AAV2-mediated striatum delivery of human CDNF prevents the deterioration of midbrain dopamine neurons in a 6-hydroxydopamine induced parkinsonian rat model
Introduction
Approximately 1% of the population aged 65 years or older is diagnosed with Parkinson's disease (PD). Occurrence is even higher in people older than 85 years; approximately 4–5% are diagnosed with this progressive neurodegenerative disorder (Farrer, 2006). The typical clinical symptoms of PD include resting tremor, rigidity, akinesia and postural instability; these symptoms become worse in PD patients as the disease progresses. The pathological hallmark of PD is the prominent loss of dopaminergic (DA) neurons of the substantia nigra (SN) that project to the striatum, and intraneuronal protein inclusions known as “Lewy bodies” may be observed (Burke, 1999). The degree of DA neuron loss has been correlated with reduced DA transporter (DAT) levels (Dauer and Przedborski, 2003). DAT is a presynaptic protein found at terminal dopaminergic neurons that regulates dopamine concentration in the synaptic cleft by allowing re-uptake of dopamine by presynaptic neurons (Nirenberg et al., 1996). Recently, chemical probes targeting DAT have been increasingly considered to be more accurate markers of DA neuronal function, especially in the PET-based neural imaging (Shih et al., 2006).
As the etiology of PD remains unclear, the current available treatments are heteropathic and aim to relieve symptoms. Therefore, novel therapies for slowing or reversing disease progression, and specifically those preventing neurodegeneration, are urgently needed (Burton et al., 2003). Neurotrophic factors, which are designated as a collective subset of growth factors that promote neuronal survival and stimulate neuritic outgrowth of specific types of neurons, are preferred to restore DA neuronal function in degenerative conditions. Recently, a new family of neurotrophic factors has been identified, which are particularly valuable for the treatment of PD. Two members of this family are mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) (Peterson and Nutt, 2008, Petrova et al., 2003, Voutilainen et al., 2009). These factors appear to be more selective and effective for dopaminergic neurons than other known trophic factors, such as glial-derived neurotrophic factor GDNF (Hoffer, 2011) and brain-derived neurotrophic factor BDNF (Nagahara and Tuszynski, 2011). CDNF is abundantly produced in various brain tissues of adult mice, such as the cortex, hippocampus, SN, cerebellum and locus coeruleus (Lindholm et al., 2007). Sf9-derived recombinant human CDNF proteins injected into the rat striatum has been shown to exhibit a substantial neuroprotective effect against 6-OHDA lesions. A single dose of CDNF introduced prior to 6-OHDA injection significantly reduced amphetamine-induced turning behavior and almost completely rescued deteriorated DA cells of the SN. Intrastriatal CDNF administration for 4 weeks following 6-OHDA injection restored dopaminergic function and significantly prevented nigral DA neuron degeneration (Lindholm et al., 2007). The report suggested that CDNF could be of great therapeutic potential in treating human PD patients.
Adeno-associated viral (AAV) vectors are predominant viral vectors in gene therapy clinical trials for neurodegenerative diseases (Lu, 2004). Neurotrophic factor-delivered by AAV vectors have shown encouraging promise for PD gene therapy in both animal studies and clinical trials (Mochizuki and Mizuno, 2003). Among the applications of AAV-mediated transgene expression in the central nervous system, the AAV2 vector is most frequently used. AAV2 is effective in transducing neurons, allowing long-term transgene expression, and is associated with minimal host immune responses (Bankiewicz et al., 2006, Yan et al., 2007). In the present study, we constructed an AAV2.CDNF overexpression vector and injected it directly into the adult rat striatum after 6-OHDA treatment to examine whether AAV-mediated CDNF delivery was able to restore long-term behavior changes induced by 6-OHDA lesions. We also examined the substantia nigra dopamine (DA) cell numbers and striatal DAT activity in 6-OHDA parkinsonian rats with or without the administration of AAV2.CDNF.
Section snippets
Plasmid construction and rAAV2 viral vector production
pAAV2-CDNF is a derivative of pSNAV2.0, which is a patent vector of Vector Gene Technology Company Limited (VGTC, Beijing, China), inserted with human CDNF at Age I/Nhe I sites. The AAV2 viruses used in this study, AAV2.eGFP and AAV2.CDNF, both used an overexpression cassette driven by a CMV promoter. Virus production and purification were contracted to the Vector Gene Technology Company Limited using BHK21 as the producer cell line. The obtained viruses were tested to verify clinical grade
Characterization of AAV2.CDNF for long term transduction in rat striatum
PCR was used to amplify the complete human CDNF coding sequence from a cDNA library; the sequence was then cloned into a pSNAV vector and packaged in the AAV2 virus using BHK21 as the producer cell line. To determine whether efficient transduction occurred following viral infection, 293T cells were infected with the obtained AAV2.CDNF virus for 24 h. Little RT-PCR product was observed in naïve cells, while high levels of CDNF mRNA were detected in pAAV2-CDNF and AAV2.CDNF treated cells (Fig. 1b
Discussion
To treat PD, neurotrophic factors are a preferred neurorestorative therapy in patients with significantly compromised nigrostriatal dopamine pathways (Lindholm and Saarma, 2010). Previously, the most promising candidates were GDNF (Aoi et al., 2001, Brizard et al., 2006) and NRTN (Oiwa et al., 2002, Rosenblad et al., 1999), both of which demonstrated neurorestorative effects in 6-OHDA intrastriatally injected rats. However, the preliminary results of clinical trials of these factors in PD
Acknowledgments
This study was supported by the Chinese National Basic Research Program (2011CB504100) and the National Natural Science Foundation of China (31000476).
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The first two authors contributed equally to this article.