An α-synuclein AAV gene silencing vector ameliorates a behavioral deficit in a rat model of Parkinson's disease, but displays toxicity in dopamine neurons.

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 9weeks, 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.

Reversal of dopaminergic degeneration in a parkinsonian rat following micrografting of human bone marrow-derived neural progenitors.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the selective loss of dopaminergic (DA) neurons in the midbrain. Various types of stem cells that have potential to differentiate into DA neurons are being investigated as cellular therapies for PD. Stem cells also secrete growth factors and therefore also may have therapeutic effects in promoting the health of diseased DA neurons in the PD brain. To address this possibility in an experimental model of PD, bone marrow-derived neuroprogenitor-like cells were generated from bone marrow procured from healthy human adult volunteers and their potential to elicit recovery of damaged DA axons was studied in a partial lesion rat model of PD. Following collection of bone marrow, mesenchymal stem cells (MSC) were isolated and then genetically modified to create SB623 cells by transient transfection with the intracellular domain of the Notch1 gene (NICD), a modification that upregulates expression of certain neuroprogenitor markers. Ten deposits of 0.5 microl of SB623 cell suspension adjusted from 6,000 to 21,000 cells/microl in PBS or PBS alone were stereotaxically placed in the striatum 1 week after the nigrostriatal projection had been partially lesioned in adult F344 rats by injection of 6-hydroxydopamine (6-OHDA) into the striatum. At 3 weeks, a small number of grafted SB623 cells survived in the lesioned striatum as visualized by expression of the human specific nuclear matrix protein (hNuMA). In rats that received SB623 cells, but not in control rats, dense tyrosine hydroxylase immunoreactive (TH-ir) fibers were observed around the grafts. These fibers appeared to be rejuvenated host DA axons because no TH-ir in soma of surviving SB623 cells or coexpression of TH and hNuMA-ir were observed. In addition, dense serotonin immunoreactive (5-HT-ir) fibers were observed around grafted SB623 cells and these fibers also appeared to be of the host origin. Also, in some SB623 grafted rats that were sacrificed within 2 h of dl-amphetamine injection, hot spots of c-Fos-positive nuclei that coincided with rejuvenated dense TH fibers around the grafted SB623 cells were observed, suggesting increased availability of DA in these locations. Our observations suggest that NICD-transfected MSC hold potential as a readily available autologous or allogenic cellular therapy for ameliorating the degeneration of DA and 5-HT neurons in PD patients.