Axonal dystrophy and degeneration associated with PD have been related with aberrant autophagic activity. Moreover, swollen neurites containing mainly autophagic vacuoles and increased number of autophagosomes have been observed in mouse and cellular models of PD. In agreement with these observations, the abnormal presence of autophagic vacuoles is evident in brains of PD patients, in contrast to the rare detection of autophagosomes in normal aged brains. The question is, why do autophagosomes build up in PD cells? We set out in our study to investigate the mechanisms that could explain the particular patterns of autophagic pathology observed in PD that will be generally relevant to sporadic PD (sPD).
Several studies have suggested mitochondria as critical players in the pathophysiology of sPD. Although recent advances in transgenic technology have made possible the development of diverse disease models, most of the models commonly used to study sPD fail to reliably describe the pathological features frequently found in PD patient brains. This, obviously, questions the relevance of some of the data obtained with these models to sPD pathology.
To overcome these difficulties, we have designed a specific approach to address mitochondrial dysfunction as a common feature of, or, possibly, the driving force in, sPD pathology. In this study, we have modeled sPD by creating cytoplasmic hybrid cell lines (cybrids) in which endogenous mitochondrial DNA (mtDNA) from sPD or control subject platelets was transferred to human neuron-like cells after complete depletion of endogenous mtDNA (Rho0 cells). By this approach it is possible to follow the effects of mtDNA heteroplasmy within a nuclear and environmentally controlled context, thus providing a rational basis for the propagation of PD-related mitochondrial dysfunction. Thus, by comparing cells bearing mitochondria from healthy individuals with cells bearing mitochondria from PD patients, we were able to assess the effect of inherent PD-related changes in mitochondria over other cellular functions such as autophagy and the interplay of this latter process with typical cellular features of sPD. We have also extended our experiments to Rho0 cells, primary cortical neurons in which mitochondria were destabilized, differentiated sPD cybrids, and PD patient lymphocytes to draw a parallel to what is happening in PD patient neurons and peripheral blood models.
To better understand the foundation for the pathological autophagy patterns in PD, we have investigated basal and induced autophagy responses (starvation or rapamycin treatment). We have also interfered with clearance of autophagosomes by (1) inhibiting lysosomal proteolysis, (2) impeding autophagosome trafficking and subsequent fusion with lysosomes, and (3) modulating microtubule-dependent autophagic transport, in order to determine the pathological causes of autophagy.
Consistently, in all of our models, autophagosomes are actively formed but some of their structural components and autophagic substrates are unable to be efficiently degraded within lysosomes. Although the presence of accumulating autophagosomes could represent an aberrant activation of autophagy, we have now provided evidence that autophagy is not overstimulated in our models but, instead, a defective clearance of autophagic vacuoles accounts for those observations.
In our study, the rapid accumulation of autophagosomes within a few hours after blocking lysosomal proteolysis, even in primary cortical neurons, demonstrates a proper basal level of autophagic activity. However, and of significant relevance to PD pathology, the data on autophagic flux indicate that formed autophagosomes are not efficiently eliminated by lysosomal degradation. This was strongly supported by EM when we found an exacerbated accumulation of autophagosomes in sPD cybrids and more obviously in Rho0 cells.
Accordingly, we did not find significant differences in the total cellular levels and subcompartmentalization of BECN1, a principal regulator of autophagosome formation. However, sPD cybrids exhibit increased basal levels of BCL2 mainly associated with increased targeting to mitochondria. Thus, the anti-autophagic activity of BCL2 was not verified, as the mild changes observed in the binding and sequestration of BECN1 by BCL2 are not expected to disturb the formation of the class III PtdIns 3-kinase complex that is critical to the induction of autophagy.
In addition, we have further established the proof of concept that autophagy failure stemming from mitochondrial dysfunction in our models of sPD occurs from an altered microtubule assembly that impairs microtubule-dependent mitochondria and autophagosomal transport toward lysosomes. When we directly analyzed autophagic vacuoles, mitochondrial transport and autophagosome-lysosome fusion by live-cell and fluorescence microscopy, we found that their motility and dynamics were significantly impaired due to the disruption of microtubule network trafficking.
In conjunction with altered mitochondrial function and motility, tubulin cytoskeleton alterations also contribute to accumulation of protein aggregates and autophagic vesicles, and/or associated substrates, as observed in our sPD cybrids model, thus promoting apoptosis.
These findings describe novel and important features in the neuropathological cascade of PD connecting mitochondria, autophagy and microtubule dysfunctions to sPD (). Although mounting evidence has recently emerged showing that lysosomal dysfunction can be an important pathogenic factor in PD, our study identifies for the first time PD-associated defects in mitochondrial function as the basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated with autophagic pathology in PD.
Figure 1. Model for autophagic pathology triggered by mitochondrial dysfunction in Parkinson disease. (A) In normally functioning neurons, mitochondria and autophagosomes enclosing damaged organelles or protein aggregates are able to travel long (more ...)