Abstract
α-synuclein (αSyn) is the main protein component of Lewy bodies, intracellular inclusions found in the brain of Parkinson’s disease (PD) patients. Neurotoxic αSyn species are broadly modified post-translationally and, in patients with genetic forms of PD, carry genetically encoded amino acid substitutions. Mutations and C-terminal truncation can increase αSyn oligomerization and fibrillization. Although several genetic mouse models based on αSyn mutations and/or truncations exist, there is still a lack of mouse models for synucleinopathies not relying on overexpression. We report here two synucleinopathy mouse models, which are based on a triple alanine to proline mutation and a C-terminal truncation of αSyn, but do not overexpress the mutant protein when compared to the endogenous mouse protein. We knocked hαSynTP or hαSynΔ119 (h stands for “human”) into the murine αSyn locus. hαSynTP is a structure-based mutant with triple alanine to proline substitutions that favors oligomers, is neurotoxic and evokes PD-like symptoms in Drosophila melanogaster. hαSynΔ119 lacks 21 amino acids at the C-terminus, favors fibrillary aggregates and occurs in PD. Knocking-in of hαSynTP or hαSynΔ119 into the murine αSyn locus places the mutant protein under the control of the endogenous regulatory elements while simultaneously disrupting the mαSyn gene. Mass spectrometry revealed that hαSynTP and hαSynΔ119 mice produced 12 and 10 times less mutant protein, compared to mαSyn in wild type mice. We show phenotypes in 1 and 1.5 years old hαSynTP and hαSynΔ119 mice, despite the lower levels of hαSynTP and hαSynΔ119 expression. Direct comparison of the two mouse models revealed many commonalities but also aspects unique to each model. Commonalities included strong immunoactive state, impaired olfaction and motor coordination deficits. Neither model showed DAergic neuronal loss. Impaired climbing abilities at 1 year of age and a deviant gait pattern at 1.5 years old were specific for hαSynΔ119 mice, while a compulsive behavior was exclusively detected in hαSynTP mice starting at 1 year of age. We conclude that even at very moderate levels of expression the two αSyn variants evoke measurable and progressive deficiencies in mutant mice. The two transgenic mouse models can thus be suitable to study αSyn-variant-based pathology in vivo and test new therapeutic approaches.
Highlights
Using embryonic stem cell technology, we generated the hαSyntm1 mouse strain that contained the loxP flanked-human αSyn (hαSyn), followed by hαSynTP and the pLAP reporter (Figure 1A, blue mouse, upper diagram). hαSyntm1 mice were crossed with CMV-Cre mice, which express Cre recombinase ubiquitously (Schwenk et al, 1995)
We corroborated the absence of hαSyn in hαSynTP mice by mass spectrometry (MS) using single reaction monitoring from hippocampal protein extracts
(61 – EQVTNVGGAV VTGVTAVAQK – 80), was reduced by a factor of 2.3 in hαSyntm1 mice (57 ± 7 fmol/μg total protein vs. 25 ± 2 fmol/μg, Figure 1C, black vs. blue). This indicates that the mouse αSyn locus (mαSyn) protein in the hippocampus of a wild type (WT) mouse was expressed at a 2.3-times higher level than hαSyn in the hαSyntm1 knock-in model
Summary
Alpha-synuclein (αSyn) is a pre-synaptic 140 amino acid long, intrinsically disordered protein (Spillantini et al, 1997; Bendor et al, 2013). αSyn is expressed ubiquitously at high levels in the brain (Lein et al, 2007; Sjöstedt et al, 2020) and it is thought to play a role in synaptic maintenance by modulating synaptic vesicle recycling (Lotharius and Brundin, 2002) and vesicle fusion (Burre et al, 2010). αSyn is the main protein component of Lewy bodies, intracellular inclusions found in the brain of Parkinson’s disease (PD) patients (Spillantini et al, 1997). Many current animal models involve human αSyn (hαSyn) or its genetic variants expressed under strong promoters in the brain of rodents (Bobela et al, 2014; Konnova and Swanberg, 2018) This up to 30-fold overexpression results in a range of neuropathological and behavioral phenotypes (Fleming et al, 2005). These gene-targeted mice tend to have slower progressing and milder phenotypes than overexpression-based models, they avoid overexpression artifacts, ectopic expression and mutations resulting from the random insertion of the transgenes (Zhu et al, 2019) This more subtle genetic manipulation may still affect protein expression, and even the phenotype, as non-coding sequences (such as promoters) may be different in mouse and human (Nair et al, 2019). Our data further suggest that the hαSynTP and hαSyn 119 mouse models can be suitable to assess therapeutic treatments, especially those aimed at early stages of PD
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