Fernández-Chacón, RafaelLópez-Begines, Santiago2025-07-082025-07-082025-03-07Rubio Pastor, F. (2025). Molecular Mechanisms of neurodegeneration upon genetic Removal of a synaptic co-chaperone in Adulthood. (Tesis Doctoral Inédita). Universidad de Sevilla, Sevilla.https://hdl.handle.net/11441/175118Neurodegenerative diseases are progressive and incurable disorders, and the molecular mechanisms underlying synaptic and neuronal degeneration are poorly understood. The synaptic co-chaperone Cysteine String Protein-?/DNAJC5 (CSP?/DNAJC5) has been linked to neurodegeneration in humans, as mutations in the DNAJC5 gene cause adult-onset autosomal dominant neuronal ceroid lipofuscinosis (Kufs disease/NCL4). In mice, CSP?/DNAJC5 protects presynaptic terminals from degeneration and its genetic deletion results in activity-dependent synaptic degeneration, particularly affecting fast-spiking GABAergic interneurons. A key step in understanding the molecular mechanisms of CSP?/DNAJC5 in preventing neurodegeneration is identifying its client proteins. However, the early mortality of CSP?/DNAJC5 knock-out mouse mice has limited investigations of its role in adulthood. To address this, we developed a conditional CSP?/DNAJC5 knock-out mouse model (UBCCreERT2:Dnajc5flox/flox) enabling time-specific genetic deletion in adulthood. Following tamoxifen-induced CSP?/DNAJC5 deletion, these mice exhibited severe, lethal symptoms and skin alterations, demonstrating that CSP?/DNAJC5 is critical for central nervous system function in adulthood and opening avenues for new research. Notably, these mice did not show pathological neuronal lipofuscinosis accumulation, supporting the idea that CSP?/DNAJC5 absence does not cause Kufs disease/NCL4. We hypothesized that CSP?/DNAJC5 deletion reduces the half-life of its client proteins in the brain. To test this hypothesis, we employed metabolic labeling with 13C6-Lys enriched diet and mass spectrometry to measure brain protein turnover. Remarkably, several proteins in the brain cortex displayed altered lifetimes, to undergo significant changes in their lifetimes, implicating CSP?/DNAJC5 in maintaining the normal operation of the proteostasis machinery and the homeostasis of lipids and cholesterol. Consistent with this, lipidomic analysis in conventional CSP?/DNAJC5 knock-out mice revealed increased cholesterol esters, and transcriptomic analysis in adult inducible CSP?/DNAJC5 knock-out mice showed significant changes in genes regulating cholesterol biosynthesis. Furthermore, single-nucleus RNA-seq analysis revealed alterations in genes associated with synaptic function, glial-neuron communication, and myelination. Lastly, we explored whether CSP?/DNAJC5 phenotypes could be prevented or rescued using engineered viral vectors designed to cross the blood-brain barrier via intravenous injection. While these vectors successfully delivered CSP?/DNAJC5 to central nervous system neurons, they were insufficient to rescue the neurological phenotypes. Overall, the findings presented in this thesis shed light on the molecular mechanisms through which CSP?/DNAJC5 protects synapses and on how its absence can trigger synaptic degeneration, offering new perspectives for further understanding and research.application/pdf307 p.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccess