Thorase Conditional Knockout (cKO) Mice Exhibits PD-like Behavior
Our previous studies demonstrated that Thorase is neuroprotective and regulates synaptic plasticity, learning and memory 14-16. Conventional Thorase knockout (KO) mice die from postnatal days 19 to 25 15. To avoid the lethality of Thorase ablation in mice, Thorase cKO mice were generated through the mating of CaMKIIα-iCre mice with floxed-Thorase mice, deleting Thorase in the brain except for in the cerebellum region in Thorasef/f mice 15. Besides the impairment of short memory, Thorase cKO mice also exhibited severe PD-like behavioral deficits beginning at around 5 months of age, including abnormal limb-clasping reflexes and tremors during the tail suspension test (Fig.1a). Additionally, some Thorase mutant mice walked on their tiptoes (Fig.1b). The open field test showed that Thorase cKO mice moved slightly slower than their wild-type (WT) littermates (Fig.1c, d). The cKO mice preferred to move along the margins of the open field and spent a reduced amount of time in the center (Fig.1c, e). Then, the gait of the Thorase cKO mice displayed a reduced stride length (Fig.1f-h). The sway length was also significantly reduced in Thorase cKO mice (Fig.1f, I, j). Therefore, we proposed that Thorase might be involved in PD. Consequently, we assessed the effects of Thorase deletion on grip strength and motor coordination. Consistent with our hypothesis, during the rotarod test, Thorase cKO mice displayed significantly decreased grip strength (Fig. 1k) and reduced latency to fall times and distances compared to their WT littermates (Fig. 1l-n). Collectively, these findings suggest that Thorase deficiency causes phenotypes that mimic PD-like behaviors.
Thorase Deficiency Results in Extensive α-synucleinopathy and Reduced TH+ Dopaminergic Neurons
Given that the behavioral deficits in Thorase cKO mice phenocopy the clinical symptoms of PD, we examined the brains of Thorase cKO mice for signs of α-synucleinopathy. Thorase cKO mice exhibited substantial α-synuclein (α-syn) accumulation that co-labeled with high levels of phosphorylated (Ser129) α-syn (pS129-α-syn), the most frequently modified form of α-syn within PD pathological inclusions, and pathogenic fibrillary aggregates (Fig. 2a). Stereological quantification analysis showed that the intensities of pS129-α-syn staining were significantly increased in the substantia nigra pars compacta (SNpc), and cortical and hippocampal regions of Thorase cKO mice compared to those of their WT littermates (Fig. 2b-d). Stereological counting of tyrosine hydroxylase (TH) staining showed a marked reduction in TH+ fiber density in the striatum (Fig. 2e, f) and a reduced number of TH+ dopaminergic neurons in the SNpc of Thorase cKO mice (Fig. 2g, h).We performed a Western blot assay and found that Thorase cKO mice exhibited significantly increased levels of high-molecular-weight pS129-α-syn, including the pS129-α-syn dimers, trimers, tetramers, and oligomers (Fig. 2i, j), which are more neurotoxic 24. We also found that the level of total α-syn was significantly increased in the brains of Thorase cKO mice compared to that of their WT littermates (Fig. 2k, l). Collectively, these results demonstrate that Thorase deletion in the brain results in extensive PD-like α-synucleinopathy. In addition, we also found an enhanced astrocytosis and more activated microglia in the brains of Thorase cKO mice. Both Western blots and immunohistochemistry assays showed significantly higher levels of GFAP and Iba1 expression in the brains of Thorase cKO mice (Fig. 2m-p), indicating an inflammatory reactivity in the brain.
Thorase Deficiency Accelerates α-Synucleinopathy and Behavioral Impairments in a Familial PD A53T Mouse Model
To further address whether Thorase has effects on the pathogenesis and progression of PD, we examined the role of Thorase in a familial PD A53T mouse model. Thorase cKO mice were crossbred with transgenic PD mice bearing the human α-synuclein gene with the A53T mutation (hA53T) mice. The levels of pS129-α-syn immunoreactivity in the SNpc, cortical and hippocampal regions of the Thorase cKO-hA53T α-syn (cKO-A53T) mice were significantly increased compared to those in the SNpc, cortical and hippocampal regions of the A53T littermate mice (Fig. 3a-d). The increase in pS129-α-syn was further verified by Western blot analysis, which revealed the largest change in the expression of the high-molecular-weight α-syn forms (Fig. 3e, f). Compared to littermate A53T mice, cKO-A53T mice also displayed a significant reduction in TH+ fiber density and a reduced number of TH+ dopaminergic neurons in striatum regions (Fig. 3g, h) and in the SNpc (Fig. 3i, j). The cKO-A53T mice exhibited a similar pattern of behavioral impairments in the open field test as the cKO mice (Fig. 3k-m). In addition, the conditional ablation of Thorase significantly exacerbated the impairments in grip strength, motor coordination and balance in A53T mice (Fig. 3n-p).
Thorase Regulates Mitochondrial Fusion/Fission Balance
Consistent with previous studies 20,25,26, Thorase predominantly localized at the outer membranes of mitochondria in neurons, mouse embryonic fibroblasts (MEFs) and HeLa cells (Supplementary Fig. 1). To examine the potential effect of Thorase dysfunction on mitochondrial morphology, we performed transmission electron microscopy (TEM) on the neurons in the brains of Thorase KO mice. The results showed a dramatic increase in vacuolated mitochondria in Thorase KO mouse brains (Fig. 4a, b). The overall mitochondrial density and coverage was increased, but the mitochondria in the brains of Thorase KO mice were smaller and rounder than those of the WT littermates (Fig. 4a, 4c-f). These findings suggest that Thorase deletion affects mitochondrial morphology and results in increased mitochondrial fragmentation. As mitochondrial dynamics also play a key role in MQC, we sought to assess whether the increase of fragmented mitochondria in Thorase KO neurons results from alterations in the balance of mitochondrial fusion and fission. We visualized mitochondria with the mitochondrial-selective fluorescent dye MitoTracker DsRed and found that Thorase KO MEFs showed more fragmented (<0.8 μm) or intermediate (0.8~3 μm) mitochondria and fewer hyperfused (>3 μm) mitochondria than WT MEFs (Fig. 4g, h), similar to those observed in vivo. Next, we investigated whether Thorase overexpression reversed this fragmentation tendency. The overexpression of a Thorase-GFP fusion protein (Thorase-GFP) induced an increase in the number of hyperfused mitochondria in WT MEFs (Fig. 4i, j). To exclude other contributory factors, we used mito-GFP with a mitochondrial targeting sequence as a control. Not surprisingly, the mito-GFP control failed to reverse the fragmented tendency (Fig. 4i, j). The same effect of Thorase overexpression was also observed in HeLa cells (Supplementary Fig. 2). We also found that the fragmentation of mitochondria in Thorase KO MEFs could be rescued by restoring the expression with Thorase-GFP protein, but not by the expression of mito-GFP (Fig. 4i, j). Collectively, these results indicate that mitochondrial dysfunction in cells lacking Thorase results in mitochondrial fission, whereas the overexpression of Thorase promotes mitochondrial fusion.
Thorase is Essential for PINK1/Parkin-mediated Mitophagy
Next, we sought to determine whether Thorase is involved in mitophagy, which selectively degrades dysfunctional mitochondria through autophagy. We applied mt-Keima, a ratiometric pH-sensitive fluorescent (561/458nm) protein that is targeted to the mitochondrial matrix, to differentially monitor mitochondria in the neutral cytoplasm (a low intensity of mt-Keima-derived fluorescence, green) or acidic lysosomal mitochondria (a high intensity of fluorescence, red) 27. The ratio of the mitochondrial signal (green)/lysosomal signal (red) within the neuronal body was used as a measure of lysosomal delivery of mitochondria (‘mitophagy index’). The primary cultured WT neurons displayed a significantly higher red mt-Keima signal that accumulated in mitochondria than Thorase KO neurons (Fig. 5a, b), suggesting a blockade of the clearance of dysfunctional mitochondria by mitophagy in Thorase KO neurons.
Since PINK1/Parkin pathway is one of major pathways that regulate mitophagy28, we applied a well-established PINK1/Parkin mitochondrial degradation assay 27 to determine whether Thorase was involved in PINK1/Parkin-mediated mitophagy. We prepared Thorase KO HeLa cells using the CRISPR/Cas9 system. In cultured WT HeLa cells overexpressing Parkin, mitochondria depolarization induced by the CCCP treatment resulted in a greater than 80% loss of mitochondria (Fig. 5c, d). However, no significant change was observed in Thorase KO HeLa cells overexpressing Parkin after the CCCP treatment (Fig. 5c, d). Then, we performed a co-immunoprecipitation assay in HEK293 cells co-transfected with Thorase-myc and PINK1-GFP. Thorase interacted with PINK1 (Fig. 5e). Based on these results, Thorase regulates PINK1/Parkin-mediated mitophagy to clear dysfunctional mitochondria.
