Biology subjects

Arvanitakis, Z.

Publications and source records attributed to Arvanitakis, Z..

3 recordsLinked to original sources

bioRxiv2025

Phosphoproteomics unveils the signaling dynamics in neuronal cells stimulated with insulin and insulin-like growth factors

BackgroundGiven the role of metabolism in brain health and disease, investigating the role of insulin (INS) and insulin-like growth factors (IGFs) as potential therapeutic strategies for neurodegenerative diseases is currently underway. Yet, the signaling pathways associated with INS and IGFs in the brain remain elusive, particularly for the human brain. Unraveling these pathways is critical for harnessing their therapeutic potential in metabolism-associated brain disorders. MethodsThis study employed phosphoproteomics using human neuroblastoma cell line, SH-SY5Y, to unravel the signaling network of INS, IGF-1, and IGF-2. Briefly, cells were stimulated at 10 and 60-minutes with the ligands, followed by protein extraction, trypsin digestion, tandem mass tag (TMT)-labelling and phosphopeptides enrichment using an immobilized metal affinity chromatography (IMAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Data was processed using R statistical software. Protein annotations were obtained from the UniprotKB database, and pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). ResultsPhosphoproteomics performed at 10 and 60 minutes identified 34358 phosphosites of which 3284 were significant at 10 min and 2374 at 60 min (p.adj <0.05) across all three ligands. Ligand stimulation induced modulation in phosphorylation at both the receptor level and downstream signaling targets at serine (S), threonine (T) and tyrosine (Y) residues. Phosphorylation of LIMA1-Y229, a regulator of actin-cytoskeletal function, was the most prominent Y phosphosite across all ligands. IPA identified Rho GTPase, the molecular switches that regulate actin cytoskeletal dynamics, as the most significantly enriched pathway, with IGF-1 predominantly driving phosphorylation of Rho GTPase effectors such as Rho Guanine nucleotide exchange factors (ARHGEFs), Rho GTPase activating proteins (ARHGAPs) and CDC42. Myocardin related transcription factor A (MRTFA), a transcriptional target of Rho GTPase, was increased in ligand-stimulated cells at 10 min, and inhibition of Rho/SRF pathway by CCG1423 prevents nuclear localization of IGF-1-induced MRTFA. ConclusionsThis study demonstrates that INS, IGF-1 and IGF-2 regulate Rho GTPase and MRTFA activation, thereby contributing to the control of actin cytoskeletal dynamics in neuronal cells. Given the role of INS and IGFs in neuronal survival and neurodegenerative conditions, elucidating the mechanisms is of critical importance, as it offers insights into disease pathogenesis and potential therapeutic targets.

biochemistry↗

bioRxiv2025

Evidence for cPLA2 activation in Alzheimer's Disease Synaptic Pathology

BackgroundSynapses are essential for learning and memory, and their loss predicts cognitive decline in Alzheimers disease (AD). Synaptic loss is associated with excitotoxicity, neuroinflammation, amyloid-{beta}, and tau pathology, but the molecular mechanisms remain unclear. There is an urgent need to identify new targets to modify the disease and slow synaptic loss and cognitive decline. This study examines if calcium-dependent phospholipase A2 (cPLA2) is implicated in AD synaptic loss. cPLA2 catalyzes membrane phospholipids to release arachidonic acid, which can be metabolized into inflammatory eicosanoids. MethodscPLA2 levels were examined in synaptosomes isolated from the postmortem frontal cortex of individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), and AD dementia from the Religious Orders Study (ROS). Eicosanoids in synaptosomes were analyzed using lipidomics. Immunofluorescent staining investigated cPLA2 interactions with synaptic markers. Human iPSCs-derived neurons were used to study cPLA2 overactivation after exposure to amyloid-{beta} 42 oligomers (A{beta}42O), its relationships with synaptic markers, and the effects of cPLA2 inhibitors. ResultsWe observed elevated cPLA2 (cPLA2 and cPLA2{beta}) in AD synaptosomes and positive correlations with postsynaptic density protein 95 (PSD-95) and cognitive dysfunction. Eicosanoids were increased in AD synaptosomes and correlated with cPLA2, indicating cPLA2 activity at synapses/synaptosomes. Phosphorylated cPLA2 (p-cPLA2) colocalized with PSD-95 in synaptosomes, and with postsynaptic Ca2+/calmodulin-dependent protein kinase II (CaMKII) and dendritic microtubule-associated protein 2 (MAP2) in NCI and AD brains, where their levels were reduced in AD. P-cPLA2 colocalizes with MAP2 at the neuronal soma associated with neuritic plaques and neurodegeneration in AD. A{beta}42O activates cPLA2 in human iPSCs-derived neurons, leading to p-cPLA2 relocation from the cytosol to synaptic and dendritic sites to colocalize with CaMKII and MAP2, resulting in their reduction. P-cPLA2 also colocalized with PSD-95 in A{beta}42O-exposed neurons, accompanied with increased PSD-95 intensity at soma membrane. These processes were reversed by the cPLA2 inhibitor ASB14780. ConclusionscPLA2 overactivation at synapses, dendrites, and excitatory neuronal somas is associated with synaptic loss, neuritic plaques and neurodegeneration, potentially contributing to cognitive decline in AD. Future research needs to explore the role of cPLA2 as a disease-modifying target for AD.

neuroscience↗

bioRxiv2024

Microglia States are Susceptible to Senescence and Cholesterol Dysregulation in Alzheimer's Disease

Cellular senescence is a major contributor to aging-related degenerative diseases, including Alzheimers disease (AD) but much less is known on the key cell types and pathways driving mechanisms of senescence in the brain. We hypothesized that dysregulated cholesterol metabolism is central to cellular senescence in AD. We analyzed whole transcriptomic data and utilized single-cell RNA seq integration techniques to unveil the convoluted cell-type-specific and sub-cell-type-state-specific senescence pathologies in AD using both ROSMAP and Sea-AD datasets. We identified that microglia are central components to AD associated senescence phenotypes in ROSMAP snRNA-seq data (982,384 nuclei from postmortem prefrontal cortex of 239 AD and 188 non-AD) among non-neuron cell types. We identified that homeostatic, inflammatory, phagocytic, lipid processing and neuronal surveillance microglia states were associated with AD associated senescence in ROSMAP (152,459 microglia nuclei from six regions of brain tissue of 138 early AD, 79 late AD and 226 control subject) and in Sea-AD (82,486 microglia nuclei of 42 dementia, 42 no dementia and 5 reference subjects) via integrative analysis, which preserves the meaningful biological information of microglia cell states across the datasets. We assessed top senescence associated bioprocesses including mitochondrial, apoptosis, oxidative stress, ER stress, endosomes, and lysosomes systems. Specifically, we found that senescent microglia have altered cholesterol related bioprocesses and dysregulated cholesterol. We discovered three gene co-expression modules, which represent the specific cholesterol related senescence transcriptomic signatures in postmortem brains. To validate these findings, the activation of specific cholesterol associated senescence transcriptomic signatures was assessed using integrative analysis of snRNA-seq data from iMGs (microglia induced from iPSCs) exposed to myelin, Abeta, and synaptosomes (56,454 microglia across two replicates of untreated and four treated groups). In vivo cholesterol associated senescence transcriptomic signatures were preserved and altered after treatment with AD pathological substrates in iMGs. This study provides the first evidence that dysregulation of cholesterol metabolism in microglia is a major driver of senescence pathologies in AD. Targeting cholesterol pathways in senescent microglia is an attractive strategy to slow down AD progression.

neuroscience↗