Targeting alpha-synuclein with a microRNA-embedded silencing vector in the rat substantia nigra: positive and negative effects.
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. Furthermore, the 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 2 m 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 2 m. Co-expression of either silencing vector also induces SN inflammation, although some recovery was observed by 2 m in hSNCA-silenced SN.


CONCLUSIONS
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.
Department of Pediatrics, Neurobiology Program, Ann and Robert H. Lurie Children's Hospital of Chicago Research Center, Feinberg School of Medicine, Northwestern University, 225 E. Chicago Ave., Box 209, Chicago, IL 60611, United States.
Christina E
CE
Khodr
Department of Pediatrics, Neurobiology Program, Ann and Robert H. Lurie Children's Hospital of Chicago Research Center, Feinberg School of Medicine, Northwestern University, 225 E. Chicago Ave., Box 209, Chicago, IL 60611, United States.
Amanda
A
Becerra
Department of Pediatrics, Neurobiology Program, Ann and Robert H. Lurie Children's Hospital of Chicago Research Center, Feinberg School of Medicine, Northwestern University, 225 E. Chicago Ave., Box 209, Chicago, IL 60611, United States.
Ye
Y
Han
Department of Pediatrics, Neurobiology Program, Ann and Robert H. Lurie Children's Hospital of Chicago Research Center, Feinberg School of Medicine, Northwestern University, 225 E. Chicago Ave., Box 209, Chicago, IL 60611, United States. Electronic address: m-bohn@northwestern.edu.
Martha C
MC
Bohn
D000818:Animals; D003342:Corpus Striatum / Q000378:metabolism / Q000473:pathology; D000229:Dependovirus / Q000235:genetics; D004298:Dopamine / Q000378:metabolism; D005552:Forelimb / Q000503:physiopathology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009043:Motor Activity / Q000502:physiology; D009069:Movement Disorders / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013378:Substantia Nigra / Q000378:metabolism* / Q000473:pathology; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000378:metabolism; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism*
D035683:MicroRNAs; D034741:RNA, Small Interfering; C497604:SNCA protein, human; C493454:Slc18a2 protein, rat; D050493:Vesicular Monoamine Transport Proteins; D051844:alpha-Synuclein; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine
Gene therapy; Neurodegeneration; RNAi; Substantia nigra; Tyrosine hydroxylase