Thorase Interacts with α-syn, and Thorase Deficiency-Induced Mitophagy Impairment Causes α-syn Accumulation
As Thorase maintains mitochondrial function by clearing mislocalized proteins 19, we next sought to assess whether Thorase directly interacts with α-syn and whether the accumulation of α-syn in the brains of Thorase KO mice results from a blockage in the mitophagy. Coimmunoprecipitation in HEK293 cells co-transfected with Thorase-myc and α-syn-GFP indicated that Thorase interacts with α-syn (Fig. 6a). We also confirmed the interaction of endogenous Thorase with α-syn in mouse brain tissues (Fig. 6b). As Thorase cKO mice exhibited substantial α-synucleinopathy in the brain, we hypothesized that the neurons lacking Thorase would display reduced degradation of α-syn. Therefore, HA-tagged ubiquitin (Ub-HA) and α-syn-GFP expression constructs were co-transfected with/without Thorase-myc into HEK293 cells. Immunoprecipitation assays showed that Thorase-myc overexpression significantly reduced the level of the high-molecular-weight smear representing polyubiquitinated α-syn-GFP (Fig. 6c). Treatment with the autophagosome-lysosome fusion inhibitor Baf blocked Thorase-mediated degradation of polyubiquitinated α-syn-GFP. In contrast, treatment with MG132, an inhibitor of the ubiquitin-proteosome system 29, did not prevent Thorase-mediated polyubiquitinated α-syn-GFP degradation (Fig. 6c).These results suggest that Thorase regulates α-syn degradation mainly through the autophagy pathway.
Thorase deficiency causes autophagy impairments resembling a late-stage autophagy block. Previous studies have shown that synthetic preformed α-syn fibrils (PFFs) seeded in primary neurons recruit endogenous mouse α-syn resulting in a Lewy body/Lewy neurite (LB/LN)-like pathology in 30,31. Thus, we examined the role of Thorase in the development of α-syn aggregates in primary cultured neurons. Exogenous human α-syn-A53T-PFFs were generated from recombinant α-syn-A53T (Supplementary Fig. 3) and applied to primary hippocampal neurons derived from Thorase KO mice and WT littermates after in vitro culture. LB-like and LN-like fibrillary aggregates in WT and KO neurons were examined by staining endogenous mouse α-syn (total α-syn) with a Syn mAb. Compared to WT neurons, the Thorase KO neurons showed an increase in the extent of fibrillary LN/LB-like inclusions either at the basal level (PBS-treated) or after PFF treatment (Fig. 6d, e). To investigate α-syn recruitment to pathologic inclusions, as demonstrated by extensive phosphorylation at Ser129 (pS129-α-syn) 30, we used pSyn#64 monoclonal antibodies specific for pS129-α-syn and found that pS129-α-syn-positive neuritic and perikaryal inclusions were significantly increased in PFF-treated Thorase KO neurons compared to WT neurons (Fig. 6f, g). These results further indicate an increased formation of pathologic α-syn aggregates in Thorase KO neurons.
Thorase Overexpression Prevents α-Synucleinopathy in PD Mouse Model A53T mice
To validate whether Thorase overexpression attenuates α-syn accumulation in vivo, A53T transgenic mice were crossed with inducible tetO-Thorase transgenic (cTg) mice to overexpress Thorase in the forebrains 16. Immunohistochemical staining of brains from 9-month-old mice showed that pS129-α-syn accumulation was significantly lower in Thorase cTg-A53T mice than A53T mice (Supplementary Fig. 4a, b). Western blot analyses also showed that hSCNA, monomeric, and high-molecular-weight pS129-α-syn accumulation was significantly reduced in Thorase cTg-A53T mouse brain lysates compared to that in A53T mouse brain lysates (Supplementary Fig. 4c, d). We also discovered that Thorase overexpression significantly prevented the accumulation of the PD-associated insoluble α-syn and pS129-α-syn fractions; soluble α-syn levels dropped only slightly but showed no significant differences (Supplementary Fig. 4e, f). Together, these findings reveal that Thorase overexpression attenuates α-syn accumulation in vivo.