Several prior lines of evidence already tie together autophagy and PD. Autophagy helps neurons clear α-synuclein pathology (Webb et al., 2003; Spencer et al., 2009; Yu et al., 2009), and an excess of wild-type α-synuclein disrupts this disposal process in mammalian cells and PD transgenic mice (Winslow et al., 2010). Moreover, PD-related LRRK2 mutations scupper autophagy in cell models and transgenic mice (Alegre-Abarrategui et al., 2009; Ramonet et al., 2011). But can disrupted autophagy lead to PD pathogenesis?

As reported in Journal of Neuroscience, first author Lauren Friedman and colleagues pursued this question by knocking out the essential autophagy gene Atg7 in dopaminergic neurons, including those in the substantia nigra that die and give rise to motor symptoms in PD. Earlier, coauthor Masaaki Komatsu at the Tokyo Metropolitan Institute of Medical Science, Japan, had shown that knocking out Atg7 across the whole mouse brain caused PD-like inclusions and neurodegeneration in the first weeks of life (ARF related news story on Komatsu et al., 2006). In the present study, the researchers directed the Atg7 deletion specifically to dopamine (and norepinephrine) neurons by crossing the previously characterized mice, which have a floxed Atg7 transgene, to transgenic mice expressing Cre recombinase in tyrosine hydroxylase (TH)-producing cells. They verified by immunofluorescent staining for Atg7 and TH that the gene was inactivated in virtually all midbrain dopaminergic neurons. Labeling of ubiquitinated inclusions showed that autophagy went awry in these cells as well

Given that autophagy started failing in the Atg7/TH conditional knockouts by the time the animals reached 30 days of age, scientists expected to see rapid cell death. Instead, they found “delayed degeneration that was reminiscent of age-dependent, late-onset PD,” Yue told ARF. The Atg7/TH mice produced less striatal dopamine and had disfigured axons by four months of age, but did not lose dopamine neurons or show significant motor problems until nine months. Furthermore, immunofluorescence and Western blots showed α-synuclein and LRRK2 accumulating in Purkinje cell neurons of CNS-wide Atg7 knockout mice, and in Atg7-deficient mouse embryonic fibroblast cells, suggesting that malfunctioning autophagy might help set the stage for PD.

Mark Cookson of the National Institute on Aging in Bethesda, Maryland, points out that the experimental approach of knocking out an essential enzyme in vulnerable neurons does not clearly establish whether autophagy is critical for PD pathogenesis. “If you have something a cell requires to survive, and you get rid of it in a group of cells, they will inevitably die,” Cookson said. “We don’t know if that thing is pathogenically important in PD.”

Nevertheless, the new paper “is very interesting and certainly further implicates autophagy as a step involved in Parkinson's pathogenesis,” David Sulzer of Columbia University Medical Campus, New York, wrote in an e-mail to Alzforum (see full comment below). Sulzer’s group also generated autophagy-deficient conditional knockouts with Atg7 deleted in dopaminergic neurons, and reported in the April 26 Neuron that these mice have abnormal presynaptic neurotransmission (Hernandez et al., 2012).

In the PNAS paper, researchers led by corresponding author Benjamin Dehay linked autophagy and PD more directly by showing that PARK9 mutations lead to loss of lysosomal function and diminished autophagosome clearance. Dehay and colleagues showed previously that the number of lysosomes drops considerably prior to dopaminergic cell death in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD (Dehay et al., 2010). Prior to that, coauthor Alfredo Ramirez and coworkers at the University of Lubeck, Germany, reported that mutations in ATP13A2—a gene in the PARK9 locus encoding a lysosomal ATPase—were linked to autosomal recessive familial parkinsonism (see ARF related news story on Ramirez et al., 2006).

To get a better handle on ATPase’s relevance to PD, Dehay and colleagues opted to study human cells and approached the German scientists for fibroblasts from PD patients who carry ATP13A2 mutations. Immunostaining, Western blot, and electron microscopy experiments revealed that lysosomes in those fibroblasts were at the wrong pH, and had trouble processing pro-apoptotic cathepsins and clearing lysosomal substrates. The researchers recapitulated this problem in ATP13A2-deficient dopaminergic neuroblastoma cells and relieved it by overexpressing wild-type ATP13A2. Consistent with their lysosomal deficits, the ATP13A2-deficient cells were unable to clear α-synuclein as well as did control cells. Dehay’s group found less ATPase in postmortem nigral tissue from sporadic PD patients than from control patients' tissue. Furthermore, immunohistochemical experiments revealed ATP13A2 in more than 90 percent of Lewy body inclusions in the PD dopaminergic neurons.

The study “makes a connection between lysosomal dysfunction, α-synuclein accumulation, and neurodegeneration,” Dehay said. “When lysosomes don’t work, we get more cathepsins released into the cytosol, which can activate apoptosis.” Malfunctioning lysosomes also cannot clear α-synuclein properly, leaving these proteins to accumulate and form Lewy body aggregates. Recent work suggests that α-synuclein and the lysosomal enzyme glucocerebrosidase (GBA) may operate in a vicious cycle, with GBA deficiency allowing α-synuclein to accumulate and α-synuclein going back to stymie lysosomal GBA activity in Gaucher’s disease, a lysosomal storage disorder with genetic association to PD (see ARF related news story). Moreover, ATP13A2 came up in a yeast screen for modifiers of α-synuclein toxicity, and overexpressing the ATPase in worms protected them from α-synuclein-induced neurodegeneration (see ARF related news story).

Building on the idea that ATP13A2 may be neuroprotective, the new data strongly suggest that the ATPase "is important for efficient lysosomal function and protein degradation” Guy Caldwell of the University of Alabama, Tuscaloosa, wrote in an e-mail to Alzforum (see full comment below).

In follow-up, the authors of both papers are pushing toward translational studies. Dehay and colleagues are screening for molecules that stimulate degradation through the autophagic-lysosomal pathway. They plan to generate ATP13A2 knockout mice and conditional knockouts with the gene deleted in dopaminergic neurons. In addition, they are trying to identify proteins that interact with the ATPase. Toward that end, researchers at Massachusetts General Hospital, Boston, in collaboration with Caldwell, identified 43 novel ATP13A2 interactors, some of which were shown to influence α-synuclein aggregation and dopaminergic neuron loss in worms (Usenovic et al., 2012). The findings were published May 29 in Human Molecular Genetics.

For their part, Yue and colleagues have identified a component of a traditional Chinese medicine that drives up autophagic activity and reduces α-synuclein levels in cultured rodent neurons and fly models for PD. They are collaborating with a biotech company to develop this compound, Yue said. Prior research has shown that autophagy-enhancing molecules can help clear aggregated huntingtin protein (Sarkar et al., 2007), and other work suggests this strategy could also help in other neurodegenerative diseases such as Alzheimer’s (see ARF related news story).—Esther Landhuis.

References:
Dehay B, Ramirez A, Martinez-Vicente M, Perier C, Canron MH, Doudnikoff E, Vital A, Vila M, Klein C, Bezard E. Loss of P-type ATPase ATP13A2/PARK9 function induces general lysosomal deficiency and leads to Parkinson disease neurodegeneration. Proc Natl Acad Sci U S A. 2012 May 30. Abstract

Friedman LG, Lachenmayer ML, Wang J, He L, Poulose SM, Komatsu M, Holstein GR, Yue Z. Disrupted Autophagy Leads to Dopaminergic Axon and Dendrite Degeneration and Promotes Presynaptic Accumulation of α-Synuclein and LRRK2 in the Brain. J Neurosci. 2012 May 30;32(22):7585-93. Abstract