable to study certain stress response mechanisms that occur in AD. Purpose: Here, we tried to develop a serum-free murine primary cortical astrocyte culture model to study endoplasmic reticulum (ER) stress and inflammation to investigate the effect of amyloid-beta (Aβ _1–42 ). Methods: Astrocytes were cultured in a controlled serum-free environment to minimise interference from serum components. Serum-free astrocytes were exposed to oligomeric Aβ and subjected to imaging, immunocytochemistry, real-time PCR and western blot analysis. Results: Using an established protocol, no significant activation of eIF2α, a key marker of ER stress, was observed under serum-free conditions, but with the removal of N-acetyl cysteine (NAC), ER stress response was enhanced after 24 hours of Aβ exposure. Subsequently, the Aβ-induced inflammatory response, assessed through TNF-α expression, which was minimal in the presence of growth factors, became pronounced when these factors were withdrawn. Concomitantly, a significant increase in astrocytes reactivity, assessed by GFAP expression upon 24 hours of Aβ exposure, was observed. Transcript analysis revealed a time-dependent shift in the expression of inflammatory molecules, with early time points showing an increase in anti-inflammatory markers, while late exposure promoting pro-inflammatory responses. Conclusion: This study identifies that NAC and growth factors impede ER stress and inflammatory responses in astrocytes upon Aβ exposure in serum-free culture. These findings also highlight the potential of a serum-free culture system for studying ER stress and inflammation in astrocytes to understand the complex role of these cells in AD pathophysiology.

Astrocytes are integral to understand Alzheimer’s disease (AD) pathology but the existing serum-supplemented in vitro astrocyte culture models are not suitable to study certain stress response mechanisms. Here, we developed a serum-free murine primary cortical astrocyte culture model to study endoplasmic reticulum (ER) stress and inflammation to see the effect of amyloid-beta (Aβ _1-42 ). Astrocytes were cultured in a controlled serum-free environment to minimize interference from serum components. Serum-free astrocytes were exposed to oligomeric Aβ to induce ER stress and inflammation. Initially no significant activation of eIF2α, a key marker of ER stress, was observed under serum-free condition but with the removal of N-acetyl cysteine ER stress response was enhanced after 24 hours of Aβ exposure. Subsequently, the inflammatory response, assessed through TNF-α expression, was minimal in the presence of growth factors but became pronounced when these factors were withdrawn. Astrocytic reactivity, assessed by GFAP expression, was observed with prolonged Aβ exposure, indicating a reactive astrogliosis response. Transcript analysis revealed a time-dependent shift in the expression of inflammatory modulators, with early time points showing increased anti-inflammatory markers and late exposure promoting pro-inflammatory responses. These findings highlight the potential of serum-free cultures for studying ER stress and inflammation together in astrocytes and offer insights into the complex role of these cells in AD pathophysiology.

Dysfunctional autophagy is a primary characteristic of Alzheimer’s disease (AD) pathogenesis. How autophagic impairment leads to cellular changes that contributes to AD pathogenesis remains unclear. To study this further, we assessed levels of autophagy related proteins in 5xFAD mice brain at different ages and found their robust upregulation in cortex and hippocampus suggesting increased induction of autophagy with disease progression but failed clearance. We have identified a transcriptional coregulator Sertad1, as a key mediator of dysfunctional autophagy in AD mice. We found a progressive elevation in Sertad1 levels in 5xFAD mice with age compared to wild-type (WT) mice. Sertad1 knockdown in 5xFAD mice brain lowered levels of autophagy related proteins and lysosome marker, LAMP1 suggesting its role in autophagy flux modulation. FoxO3a is an important transcriptional regulator of the autophagy network and lies at the nexus of autophagy-apoptosis cross-talk. We found that Sertad1 knockdown blocked nuclear translocation of FoxO3a along with a restoration in Akt activity. Further, we showed that knockdown of Sertad1 in 5xFAD mice brain improved cognitive functions along with a remarkable restoration in synaptic health and dendritic spine density. Taken together, our results demonstrated that autophagy is robustly induced with disease progression but it is impaired; Sertad1 knockdown restored autophagy defects, synaptic loss and improved learning and memory in AD models. Thus, we propose that Sertad1 acts in a multimodal manner regulating crucial cell death pathways including apoptosis and autophagy and could be an excellent target for therapeutic intervention to combat a multifactorial disorder such as AD.

Background Microgliosis is one of the early hallmarks of Alzheimer’s disease (AD) that plays a crucial role in AD-associated neuroinflammation. However, strategies to harness microglial activation without affecting its homeostatic function are currently lacking. Our recent finding revealed astrocyte secreted cytokine Intercellular adhesion molecule 1 (ICAM-1) improves memory and cognitive impairment in 5xFAD mice model of AD. Here, we investigated the possible mechanism of ICAM-1 that involves modification of microglial activation state and function. Methods We treated primary microglia cultures with amyloid-ß (Aß) in presence or absence of ICAM-1 and then assessed microglial inflammatory activation and phagocytic clearance of Aß by western blotting and confocal imaging. We further treated 5xFAD mice with ICAM-1 intraperitoneally and ICAM-1 antagonist lifitegrast intranasally to assess the beneficial effect of ICAM-1 on Aß mediated microgliosis, synaptic protein expressions and cognitive deficits. Results Here we report that, ICAM-1 inhibits microglial inflammatory activation by inhibiting ERK-STAT3 pathway which is indispensable for microglial inflammation. Moreover, ICAM-1 was found to potentiate microglia to engulf and eliminate Aß in primary culture and reduced Aß plaque load and associated microglial activation in 5xFAD mice hippocampus. This reduced plaque levels and associated microgliosis in turn refurbished synaptic protein expressions and improved cognition and memory in these mice. Interestingly, ICAM-1 mediated microglial modification along with cognitive improvement was partially lost when ICAM-1-LFA-1 interaction was inhibited. Conclusion Taken together these findings delineate the importance of ERK/STAT3 pathway in Aß mediated microglial inflammation and the modulatory role of ICAM-1 in microglial activation and phagocytosis in order to improve clearance of Aß and associated memory and cognitive impairments.

Parkinson’s disease (PD) is a predominant movement disorder caused mainly due to selective loss of the dopaminergic neurons in the substantia nigra pars compacta of the mid brain. There is currently no cure for PD barring treatments to manage symptoms. The reasons might be due to lack of precise understanding of molecular mechanisms leading to neurodegeneration. Aberrant cell cycle activation has been implicated in neuronal death pathways of various neurodegenerative diseases including PD. This study investigates the role of cell cycle regulator Cell division cycle 25A (Cdc25A) in a PD-relevant neuron death model induced by 6-OHDA treatment. We find Cdc25A is rapidly elevated, activated and is playing a key role in neuron death by regulating Rb phosphorylation and E2F1 activity. Knockdown of Cdc25A via shRNA downregulates the levels of pro-apoptotic PUMA, an E2F1 target and cleaved Caspase-3 levels, suggesting Cdc25A may regulate neuronal apoptosis through these effectors. Our work sheds light on the intricate signaling networks involved in neurodegeneration and highlights Cdc25A as a potential therapeutic target for mitigating aberrant cell cycle re-entry underlying PD pathogenesis. These novel insights into molecular mechanisms provide a foundation for future development of neuroprotective strategies to slow or prevent progression of this debilitating disease.