Our genetic study demonstrates that expression of a C-terminal truncated mutant parkin in DA neurons can elicit progressive DA neuron dysfunction and degeneration in mice. The Parkin-Q311X BAC transgenic model represents the first parkin-based genetic mouse model that exhibits such a neurodegenerative phenotype in vivo
. The BAC transgenic approach is useful for modeling the dominant toxicity of a disease protein. BACs have large genomic inserts (average about 200 kb) and are likely to confer accurate, endogenous-like transgene expression in vivo
(Yang et al., 1997
; Heintz, 2001
; Yang and Gong, 2005
). In this model, truncated mutant parkin was driven by a Slc6a3
BAC and was expressed at about 50% of the endogenous parkin level in the DA neurons. This approach excludes the possibility of nonspecific artifacts due to gross over-expression and/or positional effects (e.g.
ectopic or mosaic expression), which may occur with smaller promoter transgenic constructs. Since Parkin-Q311X mice have restricted expression of the mutant parkin in the DA neurons only, our current study was meant to investigate the cell-autonomous dominant toxic effects of this mutant parkin in DA neurons. Our study was not designed to address whether mutant parkin dominant toxic effects are selectively toward the DA neurons, or they could affect other cell types in the brain. Future mouse genetic studies with ubiquitous expression of mutant parkin, preferably driven by its endogenous promoter and regulatory elements, are necessary to address such question.
Our Parkin-Q311X mice offer a novel mammalian genetic model with which to study the mechanisms underlying an age-dependent and slowly progressive form of DA neuron degeneration. With one exception (Fleming and Chesselet, 2006
; Chesselet, 2008
), existing genetic mouse models based on known PD mutations (i.e.
parkin, Pink1, DJ-1
loss of function, and α-synuclein overexpression) recapitulate aspects of DA neuron dysfunction without exhibiting progressive degeneration of DA cell bodies. Other genetic mouse models, based on genes not linked to familial PD, do exhibit a DA neuron degeneration phenotype (e.g.
loss-of-function mutants for En1/En2, Pitx3, and Ret, and knock-down mutant for VMAT2) and/or L-DOPA-reversible Parkinsonism (e.g.
DA-neuron-specific deletion of Tfam) (Hwang et al., 2005
; Sgado et al., 2006
; Caudle et al., 2007
; Ekstrand et al., 2007
; Kramer et al., 2007
) but, in most cases, the loss of DA neurons in these models occurs early in development, and/or their direct relevance to PD remains hypothetical.
Another important finding in this study was the progressive accumulation of PK-resistant α-synuclein in the substantia nigra of Parkin-Q311X mice. The absence of LBs in the brains of certain PD patients with parkin mutation prompted the suggestion that parkin may regulate α-synuclein aggregation in LB formation (Matsumine, 1998
; Mori et al., 1998
). However, recent neuropathological studies of the brains of certain PD patients with parkin mutation (Farrer et al., 2001
; Sasaki et al., 2004
; Pramstaller et al., 2005
) and mouse genetic studies of α-synuclein transgenic mice in a parkin null background (von Coelln et al., 2006
) argue that α-synuclein aggregation and LB formation are independent of parkin function. Finally, several studies suggest that wild-type parkin function can protect primary midbrain neurons (Petrucelli et al., 2002
), Drosophila (Yang et al., 2003
) and rodent (Lo Bianco et al., 2004
) DA neurons from neurodegeneration induced by α-synuclein overexpression. Hence, previous studies suggest that at least part of the normal function of parkin is to protect against the toxic effects of α-synuclein, and loss of both copies of parkin in ARJP can result in loss of parkin-mediated protection to α-synuclein toxicity as well as other toxic insults to DA neurons (Cookson, 2003
). Since parkin loss-of-function mutant mice do not exhibit the α-synuclein pathology and DA neuron degeneration phenotypes observed in the Parkin-Q311X mice (Goldberg et al., 2003
; Itier et al., 2003
; Lockhart et al., 2004
; Palacino et al., 2004
; Von Coelln et al., 2004b
; Fleming and Chesselet, 2005
; Perez and Palmiter, 2005
), we conclude that the progressive α-synuclein accumulation represents a novel dominant toxic mechanism elicited by the truncated parkin mutant.
