Abstract
Huntington and Parkinson diseases (HD and PD) are two major neurodegenerative disorders pathologically characterized by the accumulation of the aggregate-prone proteins mutant huntingtin (in HD) and α-synuclein (in PD). Mutant huntingtin is an autophagy substrate and autophagy modulators affect HD pathology both in vitro and in vivo. In vitro, α-synuclein levels are able to modulate autophagy: α-synuclein overexpression inhibits autophagy, whereas downregulation promotes autophagy. Here, we review our recent studies showing that α-synuclein levels modulate mutant huntingtin toxicity in mouse models. This phenotypic modification is accompanied by the in vivo modulation of autophagosome numbers in mouse brains from both control and HD mice expressing different levels of α-synuclein.
HD is a fatal neurodegenerative disorder caused by the expansion of a polyglutamine tract within exon 1 of the huntingtin gene. Pathologically, it is characterized by the accumulation of mutant huntingtin in intraneuronal inclusions primarily in the brain but also peripherally. The polyglutamine-expanded mutant huntingtin protein is a well-characterized autophagy substrate, and autophagy modulators have been shown to affect HD pathology both in vitro and in vivo. Alpha-synuclein is the main component of Lewy bodies, the intracytoplasmic neuronal inclusions that are the pathological hallmark of PD. Alpha-synuclein point mutations can cause rare cases of familial PD. Moreover, rare duplications or triplications of the gene encoding α-synuclein also cause PD, implying that increasing wild type α-synuclein levels is sufficient to cause PD.
In a previous report, the Rubinsztein lab showed that α-synuclein overexpression increases mutant huntingtin aggregation and toxicity in HD cell models. Recently, we went on to demonstrate that wild-type α-synuclein overexpression inhibits autophagy both in vitro and in vivo at a very early stage of autophagosome formation. The mechanism proposed for the autophagy inhibition implicates a disruption in the homeostasis of the RAB1A protein, a GTPase involved in the regulation of secretion in yeast and mammalian cells, leading to the mislocalization of the essential autophagy protein ATG9.
Based on these previous findings, we tested the effect of modulating α-synuclein levels in two different and well-characterized mouse models of HD, the N171–82Q and the R6/1 strains. We crossed both HD models with a line overexpressing human wild-type α-synuclein as well as with a mouse line carrying a spontaneous deletion encompassing the α-synuclein locus (Snca), and compared HD-relevant phenotypes for all genotypes. In the two HD mouse lines, overexpression of wild-type α-synuclein enhances the onset of two typical HD endophenotypes: tremor onset and weight loss, but has no effect on other motor phenotypes, such as rotarod performance or overall survival. Conversely, the same two endophenotypes (tremor onset and weight loss) are ameliorated in HD mice when no α-synuclein is expressed. At the same time, working independently, the Lucas laboratory used knockout (KO) mice with a targeted deletion in the α-synuclein locus and crossed them to the R6/1 HD model. In this model, α-synuclein deficiency attenuates early motor impairments, delaying the onset of hind limb clasping and improving performance in the rotarod test at early symptomatic ages. Both sets of data are largely similar, confirming an attenuation of early HD-related phenotypes when α-synuclein is deleted.
These phenotypic modifications are mirrored by changes in the levels of LC3-II (a correlate of autophagosome numbers) in the brains of control and HD mice expressing different levels of α-synuclein. LC3-II levels (compared with actin) are reduced in HD mouse brains where human wild-type α-synuclein is overexpressed. This is consistent with our previous data showing that α-synuclein overexpression impairs autophagy. Moreover, p62, a known autophagy substrate, also accumulates in brains from HD mice overexpressing α-synuclein. Conversely, we showed that LC3-II levels are higher in the brains of mice where α-synuclein is deleted, compared with wild-type littermates. This increase in LC3-II levels is also seen in HD brains with no α-synuclein expression. Although steady-state LC3-II levels can be increased due to either an increase in autophagosome formation or decreased autophagosome degradation, one cannot deconvolute these options in vivo in mouse brains. However, these data are consistent with our previous cell-based work showing that α-synuclein knockdown enhances autophagy, increasing both LC3-II levels and LC3-II formation. Consistent with this potential in vivo increase in autophagy, the Lucas laboratory manuscript shows reduced huntingtin inclusion numbers at a late stage of disease progression in HD mouse brains lacking α-synuclein.
Our previous data in cells and Drosophila, when combined with this current study, suggest that the autophagy compromise is a very plausible contributor to the enhanced HD phenotypes seen with α-synuclein overexpression. This conclusion is supported by the demonstration by us and others, using a wide range of different cell, Drosophila and mouse systems that autophagy regulates mutant huntingtin levels, and it is known that mutant huntingtin mediates toxicity predominantly via a gain-of-function mechanism. The reduced LC3-II levels seen in HD brains overexpressing α-synuclein, together with the p62 accumulation, both reflecting altered autophagy, are compatible with autophagy being at least one major mechanism whereby α-synuclein modulates mutant huntingtin toxicity in mammalian systems. However, although it’s clear that the changes in autophagic flux due to α-synuclein status should have an effect on HD pathology, other additional mechanisms for the modifier effect cannot be ruled out.
In summary, we have shown that even physiological levels of α-synuclein seem to negatively regulate autophagy in vivo. This has potential implications for both PD and HD, but also potentially other proteinopathies or pathological conditions where autophagy enhancement has been shown to be beneficial.