T-Type Ca2+ Enhancer SAK3 Activates CaMKII and Proteasome Activities in Lewy Body Dementia Mice Model

Lewy bodies are pathological characteristics of Lewy body dementia (LBD) and are composed of α-synuclein (α-Syn), which is mostly degraded via the ubiquitin–proteasome system. More importantly, 26S proteasomal activity decreases in the brain of LBD patients. We recently introduced a T-type calcium channel enhancer SAK3 (ethyl-8-methyl-2,4-dioxo-2-(piperidin-1-yl)- 2H-spiro[cyclopentane-1,3-imidazo [1,2-a]pyridin]-2-ene-3-carboxylate) for Alzheimer’s disease therapeutics. SAK3 enhanced the proteasome activity via CaMKII activation in amyloid precursor protein knock-in mice, promoting the degradation of amyloid-β plaques to improve cognition. At this point, we addressed whether SAK3 promotes the degradation of misfolded α-Syn and the aggregates in α-Syn preformed fibril (PFF)-injected mice. The mice were injected with α-Syn PFF in the dorsal striatum, and SAK3 (0.5 or 1.0 mg/kg) was administered orally for three months, either immediately or during the last month after injection. SAK3 significantly inhibited the accumulation of fibrilized phosphorylated-α-Syn in the substantia nigra. Accordingly, SAK3 significantly recovered mesencephalic dopamine neurons from cell death. Decreased α-Syn accumulation was closely associated with increased proteasome activity. Elevated CaMKII/Rpt-6 signaling possibly mediates the enhanced proteasome activity after SAK3 administration in the cortex and hippocampus. CaMKII/Rpt-6 activation also accounted for improved memory and cognition in α-Syn PFF-injected mice. These findings indicate that CaMKII/Rpt-6-dependent proteasomal activation by SAK3 recovers from α-Syn pathology in LBD.


Introduction
Lewy body dementia (LBD) is the second most common neurodegenerative disease worldwide. In addition to motor dysfunctions such as trembling and slow movements, non-motor dysfunctions, including dementia, depression, and anxiety, are also observed in LBD patients [1]. The main pathological features of LBD are α-synuclein (α-Syn) neuronal inclusions, such as the presence of Lewy bodies (LBs) and neuronal loss [2]; α-Syn is the principal constituent of LBs [3]. There are many forms of α-Syn, such as monomers, oligomers, fibers, and other conformations [4]. Previous reports have suggested that α-Syn oligomers exhibit toxicity in vitro and in vivo [5,6]. A study using a murine model showed that the injection of α-Syn preformed fibrils (PFFs) into the striatum spread to the substantia nigra (SN) [7], similar to prion proteins, leading to the loss of dopamine neurons. Neuronal death and loss of neuronal circuits in the striatum induce cognitive and motor impairments in mice [8]. Therefore, α-Syn plays a crucial role in the pathogenesis and progression of LBD and PD.

SAK3 Improved the Reduction of Proteasome Activity through the CaMKII-Rpt6 Signaling Pathway in PFF-Injected Mice
The degradation of α-Syn is mediated by the proteasome system [41]. To assess its therapeutic potential for α-Syn degradation, we examined the effect of SAK3 on proteasomal activity. In the proteasome activity assay, we found that proteasome activity was decreased in PFF-injected mice (Suc-LLVY-3M: 53 sentative immunofluorescence images of TH in the VTA and SNc regions during the 3-month SAK3 treatment schedule. Scale bar: 500 μm. The number of TH-positive cells was counted in the (C) VTA and (D) SNc region (n = 6-8 per group). (E) The representative immunofluorescence images of TH in the VTA and SNc region during the 1-month SAK3 treatment schedule. Scale bar: 500 μm. The number of TH-positive cells was counted in the (F) VTA and (G) SNc region (n = 7-8 per group). Error bars represent SEM. *** p < 0.001 vs. vehicle-treated PBS-injected mice; # p < 0.05, ### p < 0.001 vs. vehicle-treated α-Syn PFF-injected mice. Abbreviations: Veh = vehicle; S = SAK3.