The mechanisms linking mutant parkin action, late-onset accumulation of PK-resistant α-synuclein and eventual DA neuron degeneration remain to be elucidated. However, several lines of evidence suggest that altered oxidative stress or cellular response to oxidative stress may play a role in this pathogenic process. First, oxidative stress is known to increase with aging (Lin and Beal, 2006
) hence it could account for the late-onset (>16 months) motor and pathological phenotypes in Parkin-Q311X mice. Second, overexpression of several parkin mutants, including parkin-Q311X, but not wild-type parkin, can increase oxidative stress in transfected mammalian cells (Hyun et al, 2002
) and decrease cellular protection against a variety of toxic oxidative insults (i.e.
, and HNE) (Hyun et al., 2005
). Second, in Drosophila parkin-Q311X and parkin-T240R transgenic overexpression models, the DA neuron degeneration phenotype can be partially ameliorated by overexpressing vesicular monoamine transporter (VMAT) and can be enhanced by the loss of VMAT (Sang et al., 2007
). This result suggests that the dominant toxicity of parkin mutants is sensitive to the relative levels of cytosolic vs vesicular DA, consistent with a prior hypothesis that cytosolic DA contributes to oxidative damage to DA neurons and vulnerability in PD (Sulzer et al, 2000). Our study provides further support for this idea to the extent that PK-resistant α-synuclein in substantia nigra is co-localized with 3-NT, a marker of oxidative protein damage (Gow et al., 1996
; Ischiropoulos, 1998
). Furthermore, 3-NT immunoreactive α-synuclein lesions have been observed in PD postmorterm brains (Good et al., 1998
; Duda et al., 2000
), and oxidative stress can enhance α-synuclein oligomerization and aggregation (Giasson et al., 2000
). Taken together, these data suggest that one possible dominant toxic mechanism of DA neuron insult from a parkin mutant could be increased oxidative stress leading to oligomerization and aggregation of α-synuclein.
The hypothesis that parkin mutant may exert a gain-of-function toxicity is supported by a series of human imaging (Hilker et al., 2001
; Binkofski et al., 2007
; Hagenah et al., 2007
; Schneider et al., 2008
) and genetic studies (Khan et al., 2005
; Clark et al., 2006a
; Lesage et al., 2007
). These studies suggest that heterozygous parkin mutations may manifest as clinically asymptomatic DA neuron dysfunction and degeneration or as sporadic cases of early-onset PD (West et al., 2002
; Foroud et al., 2003
; Beffert and Rosenberg, 2006
; Clark et al., 2006b
; Sun et al., 2006
). However, data derived from the human genetic studies are suggestive but not conclusive on this issue (Lincoln et al., 2003
; Khan et al., 2005
; Clark et al., 2006a
; Lesage et al., 2007
). Available experimental evidence suggests that at least a subset of parkin mutations including truncation and point mutations could exert dominant toxicity. First, overexpression of multiple parkin mutants but not wild-type parkin induces dominant effects in transfected cells, including altered substrate binding, aggregation/oligomerization and subcellular mis-localization, and induction of oxidative stress (Hyun et al., 2002
; Cookson et al., 2003
; Henn et al., 2005
; Hyun et al., 2005
; Sriram et al., 2005
; Wang et al., 2005b
; Wang et al., 2005a
). Second, in Drosophila, overexpression of three different parkin mutants, one truncation mutation (parkin-Q311X) and two point mutations (T240R and T275W), all result in progressive motor deficits and DA neuron loss (Sang et al, 2007
; Wang et al, 2007
While the present study was not designed to resolve the controversial human genetic link between heterozygous parkin carriers and PD, it provides a proof-of-principle in a mammalian model, for toxic gain of function of parkin mutants. Furthermore, our data provide a further mechanistic link between parkin dominant toxicity and two known etiological factors in PD, α-synuclein and oxidative stress. Together, these results underscore the important need to further study the potential dominant toxicity of certain parkin mutants in mammals and to perform more comprehensive human genetic studies and longitudinal clinical studies in order to conclusively assess the putative link between heterozygous parkin and PD.