In this study, we have shown that DHM and Sal B induce the degradation of α-syn aggregates and we attribute this to the observed activation of the CMA pathway both in vitro and in vivo. Firstly, low doses of DHM and Sal B could reduce the expression level of α-syn aggregates by up-regulating the CMA pathway in the SynT-aggregation cell model. We then confirmed that DHM and Sal B up-regulate LAMP-1, an important marker for the structure and function of lysosomal membranes and LAMP-2A, a key marker of CMA, in α-syn transgenic mice and decrease astrogliosis and microgliosis. This data indicates that treatment with DHM or Sal B upregulates the CMA pathway, which is known to play a key role in degrading abnormal a-syn aggregates.
Autophagy has been considered an essential mechanism in neurodegenerative diseases such as PD and Alzheimer’s disease [
39‐
41]. Increasing evidence suggests that aggregated and misfolded α-syn drives the pathology of PD. Although it has been reported that Sal B can inhibit Aβ aggregation in cultured cells [
24,
42], no evidence exists to indicate whether DHM or SalB have a regulatory effect on α-syn aggregation. Here, we observed decreased α-syn expression in cell models after DHM and Sal B treatments, as well as decreased levels of the α-syn protein in α-syn transgenic mice. Therefore, it is possible that the degradation pathway of aggregated α-syn may be directly targeted by DHM and Sal B. From a structural chemistry point of view, several studies have provided evidence that compounds which have three adjacent dihydroxy groups (e.g. DHM) or vicinal dihydroxy groups (e.g. Sal B) are effective inhibitors of α-syn oligomerization and fibrilization [
43‐
45]. Thus, the special structures of DHM and Sal B may have a direct inhibitory effect on the aggregation of α-syn.
Lysosomes are the primary compartment for the degradation of intracellular proteins via autophagy [
46]. The existence of abnormal intracellular α-syn-positive aggregates in PD indicates that the degradation capability of lysosomes may be impaired [
3,
47]. CMA exerts a protective function by selectively targeting damaged or misfolded proteins for lysosomal degradation. Dysfunction of CMA in PD is characterized by reduced expression of the membrane receptor of CMA, lysosomal-associated membrane protein (LAMP) [
4,
48,
49]. Several studies using different cell culture models of synucleinopathies have shown that the CMA pathway participates in α-syn degradation and its alteration may support α-syn mediated neurodegeneration [
3,
7,
50]. Most of the previous studies report increased accumulation of α-syn by inhibiting CMA pathway, or reduced α-syn levels by activating CMA pathway [
39,
51]. LAMP-2A plays an important role in the CMA pathway of α-syn degradation and an increased expression of LAMP-2A can activate the CMA pathway [
4,
13,
52]. Here, we observed a reduction of α-syn aggregation by DHM and Sal B
in vitro. The aggregation cell model is characterized by ThS-positive α-syn aggregates, because the dye can specifically bind to amyloid-like structures to indicate the formation of large inclusions [
36,
53]. Smaller α-syn positive aggregates generated by untagged α-syn are also found in cell models, but are not positive for ThS. Thus, our data suggest that DHM and Sal B not only enhance α-syn degradation by the CMA pathway, but also modulate α-syn aggregation. Rapamycin has been widely used to inhibit the mTOR pathway and thereby induce autophagy [
54]. Chloroquine blocks lysosomal function by raising lysosomal pH, thereby inhibiting lysosomal function [
55]. Here, blocking autophagy with CQ resulted in SynT aggregation and increased toxicity. However, ALP modulation by rapamycin did not increase the toxicity of SynT and α-syn aggregation was reduced. We can see a clear effect of DHM and Sal B treatment on LAMP-1 and LAMP-2A levels, and a similar effect on aggregation induced by rapamycin. Notably, we observed that both LAMP-1 and LAMP-2A clearly co-localized with α-syn in transgenic mice after administration of DHM and Sal B, which is in agreement with previous findings [
48,
56]. Recent studies showed that DHM and Sal B can enhance the level of autophagy by regulating the mTOR pathway [
31,
57]. Sal B can stabilize the lysosome membrane by increasing the LAMP-1 protein level by reducing lysosomal enzyme translocation to the cytosol [
58]. Levels of LAMP-1 can be increased through the regulation of the nuclear localization of Transcription factor EB (TFEB) via the mTOR signaling pathway [
59]. LAMP-2A and the mTOR complex were highly relevant
in vivo [
60]. Thus, we reasoned that DHM and Sal B may enhance the degradation of α-syn by up-regulating the level of CMA and enhancing the expression of LAMP-1 and LAMP-2A via the mTOR signaling pathway.
As previously reported, DHM exerts a more rapid effect in association with enhancement of brain-derived neurotrophic factor expression and inhibition of neuroinflammation [
61]. Administration of Sal B significantly decreased microglial activation in the central nervous system [
62], promoted autophagy and induced the clearance of inflammasome, resulting in neuroprotective actions [
63]. In our study, we demonstrated that both DHM and Sal B treatments effectively inhibited astroglia- and microglia-mediated neuroinflammation. It appears that DHM and Sal B can penetrate the blood-brain barrier and display multiple pharmacological activities, including oxidation resistance, anti-tumor properties and neuroprotection [
23,
64], indicating potential for clinical application [
65]. Both DHM and Sal B displayed a protective role towards dopaminergic neurons by exerting neuroprotective effects [
66,
67].