RESUMEN
The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc-EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc-EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington's disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases.
Asunto(s)
Envejecimiento/metabolismo , Susceptibilidad a Enfermedades , Genes Reporteros , Ratones Transgénicos , Neuronas/metabolismo , Proteostasis , Envejecimiento/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica , Hipocampo/metabolismo , Hipocampo/patología , Enfermedad de Huntington/etiología , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Ratones , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Agregado de Proteínas , Agregación Patológica de Proteínas , Pliegue de Proteína , Deficiencias en la Proteostasis/etiología , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patología , Tauopatías/etiología , Tauopatías/metabolismo , Tauopatías/patologíaRESUMEN
Dopamine transmission is involved in reward processing and motor control, and its impairment plays a central role in numerous neurological disorders. Despite its strong pathophysiological relevance, the molecular and structural organization of the dopaminergic synapse remains to be established. Here, we used targeted labelling and fluorescence activated sorting to purify striatal dopaminergic synaptosomes. We provide the proteome of dopaminergic synapses with 57 proteins specifically enriched. Beyond canonical markers of dopamine neurotransmission such as dopamine biosynthetic enzymes and cognate receptors, we validated 6 proteins not previously described as enriched. Moreover, our data reveal the adhesion of dopaminergic synapses to glutamatergic, GABAergic or cholinergic synapses in structures we named "dopamine hub synapses". At glutamatergic synapses, pre- and postsynaptic markers are significantly increased upon association with dopamine synapses. Dopamine hub synapses may thus support local dopaminergic signalling, complementing volume transmission thought to be the major mechanism by which monoamines modulate network activity.