Novel AAV-Based Rat Model of Forebrain Synucleinopathy Shows Extensive Pathologies and Progressive Loss of Cholinergic Interneurons (original) (raw)
Figure 3
Molecular and biochemical quantification of α-synuclein expression.
At 40 weeks post AAV injection, a separate group of animals was sacrificed for molecular and biochemical assessments. Fresh brain tissue was rapidly dissected into five regions where α-synuclein expression had been observed in the histological observations (Figure 1); striatum, motor cortex, frontal cortex, olfactory bulb and substantia nigra. Using trizol, mRNA and protein were extracted from each region and assessed using quantitative RT-PCR (A–B) and western blot respectively (C–D). For qPCR, two new primer pairs were designed, one specific for human α-synuclein (A) and one pair for rat α-synuclein (B). Both displayed very high specificity without cross-amplification >47000∶1). Human α-synuclein mRNA was robustly expressed in all regions tested in the AAV6|a-Syn animals without any detection in the control groups. Interestingly α-synuclein mRNA was also detected in the olfactory bulb, further confirming the observation that neuroblasts in the RMS were transduced in the neonatal brain. No changes were observed in the mRNA expression of endogenous α-synuclein (B). All data in A and B are normalized to three housekeeping genes (Gap-DH, TBP and β-actin). Total α-synuclein protein levels were quantified using quantitative western blot (C–D). The known amounts of human monomeric α-synuclein was loaded on each blot to create a standard curve (C′) and total α-synuclein was detected using an antibody previously shown to have comparable affinity to rodent and human α-synuclein in WB. Final expression levels were finally normalized to β-actin. Blot in (C) shows a representative blot of tissue from the motor cortex. Interestingly, despite samples boiled in SDS and run on SDS containing gels, samples from the AAV6|a-Syn animals displayed a immunoreactive band at exactly double the size of monomeric α-synuclein (arrow in C), an observation seen in all regions. This correlated with intensity of monomeric expression and was never found in any of the control animals. Total α-synuclein levels were increased up to four-fold in regions of the cortex near the injection site (D) with lower, although still significant, increase in the frontal cortex. (* = significant increase over control groups p<0.05 in Mann–Whitney U test). Data is expressed as mean ± SEM.