title: AAV.shRNA-mediated downregulation of ROCK2 attenuates degeneration of dopaminergic neurons in toxin-induced models of Parkinson's disease in vitro and in vivo.
abstract: Parkinson's disease (PD) is a neurodegenerative disorder with prominent neuronal cell death in the substantia nigra (SN) and other parts of the brain. Previous studies in models of traumatic and neurodegenerative CNS disease showed that pharmacological inhibition of Rho-associated kinase (ROCK), a molecule involved in inhibitory signaling in the CNS, by small-molecule inhibitors improves neuronal survival and increases regeneration. Most small-molecule inhibitors, however, offer only limited target specificity and also inhibit other kinases, including both ROCK isoforms. To establish the role of the predominantly brain-expressed ROCK2 isoform in models of regeneration and PD, we used adeno-associated viral vectors (AAV) to specifically knockdown ROCK2 in neurons. Rat primary midbrain neurons (PMN) were transduced with AAV expressing short-hairpin-RNA (shRNA) against ROCK2 and LIM-domain kinase 1 (LIMK1), one of the downstream targets of ROCK2. While knock-down of ROCK2 and LIMK1 both enhanced neurite regeneration in a traumatic scratch lesion model, only ROCK2-shRNA protected PMN against 1-methyl-4-phenylpyridinium (MPP+) toxicity. Moreover, AAV.ROCK2-shRNA increased levels of the pro-survival markers Bcl-2 and phospho-Erk1. In vivo, AAV.ROCK2-shRNA vectors were injected into the ipsilateral SN and a unilateral 6-OHDA striatal lesion was performed. After four weeks, behavioral, immunohistochemical and biochemical alterations were investigated. Downregulation of ROCK2 protected dopaminergic neurons in the SN from 6-OHDA-induced degeneration and resulted in significantly increased TH-positive neuron numbers. This effect, however, was confined to nigral neuronal somata as striatal terminal density, dopamine and metabolite levels were not significantly preserved. Interestingly, motor behavior was improved in the ROCK2-shRNA treated animals compared to control after four weeks. Our studies thus confirm ROCK2 as a promising therapeutic target in models of PD and demonstrate that neuron-specific inhibition of ROCK2 promotes survival of lesioned dopaminergic neurons.
authors.affiliation: Department of Neurology, University Medicine Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.
authors.forename: Kim-Ann
authors.initials: KA
authors.lastname: Saal
authors.affiliation: Department of Neurology, University Medicine Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.
authors.forename: Jan C
authors.initials: JC
authors.lastname: Koch
authors.affiliation: Department of Neurology, University Medicine Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Tatenhorst
authors.affiliation: Department of Neurodegeneration and Restorative Research, University of Göttingen, Waldweg 33, 37073 Göttingen, Germany.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Szegő
authors.affiliation: Department of Neurology, University Medicine Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.
authors.forename: Vinicius Toledo
authors.initials: VT
authors.lastname: Ribas
authors.affiliation: Department of Neurology, University Medicine Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Michel
authors.affiliation: Department of Neurology, University Medicine Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany; Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Bähr
authors.affiliation: Department of Neurology, University Medicine Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany; Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Tönges
authors.affiliation: Department of Neurology, University Medicine Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany; Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany. Electronic address: plingor@gwdg.de.
authors.forename: Paul
authors.initials: P
authors.lastname: Lingor
mesh_terms: D015102:3,4-Dihydroxyphenylacetic Acid / Q000378:metabolism; D018663:Adrenergic Agents / Q000633:toxicity; D000818:Animals; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D059290:Dopaminergic Neurons / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors / Q000502:physiology; D006719:Homovanillic Acid; D054461:Lim Kinases / Q000235:genetics / Q000378:metabolism; D009410:Nerve Degeneration / Q000209:etiology* / Q000473:pathology*; D016627:Oxidopamine / Q000633:toxicity; D010300:Parkinson Disease / Q000150:complications* / Q000209:etiology; D011597:Psychomotor Performance; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013378:Substantia Nigra / Q000473:pathology; D014446:Tyrosine 3-Monooxygenase; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018663:Adrenergic Agents; D034741:RNA, Small Interfering; D015102:3,4-Dihydroxyphenylacetic Acid; D016627:Oxidopamine; D014446:Tyrosine 3-Monooxygenase; D054461:Lim Kinases; C515894:Limk1 protein, rat; D054460:rho-Associated Kinases; D006719:Homovanillic Acid
keywords: 6-OHDA; AAV-shRNA; Bcl-2; Dopaminergic degeneration; Dopaminergic midbrain neurons; Erk1; LIMK1; MPP(+); Parkinson's disease; ROCK2