Caspase-3 Mediates Apoptosis of Striatal Cells in GA Ⅰ Rat Model

In previous study,glutaric acid (GA) induced apoptosis of primary striatal neuron in vitro.In order to investigate the neurotoxic effects of GA on neonatal rat corpus striatum and the possible mechanism,34 male pups were randomly assigned to NS group,low dose GA (LGA,5 μmol GA/g body weight) group and high dose GA (HGA,10 μmol GA/g body weight) group.These pups were subcutaneously administered with three injections from postnatal day 3 to 22 at 7:30 am,15:00 pm and 22:30 pm and killed 12 h after the last injection.Microscopic pathology in corpus striatum was evalu-ated by HE staining.The apoptotic cells were identified by TUNEL staining.The transcript levels of caspase-3,8,9,Bax,Bcl-2 were detected by using real-time PCR and the protein levels of procaspase-3 and the active fraction were evaluated by Western blotting.In LGA and HGA groups,ventricle collapse,cortical atrophy by a macroscope and interstitial edema,vacuolations,widened perivascular space of bi-lateral striatum by a microscope were observed.TUNEL assay revealed that the apoptotic cells were in-creased in LGA and HGA groups.The transcript of caspase-3 was up-regulated to 2.5 fold,accompanied by the up-regulation of caspase-9,Bax and down-regulation of Bcl-2.The protein levels of procaspase-3 and the active fraction were up-regulated in LGA and HGA groups.The rat model for GA Ⅰ showed mitochondrial apoptotic pathway may be involved in the GA-induced striatal lesion.Further studies should be taken to investigate the underlying mechanisms.

Treatment of glutaric aciduria type I(GA-I)via intracerebroventricular delivery of GCDH

Glutaric aciduria type I(GA-I)is an autosomal recessive genetic disorder caused by a deficiency in glutaryl-CoA dehydrogenase(GCDH).Patients who do not receive proper treatment may die from acute encephalopathic crisis.Current treatments for GA-I include a low-lysine diet combined with oral supplementation of L-carnitine.A mouse model of Gcdh^(c.422_428del/c.422_428del)(Gcdh^(−/−))was generated in our laboratory using CRISPR/Cas9.Gcdh^(−/−)mice had significantly higher levels of glutaric acid(GA)in the plasma,liver,and brain than those in wild-type C57BL/6 mice.When given a high-protein diet(HPD)for two days,approximately 60%of Gcdh^(−/−)mice did not survive the metabolic stress.To evaluate whether GCDH gene replacement therapy could be used to provide sustained treatment for patients with GA-1,we prepared a recombinant adeno-associated virus(rAAV)carrying a human GCDH expression cassette and injected it into Gcdh^(−/−)neonates for a proof-of-concept(PoC)study.Our study demonstrated that delivering rAAV to the central nervous system(CNS),but not the peripheral system,significantly increased the survival rate under HPD exposure.Our study also demonstrated that rAAVPHP.eB mediated a higher efficiency than that of rAAV9 in increasing the survival rate.Surviving mice showed dose-dependent GCDH protein expression in the CNS and downregulation of GA levels.Our study demonstrated that AAV-based gene replacement therapy was effective for GA-I treatment and provided a feasible solution for this unmet medical need.