Targeting alpha-synuclein with a microRNA-embedded silencing vector in the rat substantia nigra: Positive and negative effects

Abstract

Background Alpha-synuclein (SNCA) downregulation shows therapeutic potential for synucleinopathies, including Parkinson’s disease (PD). Previously we showed that human (h)SNCA gene silencing using a short hairpin (sh)RNA in rat substantia nigra (SN) protects against a hSNCA-induced forelimb deficit, but not dopamine (DA) neuron loss. Further, the mir-embedded hSNCA gene silencing shRNA increases cell death in vitro, but the same target sequence embedded in a microRNA30 transcript (mir30-hSNCA) does not. Objective Examine hSNCA gene silencing using mir30-hSNCA in vivo. Methods Rats were stereotaxically injected into one SN with adeno-associated virus serotype 2/8 (AAV)-hSNCA, AAV-hSNCA plus AAV-mir30-SNCA or AAV-hSNCA plus a control non-silencing mir30-embedded siRNA and DA neuron markers and associated behavior were examined. Results AAV2/8-mediated SN hSNCA expression induces a forelimb deficit and tyrosine hydroxylase-immunoreactive (TH-IR) neuron loss. hSNCA gene silencing using mir30-hSNCA protects against this forelimb deficit at 2m and ameliorates TH-IR neuron loss. Striatal (ST) TH-IR fiber density and DA markers, assessed by western blot, are unaffected by AAV-hSNCA alone. Co-expression of either silencing vector reduces ST TH-IR fibers, panTH in SN and Ser40 phosphorylated TH in SN and ST, but does not affect vesicular monoamine transporter-2. However, hSNCA gene silencing promotes partial TH-IR fiber recovery by 2m. Co-expression of either silencing vector also induces SN inflammation, although some recovery was observed by 2m in hSNCA-silenced SN. Conclusion hSNCA gene silencing with AAV-mir30-hSNCA has positive effects on forelimb behavior and SN DA neurons, which are compromised by inflammation and reduced TH expression, suggesting that AAV2/8-mir30-hSNCA-mediated gene silencing, although promising in vitro, is not a candidate for therapeutic translation for PD.

Protein aggregation and neurodegeneration in prototypical neurodegenerative diseases: Examples of amyloidopathies, tauopathies and synucleinopathies

“…This mutation did not however induce nigrostriatal degeneration (Nakata et al, 2012). While full SNCA knockout mice do not present dopaminergic cell loss (Abeliovich et al, 2000;Cabin et al, 2002), silencing of a-syn in adult animals has yielded contradictory results (Gorbatyuk et al, 2010;Khodr et al, 2011Khodr et al, , 2014McCormack et al, 2010). The discrepancy between a presumed non-essential function on one hand and the clear impact of mutation on the other hand leads to at least two hypotheses.…”

“…This plausible therapeutic approach was applied with RNAi to reduce the synthesis in vitro and in vivo (Fountaine and Wade-Martins, 2007;Liu et al, 2008;Sapru et al, 2006). To date, studies have yielded conflicting results regarding the effectiveness and tolerability of this strategy, with different groups reporting nigrostriatal degeneration (Gorbatyuk et al, 2010;Khodr et al, 2011Khodr et al, , 2014 and deleterious inflammation (Khodr et al, 2014) after depleting a-syn in the rat brain. Conversely, no effects have been reported in nonhuman primate (McCormack et al, 2010).…”

Targeting α-synuclein for treatment of Parkinson's disease: mechanistic and therapeutic considerations

“…two strategies: (i) reducing the synthesis or (ii) increasing the clearance. Silencing of SNCA in adult animals using small hairpin RNA leads to contrasting results: degeneration 75, 76 and inflammation activation 77 have been reported in rodent models while no effects were reported in squirrel monkey 78 . Several explanations could underlie the apparent inconsistency, including the fact that experiments involved different animal species (rodent vs. primates) and may be dependent upon the extent and/or duration of α-syn deficiency: i.e.…”

Nucleic Acid–Based Therapeutics for Parkinson's Disease

“…For example, a therapeutic approach using ribozyme expressed by adeno-associated virus (AAV) has been attempted to degrade SNCA mRNA and allowed the survival of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra of rats challenged with the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP + ) [4]. In addition, other methods that use short hairpin RNA (shRNA) and short interfering RNA (siRNA) to suppress SNCA expression have been tested [5][6][7][8][9][10][11][12][13]. For example, partial silencing of αsynuclein expression in striatal neurons can be achieved using a lentiviral vector expressing SNCA-targeting shRNA [5].…”