Unraveling the molecular mechanisms of neuronal and astrocytic proteostasis in human models of Alzheimer’s disease

Abstract

Cellular proteostasis is the integrated regulation of protein synthesis, folding, trafficking, and degradation that sustains the integrity of the proteome. By balancing these processes, cells preserve homeostasis under both basal conditions and during periods of physiological stress. This regulation is especially critical in post-mitotic cells such as neurons, as well as in other brain cell types including astrocytes. In the brain, disruption of the proteostasis network promotes the accumulation of misfolded or aggregated proteins that drive the pathogenesis of neurodegenerative disorders, including Alzheimer’s disease (AD). Of particular relevance to neurodegeneration is the degradation arm of proteostasis, as impairments in autophagy and proteasomal pathways result in the accumulation of toxic protein aggregates that are hallmarks of disease. The autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS) represent the two major degradative mechanisms that sustain cellular proteostasis. Together, these systems form the core of the protein quality control network, ensuring the clearance of damaged, misfolded, or aggregated proteins, as well as dysfunctional organelles. While the UPS primarily targets short-lived soluble proteins, the ALP provides both bulk and selective degradation routes capable of removing larger protein aggregates and organelles, a particularly important process for mitigating the toxic effects of aggregation prone proteins in neurodegenerative diseases. While autophagy can be both a bulk and selective degradation process, selective autophagy itself depends on molecular chaperones and adaptor proteins that identify substrates and recruit them to degradative machinery. Dysfunction in these pathways has been strongly implicated in AD pathogenesis, highlighting the importance of understanding how some of these adaptor proteins and chaperones might regulate ALP activity and influence disease pathophysiology. Optineurin (OPTN) is one such autophagy adaptor protein with established roles in selective autophagy. Pathogenic mutations in OPTN have been linked to amyotrophic lateral sclerosis, frontotemporal dementia, and glaucoma, but its contribution to AD and neuronal function remains unclear. To investigate the role of OPTN in neuronal proteostasis and AD, we utilized induced pluripotent stem cell (iPSC)-derived neuron (iN) and astrocyte (iA) models. Analyses revealed a negative correlation between OPTN and specific pTau epitopes in neurons, as well as a decrease in OPTN protein abundance in brain tissues of individuals with AD. Given these findings, we generated OPTN knockout (KO), heterozygous (HET), and wildtype (WT) iNs and iAs using CRISPR/Cas9 editing in two genetic backgrounds. Loss of OPTN in iNs increased specific pTau proteoforms without substantially affecting autophagy processes or mitochondrial respiration. Despite no clear effect on mitochondrial function, several mitochondrial proteins, including OXCT1, were enriched in an unbiased analysis of the OPTN interactome in iNs, as well as proteins involved in intracellular trafficking. Proteomic analyses further identified intracellular Clusterin (CLU), an AD risk gene, as significantly upregulated in OPTN KO iNs, suggesting OPTN may influence its intracellular processing. Our model system demonstrates modest roles for OPTN in certain neuronal biological processes and potential implications for AD pathogenesis. These findings also suggest that OPTN may exhibit functional redundancy with other autophagy adaptor proteins in human neurons, leading to relatively mild phenotypic changes with complete loss of OPTN. Another important form of selective autophagy relevant to neurodegenerative disease is chaperone-assisted selective autophagy (CASA). Bcl-2-associated athanogene 3 (BAG3) is a mediator of CASA, and given the genetic and pathological links of BAG3 to proteostasis and neurodegenerative diseases, we investigated how BAG3 contributes to cellular function and Alzheimer's disease (AD) in both human neurons and astrocytes. We first utilized a large panel of iPSCs from deeply phenotyped cohorts to interrogate genetic contributions to baseline autophagic flux and UPS activity in human neurons, and protein turnover was assessed using SILAC-based quantitative proteomics. Across our panel of neurons, we observed substantial inter-individual differences in autophagic flux, which was inversely correlated with UPS activity. This reciprocal relationship extended to tau homeostasis, where higher autophagic flux resulted in reduced accumulation of aggregated, phosphorylated tau. Proteomic analyses revealed that global protein turnover dynamics stratified based on degradation pathway activity and could predict pathway-specific substrate dependencies. Interestingly, BAG3 emerged as a dynamically regulated autophagy chaperone, responsive to pharmacological inhibition of both the UPS and ALP. BAG3 knockout in neurons decreased autophagic flux and increased levels of high-molecular-weight phosphorylated tau. Notably, familial APP AD mutations and Aβ exposure induced BAG3 expression in neurons, while elevated BAG3 levels in human brain tissue were associated with higher neuropathological burden and disease progression. While an elevation of BAG3 was observed in the AD brain, it was unclear which brain cell type might be contributing most to this upregulation. We found that in human brain and iPSC models, BAG3 was most highly expressed in astrocytes. Further, BAG3 loss in our iPSC model system caused greater proteomic disruption in astrocytes than in neurons. In the absence of BAG3, astrocytes showed reduced autophagy, diminished lysosome abundance and activity, and decreased proteasome function. To uncover molecular binding partners of BAG3 that might influence these phenotypes, we performed co-immunoprecipitation, revealing interactions with HSPB8 and other heat shock proteins, proteasome regulators (PSMD5, PSMF1), and the retromer component, VPS35. Integration of BAG3 KO transcriptomic and proteomic datasets pinpointed AD-relevant proteins under post-translational control of BAG3, which included GFAP, BIN1, and HSPB8. HSPB8 levels were markedly reduced in BAG3-deficient astrocytes with overexpression partially rescuing its levels. Loss of astrocytic BAG3 impaired Aβ clearance in co-culture with APP/PSEN1 mutant neurons, directly linking BAG3 to a disease-relevant astrocyte function. Finally, analysis of postmortem brain tissue revealed BAG3 marks a stress-responsive astrocyte subtype in the brain of aged individuals with AD. Collectively, these studies define complementary and cell-type specific contributions of OPTN and BAG3 to proteostasis in the human brain and AD. They reveal how adaptor proteins and chaperones regulate neuronal and glial protein quality control, highlight BAG3 as a central regulator responsive to genetic and pathological stress, and establish mechanistic links between proteostasis dysfunction and AD pathogenesis. Together, this work advances our understanding of proteostasis networks in the brain and identifies potential therapeutic nodes within these pathways for combatting neurodegenerative disease.