RESUMEN
The etiology of Parkinson's disease (PD) converges on a common pathogenic pathway of mitochondrial defects in which α-Synuclein (αSyn) is thought to play a role. However, the mechanisms by which αSyn and its disease-associated allelic variants cause mitochondrial dysfunction remain unknown. Here, we analyzed mitochondrial axonal transport and morphology in human-derived neurons overexpressing wild-type (WT) αSyn or the mutated variants A30P or A53T, which are known to have differential lipid affinities. A53T αSyn was enriched in mitochondrial fractions, inducing significant mitochondrial transport defects and fragmentation, while milder defects were elicited by WT and A30P. We found that αSyn-mediated mitochondrial fragmentation was linked to expression levels in WT and A53T variants. Targeted delivery of WT and A53T αSyn to the outer mitochondrial membrane further increased fragmentation, whereas A30P did not. Genomic editing to disrupt the N-terminal domain of αSyn, which is important for membrane association, resulted in mitochondrial elongation without changes in fusion-fission protein levels, suggesting that αSyn plays a direct physiological role in mitochondrial size maintenance. Thus, we demonstrate that the association of αSyn with the mitochondria, which is modulated by protein mutation and dosage, influences mitochondrial transport and morphology, highlighting its relevance in a common pathway impaired in PD.
Asunto(s)
Homeostasis , Mitocondrias/metabolismo , Neuronas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Transporte Axonal , Células Madre Embrionarias Humanas/metabolismo , Humanos , Membranas Mitocondriales/metabolismo , Proteínas Mutantes/metabolismo , Tamaño de los Orgánulos , Dominios Proteicos , alfa-Sinucleína/químicaRESUMEN
Tau, as a microtubule (MT)-associated protein, participates in key neuronal functions such as the regulation of MT dynamics, axonal transport, and neurite outgrowth. Alternative splicing of exon 10 in the tau primary transcript gives rise to protein isoforms with three (3R) or four (4R) MT binding repeats. Although tau isoforms are balanced in the normal adult human brain, imbalances in 3R:4R ratio have been tightly associated with the pathogenesis of several neurodegenerative disorders, yet the underlying molecular mechanisms remain elusive. Several studies exploiting tau overexpression and/or mutations suggested that perturbations in tau metabolism impair axonal transport. Nevertheless, no physiological model has yet demonstrated the consequences of altering the endogenous relative content of tau isoforms over axonal transport regulation. Here, we addressed this issue using a trans-splicing strategy that allows modulating tau exon 10 inclusion/exclusion in differentiated human-derived neurons. Upon changes in 3R:4R tau relative content, neurons showed no morphological changes, but live imaging studies revealed that the dynamics of the amyloid precursor protein (APP) were significantly impaired. Single trajectory analyses of the moving vesicles showed that predominance of 3R tau favored the anterograde movement of APP vesicles, increasing anterograde run lengths and reducing retrograde runs and segmental velocities. Conversely, the imbalance toward the 4R isoform promoted a retrograde bias by a significant reduction of anterograde velocities. These findings suggest that changes in 3R:4R tau ratio has an impact on the regulation of axonal transport and specifically in APP dynamics, which might link tau isoform imbalances with APP abnormal metabolism in neurodegenerative processes. SIGNIFICANCE STATEMENT: The tau protein has a relevant role in the transport of cargos throughout neurons. Dysfunction in tau metabolism underlies several neurological disorders leading to dementia. In the adult human brain, two tau isoforms are found in equal amounts, whereas changes in such equilibrium have been associated with neurodegenerative diseases. We investigated the role of tau in human neurons in culture and found that perturbations in the endogenous balance of tau isoforms were sufficient to impair the transport of the Alzheimer's disease-related amyloid precursor protein (APP), although neuronal morphology was normal. Our results provide evidence of a direct relationship between tau isoform imbalance and defects in axonal transport, which induce an abnormal APP metabolism with important implications in neurodegeneration.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Transporte Axonal/fisiología , Neuronas/metabolismo , Proteínas tau/metabolismo , Animales , Células Cultivadas , Humanos , Ratones , Neuronas/ultraestructura , Isoformas de Proteínas , Tauopatías/metabolismoRESUMEN
Fusion of fluorescent probes to axonally transported proteins represents an established approach that enables live imaging of axonal transport. In this approach, in vivo examination of fluorescent particle dynamics provides information about the length, directionality, and the velocity by which axonally transported proteins travel along axons. Analysis of these parameters provides information about the distribution of axonal proteins and their dynamics in and between different subcellular compartments. Establishing the movement behavior of amyloid precursor protein within axons indicated that live imaging approaches offer the opportunity to significantly enhance our understanding of the biology as well as pathology of axonal transport. This chapter provides a fluorescence-based procedure for measuring axonal transport of APP in cultured newborn mouse hippocampal neurons.