The present study demonstrates that NH018-1 exhibits beneficial effects on MSG-induced excitotoxicity, SCA17
expanded-polyQ-mediated cell death, and SCA17 transgenic mice. The results show several pieces of evidences that NH018-1 inhibited
glutamatergic cytotoxicity by deducing downstream mitochondrial apoptotic pathway. Moreover, NH018-1 ameliorated glutamatergic excitotoxicity by down-regulating Bax and up-regulating Bcl-2
expressions, resulting in increasing Bcl-2/Bax ratio. NH018-1 inhibited MSG-mediated cytochrome C release and activation of downstream Caspase-9, Caspase-3, and PARP. In addition, NH018-1 decreased the levels of Calpain-2 and its substrate SBDP induced by MSG. Besides, MSG-induced cell apoptosis and production of ROS were decreased by NH018-1 treatment. Furthermore, application of NH018-1 to SCA17 cell model revealed that NH018-1 alleviated expanded polyQ-mediated cytotoxicity by decreasing the expression of cleaved-Caspase-9, -Caspase-3, and -PARP fragments. Importantly, NH018-1 treatment improved the performance of motor coordination in SCA17 transgenic mice.
In this study, by screening nine CHMs and nine compounds isolated from CHMs, and NH018 and compound NH018-1 were found to have high capability to increase cell viability of MSG-induced SH-SY5Y cell
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death (figure 1 and 2). In addition, NH018-1 inhibited the release of LDH into the culture medium after SH-SY5Y cell damage caused by MSG (figure 3). It was reported previously that the binding of glutamate to NMDA receptor leads to the influx of Ca2+ into the cells, and the
elevation of intracellular Ca2+ levels in CNS neurons 43, 48. The excess of Ca2+ inside cells is related to variety pathological phenomena such as neuronal toxicity 15. Thus, Ca2+ is an important indicator in several neurondegenerative disorders. Higher intracellular Ca2+ may disrupt the mitochondrial membrane potential in hippocampal neurons, cortical neurons 32, and cerebellar granule neurons 73. Bcl-2 could block loss of mitochondrial membrane potential to reduce the extent of apoptosis 55. The mitochondrial apoptosis pathway is mediated by the Bcl-2 family of proteins, and apoptosis will be inhibited at high levels of Bcl-2. In
contrast, the initiation of apoptosis is associated with an increased expression of Bax 36. Our results showed that NH018-1 increased the expression of Bcl-2 but decreased that of Bax after glutamatergic treatment (figure 4) in SH-SY5Y cells. However, the changes of
intracellular Ca2+ concentration and mitochondrial membrane potential need to be further examined.
The regulation of the mitochondrial permeability transition plays an important role in the mitochondria-mediated apoptosis pathway 27, 34, 59. In this process, cytochrome C releases from the intermembrane into
cytoplasm 6 and binds Apaf-1, leading to the activation of protease
caspase-9, and then caspase-3. Our data indicated that NH018-1 inhibited
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cytochrome C release (figure 5) and decreased the expression of
cleaved-Caspase-9, cleaved-Caspase-3, and cleaved PARP (figure 6) in SH-SY5Y cells, suggesting that NH018-1 could prevent
mitochondria-mediated apoptosis.
The calcium-dependent protein, calpain, had been known to directly participate in tau proteolysis and degradation 31, and inhibition of calpains was found to ameliorate synaptic transmission and memory in AD mouse
62. The Cyclin-dependent kinase (cdk5) activity was regulated by p25, which is generated upon the cleavage of p35 by activated calpain increased cdk5 kinase activity 51 enhances tau phosphorylation 4, and finally causes cell death. On the other hand, Ca2+ may trigger the
calpain-dependent formation of polyglutamine containing fragments in neuroblastoma cells, and ALLN or calpeptin (both are calpain inhibitors) treatment can lead to complete disappearance of aggregates. Additionally, overexpression of calpastatin (endogenous calpain inhibitor) could inhibit cleavage and aggregation of mutant ataxin-3 26. Our findings showed that NH018-1 decreased the expression of Calpain-2 and Calpain
specific-SBDP (figure 7) in SH-SY5Y cells, which indicates that NH018-1 might affect calpain/p35/p25/cdk5 pathway and decrease neuronal cell death.
High concentrations of extracellular glutamate can block cystine uptake into cells, leading to depletion of intracellular cysteine and antioxidant glutathione 25, then inducing ROS accumulation, oxidative
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stress, and subsequent neuronal cell death. Thus, ROS plays an important role in neurodegenerative diseases, and our results showed that NH018-1 reduced MSG-induced ROS production (figure 9) in SH-SY5Y cells.
The neuroprotective effects of NH018-1 on AD are well defined.
NH018-1 could suppress hydrogen peroxide-, β-amyloid protein-,
hypoxic-ischemic-, and glutamate-induced cytotoxicity 69. Nevertheless, the effects of NH018-1 on neurodegenerative diseases in addition to AD are rarely defined. Therefore, this study was designed to investigate the effects of NH018-1 on SCA17 cell and animal model. Present study showed that NH018-1 increased cell viability by inhibiting
mitochondrial-mediated cell death pathway, it indicates that NH018-1 might rescue the cell death of SCA17 (79Q) by the same way. Our results showed that NH018-1 increased cell viability (figure 10) and decreased the expressions of cleaved-Caspase-9, cleaved-Caspase-3, and
cleaved-PARP (figure 11) after induction of SCA17-79Q expression in SCA17 cells. The SCA17 transgenic mice used here recapitulate SCA17 phenotypes by exhibiting Purkinje cell loss, cerebellar atrophy, and worse performance on the rotarod 82. We found NH018-1 improved the motor performance on an accelerating rotarod (figure 12) and ameliorated gait abnormalities such as reducing the front/hind footprint overlaps as well as increasing the front/hind stride and parallel length (figure 13). NH018-1 also decreased the expression of TBP (N12), Calpain-2, and
cleaved-Caspase-3 in cerebellum of SCA17 mice (figure 14).
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Besides, previous studies indicated that NH018-1 is a reversible and selective inhibitor of AChE 14 and the potency of AChE inhibition is similar or better to physostigmine, galanthamine, donepezil and tacrine 66,
70. NH018-1 could inhibit the AChE activity to improve memory and cognitive function in AD cerebrum. As shown in figure 15, our findings showed that cerebral AChE acitivities are similar between wild type and SCA17 transgenic mice, but the AChE activity is significantly increased in the cerebellum of SCA17 transgenic mice comparing to wild type mice.
However, no significant changes were observed after treating SCA17 transgenic mice with NH018-1. According the above-mentioned results, it suggests that NH018-1 improves the SCA17 disorder through the
mitochondrial apoptosis pathway but not through the inhibition of AChE activity.
Taken together, NH018-1 has protective effects on MSG-induced cell death of SH-SY5Y cells and SCA17-79Q -induced cell death, and improved the motor coordination of SCA17 animal model via inhibition of glutamate excitotoxicity. It is the first time to demonstrate the effects of NH018-1 on SCA17 cell and animal models, therefore, NH018-1 could be a potential drug to improve SCA17 disorder.
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