Recombinant adeno-associated virus 2 (rAAV2) has been shown to deliver genes to neurons effectively in the brain, retina, and spinal cord. The characterization of new AAV serotypes has revealed that they have different patterns of transduction in diverse tissues. We have investigated the tropism and transduction frequency in the central nervous system (CNS) of three different rAAV vector serotypes. The vectors contained AAV2 terminal repeats flanking a green fluorescent protein expression cassette under the control of the synthetic CBA promoter, in AAV1, AAV2, or AAV5 capsids, producing the pseudotypes rAAV2/1, rAAV2/2, and rAAV2/5. Rats were injected with rAAV2/1, rAAV2/2, or rAAV2/5 into selected regions of the CNS, including the hippocampus (HPC), substantia nigra (SN), striatum, globus pallidus, and spinal cord. In all regions injected, the three vectors transduced neurons almost exclusively. All three vectors transduced the SN pars compacta with high efficiency, but rAAV2/1 and rAAV2/5 also transduced the pars reticulata. Moreover, rAAV2/1 showed widespread distribution throughout the entire midbrain. In the HPC, rAAV2/1 and rAAV2/5 targeted the pyramidal cell layers in the CA1-CA3 regions, whereas AAV2/2 primarily transduced the hilar region of the dentate gyrus. In general, rAAV2/1 and rAAV2/5 exhibited higher transduction frequencies than rAAV2/2 in all regions injected, although the differences were marginal in some regions. Retrograde transport of rAAV1 and rAAV5 was also observed in particular CNS areas. These results suggest that vectors based on distinct AAV serotypes can be chosen for specific applications in the nervous system.

Recombinant adeno-associated viral vectors display efficient tropism for transduction of the dopamine neurons of the substantia nigra. Taking advantage of this unique property of recombinant adeno-associated viral vectors, we expressed wild-type and A53T mutated human α-synuclein in the nigrostriatal dopamine neurons of adult rats for up to 6 months. Cellular and axonal pathology, including α-synuclein-positive cytoplasmic inclusions and swollen, dystrophic neurites similar to those seen in brains from patients with Parkinson's disease, developed progressively over time. These pathological alterations occurred preferentially in the nigral dopamine neurons and were not observed in other nondopaminergic neurons transduced by the same vectors. The degenerative changes were accompanied by a loss of 30–80% of the nigral dopamine neurons, a 40–50% reduction of striatal dopamine, and tyrosine hydroxylase levels that was fully developed by 8 weeks. Significant motor impairment developed in those animals in which dopamine neuron cell loss exceeded a critical threshold of 50–60%. At 6 months, signs of cell body and axonal pathology had subsided, suggesting that the surviving neurons had recovered from the initial insult, despite the fact that α-synuclein expression was maintained at a high level. These results show that nigral dopamine neurons are selectively vulnerable to high levels of either wild-type or mutant α-synuclein, pointing to a key role for α-synuclein in the pathogenesis of Parkinson's disease. Targeted overexpression of α-synuclein in the nigrostriatal system may provide a new animal model of Parkinson's disease that reproduces some of the cardinal pathological, neurochemical, and behavioral features of the human disease.

Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for CoREST interaction. Mutations of the motif that disrupt binding also abrogate repression. When fused to a Gal4 DNA-binding domain, CoREST functions as a repressor. CoREST is present in cell lines that express REST, and the proteins are found in the same immunocomplex. CoREST contains two SANT (SW13/ADA2/NCoR/TFIIIB B) domains, a structural feature of the nuclear receptor and silencing mediator for retinoid and thyroid human receptors (SMRT)-extended corepressors that mediate inducible repression by steroid hormone receptors. Together, REST and CoREST mediate repression of the type II sodium channel promoter in nonneural cells, and the REST/CoREST complex may mediate long-term repression essential to maintenance of cell identity.

We used a high-titer recombinant adeno-associated virus (rAAV) vector to express WT or mutant human α-synuclein in the substantia nigra of adult marmosets. The α-synuclein protein was expressed in 90–95% of all nigral dopamine neurons and distributed by anterograde transport throughout their axonal and dendritic projections. The transduced neurons developed severe neuronal pathology, including α-synuclein-positive cytoplasmic inclusions and granular deposits; swollen, dystrophic, and fragmented neuritis; and shrunken and pyknotic, densely α-synuclein-positive perikarya. By 16 wk posttransduction, 30–60% of the tyrosine hydroxylase-positive neurons were lost, and the tyrosine hydroxylase-positive innervation of the caudate nucleus and putamen was reduced to a similar extent. The rAAV-α-synuclein-treated monkeys developed a type of motor impairment, i.e., head position bias, compatible with this magnitude of nigrostriatal damage. rAAV vector-mediated α-synuclein gene transfer provides a transgenic primate model of nigrostriatal α-synucleinopathy that is of particular interest because it develops slowly over time, like human Parkinson's disease (PD), and expresses neuropathological features (α-synuclein-positive inclusions and dystrophic neurites, in particular) that are similar to those seen in idiopathic PD. This model offers new opportunities for the study of pathogenetic mechanisms and exploration of new therapeutic targets of particular relevance to human PD.

Overexpression of human α-synuclein (α-syn) using recombinant adeno-associated viral (rAAV) vectors provides a novel tool to study neurodegenerative processes seen in Parkinson's disease and other synucleinopathies. We used a pseudotyped rAAV2/5 vector to express human wild-type (wt) α-syn, A53T mutated α-syn, or the green fluorescent protein (GFP) in the primate ventral midbrain. Twenty-four adult common marmosets (Callithrix jacchus) were followed with regular behavioural tests for 1 year after transduction. α-Syn overexpression affected motor behaviour such that all animals remained asymptomatic for at least 9 weeks, then motor bias comprising head position bias and full body rotations were seen in wt-α-syn expressing animals between 15 and 27 weeks; in the later phase, the animals overexpressing the A53T α -syn, in particular, showed a gradual worsening of motor performance, with increased motor coordination errors. Histological analysis from animals overexpressing either the wt or A53T α -syn showed prominent degeneration of dopaminergic fibres in the striatum. In the ventral midbrain, however, the dopaminergic neurodegeneration was more prominent in the A53T group than in the WT group suggesting differential toxicity of these two proteins in the primate brain. The surviving cell bodies and their processes in the substantia nigra were stained by antibodies to the pathological form of α-syn that is phosphorylated at Ser position 129. Moreover, we found, for the first time, ubiquitin containing aggregates after overexpression of α-syn in the primate midbrain. There was also a variable loss of oligodendroglial cells in the cerebral peduncle. These histological and behavioural data suggest that this model provides unique opportunities to study progressive neurodegeneration in the dopaminergic system and deposition of α-syn and ubiquitin similar to that seen in Parkinson's disease, and to test novel therapeutic targets for neuroprotective strategies.