The aim of this study was to generate a mouse model for PD that develops behavioural deficits and histopathological hallmarks resembling the human disease within a practical experimental time frame. We demonstrated that unilateral injection of AAV1/2 containing human mutated A53T-aSyn into the SN induced a widespread overexpression of A53T-aSyn in dopaminergic SN neurons that led to neurodegeneration, dopaminergic fiber loss and decrease of dopaminergic neurotransmitter in the striatum within 10 weeks. Moreover, the degeneration of the nigrostriatal system was concomitant with early motor impairment in the cylinder test at 5 weeks after AAV1/2 injection. This was accompanied by accumulation of human aSyn aggregates in the SN. The novelty of this model though is the presence of structures that are similar to Lewy bodies and neurites from human PD brain, from a morphological perspective.
Our findings that AAV1/2 can induce A53T-aSyn overexpression in dopaminergic SN neurons are comparable to previous data from the PD rat model generated by SN injection of the same AAV1/2 viral vector. Moreover, as with the related rat PD model, transport of the A53T-aSyn from the SN neurons to the striatum was observed in AAV1/2-A53T-aSyn injected mice, indicating that the A53T-aSyn protein has the capability to be transported along the nigrostriatal tract [14]. Thus, as in the AAV1/2-A53T-aSyn rat model, we could show that the AAV1/2 construct was highly effective in mice regarding the coverage of the SN DA neurons and its axonal transport along the nigrostratal tract.
There is high construct validity in the AAV-aSyn models of PD. They produce degeneration and dysfunction based on the core molecular feature of the clinical disease: toxicity associated with accumulation of aberrant aSyn. The definitive detection of pathological aSyn or Lewy bodies/neurites in the SN and other structures (e.g. amygdala, locus coeruleus) involves applying a gold standard histological methodology [2] utilizing proteinase K digestion [20] and LB509 antibody labeling procedures. In human, a positive result in this assay provides support for the postmortem diagnosis of PD. We applied this method of pathological aSyn detection in midbrain tissue sections derived from the mouse model alongside a confirmed human PD case and an aged matched control. We show that the aged-matched control case did not show any detectable levels of aSyn, likely due to clearing of physiological aSyn by proteinase K digestion. In contrast, the PD case showed abundant aSyn positive inclusions within melanized SN neurons and the presence of aSyn positive neurites. Similarly, the mouse tissue showed extensive aSyn accumulation in structures resembling both SN neurons and neurites. Thus, we demonstrate that in the AAV1/2-A53T-aSyn mouse, the SN develops proteinase K resistant structures with a morphology resembling Lewy pathology observed in humans, highlighting the histopathological similarity of aggregates induced in this mouse PD model to those in human PD. Although cytoplasmic aSyn aggregates of cell bodies have been observed in other PD mouse models, histological insolubility of these aggregates and explicit morphological similarity to human PD Lewy pathology was not shown [21, 24]. Phosphorylation of aSyn at Ser129 is considered a specific marker of ?-synucleinopathies that promotes aSyn fibril formation [11]. Additionally, it has been shown that misfolding and hyperphosphorylation of aSyn might lead to central locomotor dysfunction in the (Thy1)- h[A30P]?Syn transgenic mouse model for an ?-synucleinopathy [19]. We therefore analyzed the AAV1/2-A53T-aSyn mouse for Ser129 aSyn phosphorylation and found positive profiles in the SN again demonstrating the authenticity of pathologic aSyn aggregates in this mouse PD model.
