Effects of silencing ectopically expressed hSNCA in rat substantia nigra (SN) were examined as a novel therapeutic approach to Parkinson’s disease (PD). AAV-hSNCA with or without an AAV harboring a short-hairpin (sh)RNA targeting hSNCA or luciferase was injected into one SN. At 9wks, hSNCA-expressing rats had reduced SN dopamine (DA) neurons and exhibited a forelimb deficit. AAV-shRNA-SNCA silenced hSNCA and protected against the forelimb deficit. However, AAV-shRNA-SNCA also led to DA neuron loss suggesting undesirable effects of chronic shRNA expression. Effects on nigrostriatal-projecting neurons were examined using a retrograde tract tracer. Loss of striatal-projecting DA neurons was evident in the vector injection site, whereas DA neurons outside this site were lost in hSNCA-expressing rats, but not in hSNCA-silenced rats. These observations suggest that high levels of shRNA-SNCA were toxic to DA neurons, while neighboring neurons exposed to lower levels were protected by hSNCA gene silencing. Also, data collected on DA levels suggest that neurons other than or in addition to nigrostriatal DA neurons contributed to protection of forelimb use. Our observations suggest that while hSNCA gene silencing in DA neurons holds promise as a novel PD therapy, further development of silencing technology is required.
Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons.
Experimental models of Parkinson’s disease (PD) created by aberrant expression of the alpha-synuclein (SNCA) coding region have been reported. However, non-coding regions function in normal physiology and recent in vitro studies have shown that microRNAs-7 and -153 regulate SNCA expression by binding the 3′UTR. Here, effects of different hSNCA forms were examined in vivo. Adult, male rats were injected into one substantia nigra (SN) with AAV-wtSNCA, AAV-S129A hSNCA, or AAV-S129DhSNCA either with or without a portion of the native 3′UTR. DA neurons in SN that maintained striatal (ST) projections at the end of treatment were retrogradely labeled by bilateral ST fluorogold (FG) injections and FG-positive DA neurons in SN were counted. At 5wks, hSNCA coding vectors reduced numbers of FG-positive neurons in injected SN compared to uninjected SN (wtSNCA, p≤0.05; S129A/D hSNCA, p≤0.01). At 7 and 9wks, wtSNCA- and S129D hSNCA-treated rats exhibited recovery, but S129A hSNCA-injected rats did not (p≤0.01). In contrast, numbers of FG-positive neurons were unaffected by hSNCA expression when the 3′UTR was included. When FG-positive neurons were expressed as the ratio of numbers in injected to uninjected sides, the S129AhSNCA coding vector resulted in the highest decrease at 9wks versus wtSNCA (p≤0.05) or S129D hSNCA (p≤0.01). Inclusion of the 3′UTR resulted in no significant differences in FG-positive neuron ratios. These data suggest that inclusion of the 3′UTR protects against S129A hSNCA-induced loss of nigrostriatal-projecting DA neurons in vivo and that mis-regulation of hSNCA expression and function at non-coding regions contribute to PD pathogenesis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.