SAK3 Ameliorated Motor and Cognitive Impairments in PFF-Injected Mice
Behavioral tests were conducted following the 3-month SAK3 treatment schedule ( Figure 1C) after PFF injection. Motor and cognitive functions were investigated at 4, 8, and 12 weeks after PBS or α-Syn injection.

Discussion
In this study, because oral SAK3 administration inhibits amyloid β plaque formation in APP-KI mice by activating the proteasome activity [20,21], we investigated whether SAK3 promotes the degradation of fibril α-Syn in PFF-injected LBD model mice. If SAK3 administration promotes the degradation of misfolded proteins, the therapeutics have the potential to solve the problems of diverse protein misfolding diseases such as PD, LBD, tauopathies, and Huntington diseases in addition to AD. As expected, chronic SAK3 administration rescued the impaired CaMKII-Rpt6 signaling in LBD model mice after injection of PFF. However, even after the onset of cognitive impairment, SAK3 administration significantly prevented the progression of LBD behaviors in both motor dysfunction and cognition.
Although the mechanism of α-Syn toxicity in LBD is controversial, α-Syn oligomers can exhibit toxicity [5,6,42] via mitochondrial dysfunction by altering cell membrane permeability [43], or by inducing lysosomal leakage [44]. There are several ways to convert monomers of α-Syn to oligomers, such as the phosphorylation (Ser129) [45] and oxidative modification of α-Syn [46]. For example, less than 4% of α-Syn are phosphorylated at Ser129 in healthy human brains, while more than 90% of α-Syn are phosphorylated in the brains of PD patients [47,48]. The formation of insoluble filaments was checked in vitro, and the phosphorylation of α-Syn at Ser129 was found to have a causative function in fibrillization compared to non-phosphorylated α-Syn [47]. In a Drosophila model study, mutating Ser129 revealed a complete inhibition of inclusion body formation and neuronal death following human α-Syn expression [49]. The phosphorylation levels of α-Syn (Ser129) are closely associated with the generation of LBs and neurodegenerative changes in dopamine neurons in the present study. SAK3 administration reduced the levels of phosphorylated α-Syn in dopaminergic neurons in the SNc. Although SAK3 directly mediated the inhibition of α-Syn phosphorylation, the degradation of α-Syn fibrils primarily resulted in reduced phosphorylation.
The 26S proteasome is a cylindrical complex that consists of a 20S core particle and a 19S regulatory particle [50], composed of α and β subunits [51]. In PD, it has been shown that the expression of mutations or wild α-Syn, particularly in the conformation of soluble oligomers and aggregates, inhibits the activity of 20S or 26S proteasomes [52]. The report also showed that the function of UPS and 19S proteasome ATPase Rpt6 decreased in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated PD model mice [53,54]. Although the mechanism remains unclear, the authors speculate that mitochondrial dysfunction induced by MPTP affects the UPS function, a system responsible for the selective proteolytic degradation of misfolded proteins from various intracellular compartments, including mitochondria [55]. The α-Syn oligomers have the potential to unfold through the open-gated channel of the 26S proteasome and insert themselves into the catalytic chamber, directly inhibiting the proteolytic active site of the β subunit of the 20S proteasome [30].
Likewise, aggregated α-Syn selectively binds with the proteasomal protein S6, a subunit of the 19S cap [31]. Reduced Rpt6 subunit expression was found in three brain regions of LBD, PD, and AD patients and was associated with reduced proteasome activity [35]. However, the reason for the reduced expression of the Rpt6 subunit is not clear. More importantly, strong associations were observed between Rpt6 levels and cognitive impairment [35]. We observed that Rpt6, a component of the proteasome 19S subunit, is phosphorylated by CaMKII (Figure 6) in the brains of PFF-injected mice. Therefore, proteasome activity, which is regulated by the CaMKII-Rpt6 pathway, may be a new target for neurodegenerative disease therapy.
