Regular articleRecombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain
Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disease and currently impacts approximately 4.6 million individuals worldwide, with the prevalence expected to increase in the coming decades with longer life expectancies (Dorsey et al., 2007). The cardinal symptoms of PD are motor deficits resulting from the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the accompanying loss of dopamine neurotransmission within the striatum. Although it is still unclear what causes PD, aging is known to be a primary risk factor for this disease, with the vast majority of idiopathic cases occurring in patients over the age of 65 years (Collier et al., 2011). Currently, there are no therapies that halt, slow, or reverse the progression of neurodegeneration in PD (Gombash et al., 2014). However, viral vector-mediated gene therapy is a promising therapeutic avenue because of its ability to continuously replenish diminished proteins or overexpress neuroprotective factors that alleviate symptoms or alter disease progression.
Preclinical studies using viral vector-mediated gene transfer have been successful in ameliorating symptoms and rescuing nigral dopamine neuron loss in PD models (Emborg et al., 2009, Gash et al., 1996, Gasmi et al., 2007, Gombash et al., 2012, Herzog et al., 2008, Kordower et al., 2000), yet human clinical trials have not experienced similar success (Bartus et al., 2011, ClinicalTrials.gov, 2013, Marks et al., 2010, Stocchi and Olanow, 2013). This discrepancy may be, in part, attributable to the almost exclusive use of young adult animals in preclinical studies that fail to recapitulate the aged host brain environment typical of most PD patients. For example, previous studies demonstrated that fetal dopamine neuron grafts completely ameliorated motor impairments in young rats yet produced no improvement in aged rats because of dramatically decreased survival and neurite extension in the aged host (Collier et al., 1999, Sortwell et al., 2001). Thus, experimental results derived using young adult animals may have contributed to the overly optimistic expectations of clinical efficacy.
Several groups have reported successful viral vector-mediated gene transfer to the aged brain of rodents and nonhuman primates (Bartus et al., 2011, Emborg et al., 2009, Klein et al., 2010, Kordower et al., 2000, Wu et al., 2004). However, only 2 of these reports directly compared transduction efficiency between aged and young animals, with contradicting results (Klein et al., 2010, Wu et al., 2004). Klein et al. (2010) reported equally efficient gene transfer in aged and young adult rat brains utilizing recombinant adeno-associated virus serotype 2/9 (rAAV2/9), whereas Wu et al. (2004) concluded that serotype 2/2 (rAAV2/2) was less efficient in the aged brain as compared with the young adult brain. These results were secondary to the primary findings of the study and, consequently, additional experiments comparing viral vector efficiency between young adult and aged brains were not conducted.
It is well established that certain cellular processes that are normally altered in the aged brain (D'Angelo et al., 2009, Gao et al., 2013, Lee et al., 2000, Ryazanov and Nefsky, 2002, Smith et al., 1995) are also utilized by viral vectors for transduction (Schultz and Chamberlain, 2008). The present study sought to determine whether viral vector-mediated transgene expression was reduced in the aged rat nigrostriatal system as compared with the young adult. We utilized the rAAV2/5 serotype to examine this question because of its efficient transduction of nigral neurons (Burger et al., 2004, Gombash et al., 2013). Intranigral injections of rAAV serotype 2/5 expressing green fluorescent protein (rAAV2/5 GFP) resulted in significantly reduced exogenous protein expression in the aged rat brain as compared with the young adult brain. Significantly, fewer cells throughout the aged rat midbrain expressed the transgene delivered by rAAV2/5 GFP, in addition to producing significantly less GFP protein and GFP messenger RNA (mRNA). Collectively, our results indicate that rAAV2/5-mediated gene transfer is compromised in the aged rat brain environment.
Section snippets
Experimental overview
A cohort of 21 male Sprague-Dawley (SD) rats, 11 young adult (3 months old) and 10 aged (20 months old), were unilaterally injected with rAAV2/5 expressing GFP into the substantia nigra (SN). Six young and 5 aged SD rats were sacrificed 11 days postinjection and 5 young and 5 aged SD rats were sacrificed 3 months post-injection. Similarly, a cohort of 12 male Fischer344 (F344) rats, 6 young adult and 6 aged, were unilaterally injected with rAAV2/5 expressing GFP in the SN and sacrificed 12 days
Aging and rAAV2/5 GFP injection do not impact SNpc-THir neuron number
SD rats are classified as young adult from the time of sexual maturity (P35) until 6 months, middle aged between 6 and 18 months, old (aged) after 18 months, and senile after 24 months with an average life span of 26–32 months (Alemán et al., 1998, Gao et al., 2011). Importantly, after 22–24 months, SD rats exhibit a 13% reduction in SN dopamine neurons (Gao et al., 2011). In the present study, we used 3-month-old young adult rats and 20-month-old aged rats (Fig. 1). To verify that no
Discussion
In the present study, we investigated the ability of rAAV2/5 to transduce neurons of the aged rat brain as compared with the young adult rat brain. We first verified, as demonstrated previously, that our rAAV2/5 expressing GFP is not toxic to nigral dopaminergic neurons of the SNpc (Gombash et al., 2013). Age-related reductions in total nigral dopaminergic neurons are not a factor in our present results as equivalent numbers of nigral THir neurons are present in the SD rat at 20 months as
Disclosure statement
The authors have no conflicts of interest to report.
Acknowledgements
This research was supported by the Michael J. Fox Foundation (Caryl E. Sortwell), the Graduate School of Michigan State University (Nicole K. Polinski), Mercy Health Saint Mary's (Fredric P. Manfredsson), and the Morris K. Udall Center of Excellence for Parkinson's Disease Research at Michigan State University NS058830 (Timothy J. Collier).
References (47)
- et al.
Age-related regulation of dendritic endocytosis associated with altered clathrin dynamics
Neurobiol. Aging
(2003) - et al.
Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1,2, and 5 display differential efficacy and cell tropism after delivery to different regions of the central nervous system
Mol. Ther.
(2004) - et al.
Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells
Cell
(2009) - et al.
Response of aged parkinsonian monkeys to in vivo gene transfer of GDNF
Neurobiol. Dis.
(2009) - et al.
AAV2-mediated delivery of human neurturin to the rat nigrostriatal system: long-term efficacy and tolerability of CERE-120 for Parkinson's disease
Neurobiol. Dis.
(2007) - et al.
Striatal pleiotrophin overexpression provides functional and morphological neuroprotection in the 6-hydroxydopamine model
Mol. Ther.
(2012) - et al.
Infection of purified nuclei by adeno-associated virus 2
Mol. Ther.
(2001) - et al.
Pronounced microgliosis and neurodegeneration in aged rats after tau gene transfer
Neurobiol. Aging
(2010) - et al.
Gene delivery of AAV2-neurturin for Parkinson’s disease: a double-blind randomized, controlled trial
Lancet
(2010) - et al.
Heparin-binding determinants of GDNF reduce its tissue distribution but are beneficial for the protection of nigral dopaminergic neurons
Exp. Neurol.
(2009)