We also found a significant degeneration of dopaminergic SN neurons, with a 33% reduction in AAV1/2-A53T-aSyn treated mice as compared to the EV control group. This was accompanied by significant loss of nigrostriatal dopaminergic fibers measured by striatal TH staining and DAT binding, reduced by 20% and 29%, respectively, thus recapitulating a histopathological hallmark of human PD and providing support for the model showing a high degree of face validity. Interestingly, in human PD, the onset of nigrostriatal degeneration precedes the occurrence of motor impairment with an estimated ~30% loss of SN neurons, and an even higher loss of striatal terminals, evident prior to motor symptom onset [15] (for review see Cheng et al., 2010 [6]). In contrast, in rodent models of PD, motor features can develop in the absence of significant overt nigrostriatal degeneration [13], perhaps due to the relatively intense burden of synucleinopathy in the otherwise intact nigrostriatal dopaminergic system. Indeed, although several AAV overexpression-based PD mouse models exist, either with overexpression of mutated or non-mutated aSyn, one of the main problems most of these models face so far is that they express, at best, limited nigrostriatal degeneration. Thus, two AAV models overexpressing either mutated A53T-aSyn (serotype not mentioned) [8] or human wt-aSyn (AAV2) [28] did not show degeneration of SN dopaminergic neurons even after 7 and 12 weeks post AAV injection. Similarly, in St. Martin et al. (2006), expression of human wt-aSyn by AAV (serotype not mentioned), produced no significant dopaminergic SN neuron loss after 12 weeks nor striatal dopaminergic fiber denervation of the striatum after 24 weeks [25]. In contrast, Yasuda et al. (2009), however, were able to show that AAV1-produced human wt-aSyn, delivered to the mouse SN, resulted in a significant loss in SN DA neurons at 8 weeks, however, no deficits were shown at the level of the striatum [30]. These reports are opposed to the finding in our PD model with a decrease of 38% in DA and 33% in DOPAC levels in the AAV1/2-A53T-aSyn delivered hemisphere compared to control EV injected mice. Two AAV aSyn based PD mouse models, with an AAV2 component, have, in a manner similar to the model we present here, presented a clear loss of dopaminergic SN neurons in a relatively short time frame after AAV administration. In the first study, Oliveras-Salva et al. (2013) showed, in a viral concentration dependent manner, that AAV2/7 (wt and A53T-aSyn) delivery resulted in loss of TH+ SN cells that was observed as early as 4 weeks (by 57%) with a maximal loss at 8 weeks after injection (by 82%). This was accompanied by a reduction in TH+ immunoreactivity in the striatum [21]. In the second study, Song et al. (2015) showed that AAV2/1-human wt-aSyn exposed mice showed a significant reduction in SN DA neurons after 8 weeks (by 34%) and 12 weeks (by 50%) that was accompanied by a decrease of striatal TH+ fibers and DA levels in the affected hemisphere, compared to AAV2/1-GFP controls [24]. Interestingly, and in contrast to our own report here, in the AAV2/7 and AAV2/1 models, a significant deficit in behavior was only found at later stages, from 12 weeks on, i.e. after the degeneration had occurred, and indeed, at a level exceeding what would be expected in PD patients upon first observation of motor symptoms. This is reminiscent of the 6-hydroxydopamine and 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridin toxin based models which require such levels of degeneration in order to produce spontaneous motor impairments.
There are limitations to this study. Firstly, the model necessitates a level of expertise to be able to deliver AAV1/2, by intracerebral injection, to the mouse SN, thus in our hands we mistargeted the SN in 16.3% of the cases. Secondly, reversibility of behavioural deficits using clinically defined treatments (e.g. L-DOPA, ropinirole) was not conducted and thereby the models predictive validity has yet to be determined. Thirdly, this study does not include a control group expressing a control protein. It has already been shown in the AAV1/2-A53T-aSyn rat PD model that a high titer of AAV1/2 containing green fluorescent protein (AAV1/2-GFP) causes neurodegeneration but significantly less than compared to AAV1/2-A53T-aSyn treated rats. Moreover, no loss of striatal TH-immunoreactivity was observed in AAV1/2-GFP rats, thus indicating that the toxicity of AAV1/2-GFP was not responsible for all the A53T-aSyn induced damage [14]. Nonetheless, it cannot be excluded that in the AAV1/2-A53T-aSyn mouse model the demise of dopaminergic neurons is at least to some extent independent from pathologic A53T-aSyn. Finally, although LB509 positive dystrophic neurites have been detected in the striatum of AAV1/2-A53T-aSyn mice, pathological, insoluble aSyn deposition in presynapses, that have been seen in A53T-aSyn transgenic mice [26], have not been addressed in this work.