We previously discovered the presence of CaMKII in rat brain [56,57], and its activity strengthened the function of neuronal networks in memory by inducing hippocampal long-term potentiation [58]. We further developed a novel T-VGCC enhancer, SAK3, which antagonizes the reduction of CaMKII phosphorylation levels via T-VGCC enhancement, and improved memory deficits in olfactory bulbectomized mice [19]. Moreover, SAK3 improved memory and cognitive function by increasing the autophosphorylation of CaMKII and restoring spine abnormalities in APP NL-G-F knock-in mice [20]. Moreover, SAK3 inhibited amyloid-beta (Aβ) accumulation and aggregation in APP NL-G-F knock-in mice [59]. The improved proteasome activity was mediated by the CaMKII/Rpt6 signaling pathway in APP transgenic mice [20].
This study successfully demonstrated that SAK3 reduced the levels of aggregated α-Syn in PFF-injected mice and increased proteasome activity through Rpt6 phosphorylation. We hypothesized that the T-type calcium enhancer SAK3 may work in PD, as shown in Figure 9. SAK3 triggers intracellular calcium influx and promotes Glu release by enhancing T-type calcium channels and increases proteasome activity via the CaMKII/Rpt6 signaling pathway. Moreover, proteasome activity contributes to the aggregation of α-Syn degradation and spine improvement. In conclusion, proteasome activity is promoted by SAK3 by mediating CaMKII activity. Because the detailed mechanism underlying the CaMKII-mediated degradation of aggregated α-Syn is still unclear, we will solve these problems in future studies. PD therapies focus on improving motor function. For instance, levodopa can ameliorate PD symptoms by increasing dopamine levels [60]. However, levodopa did not improve the cognitive function in MPTP-treated PD model mice [61]. Therefore, a new kind of drug such as SAK3, which can degrade the aggregated α-Syn by activating proteasome activity, will greatly impact PD and LBD therapies.
SAK3 inhibited amyloid-beta (Aβ) accumulation and aggregation in APP knock-in mice [59]. The improved proteasome activity was mediated by the CaMKII/Rpt6 signaling pathway in APP transgenic mice [20].
This study successfully demonstrated that SAK3 reduced the levels of aggregated α-Syn in PFF-injected mice and increased proteasome activity through Rpt6 phosphorylation. We hypothesized that the T-type calcium enhancer SAK3 may work in PD, as shown in Figure 9. SAK3 triggers intracellular calcium influx and promotes Glu release by enhancing T-type calcium channels and increases proteasome activity via the CaMKII/Rpt6 signaling pathway. Moreover, proteasome activity contributes to the aggregation of α-Syn degradation and spine improvement. In conclusion, proteasome activity is promoted by SAK3 by mediating CaMKII activity. Because the detailed mechanism underlying the CaMKII-mediated degradation of aggregated α-Syn is still unclear, we will solve these problems in future studies. PD therapies focus on improving motor function. For instance, levodopa can ameliorate PD symptoms by increasing dopamine levels [60]. However, levodopa did not improve the cognitive function in MPTP-treated PD model mice [61]. Therefore, a new kind of drug such as SAK3, which can degrade the aggregated α-Syn by activating proteasome activity, will greatly impact PD and LBD therapies. Figure 9. Schematic illustration of the action mechanism of SAK3 in PD pathology. SAK3 triggers intracellular calcium influx and promotes Glu release by enhancing T-type calcium channels. Proteasome activity is increased via the CaMKII/Rpt6 signaling pathway. Facilitated proteasomal activity by SAK3 contributes to the degradation of α-Syn aggregates and potentiates synaptic plasticity. AMPAR, glutamate α-amino-4-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; NMDAR, glutamate N-methyl-D-aspartate receptor; nAChR, nicotinic acetylcholine receptor. Figure 9. Schematic illustration of the action mechanism of SAK3 in PD pathology. SAK3 triggers intracellular calcium influx and promotes Glu release by enhancing T-type calcium channels. Proteasome activity is increased via the CaMKII/Rpt6 signaling pathway. Facilitated proteasomal activity by SAK3 contributes to the degradation of α-Syn aggregates and potentiates synaptic plasticity. AMPAR, glutamate α-amino-4-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; NMDAR, glutamate N-methyl-D-aspartate receptor; nAChR, nicotinic acetylcholine receptor.

Animals and Murine Model
Adult male C57BL/6J mice (8-12 weeks old) were obtained from Clea Japan, Inc. (Tokyo, Japan) and housed under conditions of constant temperature (23 ± 2 • C) and humidity (55 ± 5%) on 12 h light and dark cycles (lights on 9:00 a.m.-9:00 p.m.). Animals were provided with food and water ad libitum. All animal studies were conducted in accordance with the Institutional Animal Care and Use Committee of the Tohoku University Environmental and Safety Committee [2019PhLM0-021 (approval date: 1 December 2019) and 2019PhA-024 (approval date: 1 April 2019)]. The mice were operated as described previously [18]. In brief, α-Syn PFF (5 µg/each area) was injected into the bilateral striatum (anterior, 0.7 mm; lateral, ± 1.9 mm; depth −2.5 mm [62] relative to the bregma). Mice were injected with PBS as a control group for comparison.

Drug Administration and Experimental Design
SAK3 was dissolved in distilled water. As shown in Figure 1, animals were administered SAK3 orally once daily at 0.1, 0.5, or 1.0 mg/kg of the drug at 10 mL/kg, or the same volume of distilled water (for controls), for 1 or 3 months. Mice were subjected to behavioral tests at 4, 8, and 12 weeks after PFF injection to assess motor function (including rotarod task and beam-walking task) and cognitive function (including Y-maze task, novel object recognition task, and step-through passive avoidance task).

Rotarod Task
The rotarod task was performed as described previously [62]. The rotarod apparatus consisted of a base platform and a non-slippery rod (diameter, 3 cm; length, 30 cm). Before PFF injection, trained mice were placed on a rod rotating at 20 rpm, and the process was repeated until the fall latency exceeded 100 s. In the test session, the falling latency was measured for 300 s.

Beam-Walking Task
The beam-walking task was performed according to a previously described method [63]. The apparatus consisted of a rectangular beam (length, 870 mm; width, 5 mm) and a goal box (155 mm × 160 mm × 5 mm). Before PFF injection, trained mice were placed 10 cm away from the goal and allowed to reach the goal box. The number of foot slips (missteps) from the end of the beam to the goal box was recorded.

Y-Maze Task
Short-term spatial memory was investigated using the Y-maze task. As previously described [18,63], a mouse was placed at the end of the arm of a Y-maze and explored freely for 8 min. After each session, the objects and box were cleaned with 70% ethanol to prevent odor recognition. Alternations were defined as entries into all three arms on consecutive choices. The maximum number of alternations was defined as the total number of arms. The percentage of alternations was calculated as the actual alternations/maximum alternations ×100.

Novel Object Recognition Task
Cognitive function was evaluated using the novel object recognition task according to a previously described method [18,62]. In the trial session, the mice were exposed to two similar objects (consisting of a wooden block) placed symmetrically at the center of the open field box for 10 min. After 1 h, one object was replaced by a novel object, and exploratory behavior was monitored again for 5 min during the test session. After each session, the objects and box were cleaned with 70% ethanol to prevent odor recognition. The timing of object exploration was defined by behaviors such as rearing on, touching, and sniffing. The discrimination index was analyzed using the ratio of exploratory contacts to familiar and novel objects.

4.4.5.
Step-through Passive Avoidance Task The step-through passive avoidance task was conducted as described previously [18,63]. Briefly, after a mouse moved from the light to the dark compartment of a box, it received an electric shock (0.5 mA for 2 s) when passing through the floor, completing a trial session. To evaluate retention levels, mice were placed in the light compartment, and step-through latency times were recorded over 300 s after 24 h. To avoid stress effects, the step-through passive avoidance task was performed only 12 weeks after α-Syn PFF injection.

Immunohistochemistry
Immunohistochemistry was performed as described previously [63]. Mice were anesthetized, perfused with PBS, and fixed with 4% paraformaldehyde. After 24 h of

Statistical Analysis
All data are presented as mean ± standard error of the mean (SEM). Comparisons among multiple groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test using GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA).
Author Contributions: Investigation, J.X., H.I.; data analyses, I.K.; writing-original draft preparation, J.X.; writing-review and editing, K.F.; supervision and funding acquisition, K.F. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.