Induction of Neuron-Specific Degradation of Coenzyme A Models Pantothenate Kinase-Associated Neurodegeneration by Reducing Motor Coordination in Mice

Abstract

BackgroundPantothenate kinase-associated neurodegeneration, PKAN, is an inherited disorder characterized by progressive impairment in motor coordination and caused by mutations in PANK2, a human gene that encodes one of four pantothenate kinase (PanK) isoforms. PanK initiates the synthesis of coenzyme A (CoA), an essential cofactor that plays a key role in energy metabolism and lipid synthesis. Most of the mutations in PANK2 reduce or abolish the activity of the enzyme. This evidence has led to the hypothesis that lower CoA might be the underlying cause of the neurodegeneration in PKAN patients; however, no mouse model of the disease is currently available to investigate the connection between neuronal CoA levels and neurodegeneration. Indeed, genetic and/or dietary manipulations aimed at reducing whole-body CoA synthesis have not produced a desirable PKAN model, and this has greatly hindered the discovery of a treatment for the disease.Objective, Methods, Results and ConclusionsCellular CoA levels are tightly regulated by a balance between synthesis and degradation. CoA degradation is catalyzed by two peroxisomal nudix hydrolases, Nudt7 and Nudt19. In this study we sought to reduce neuronal CoA in mice through the alternative approach of increasing Nudt7-mediated CoA degradation. This was achieved by combining the use of an adeno-associated virus-based expression system with the synapsin (Syn) promoter. We show that mice with neuronal overexpression of a cytosolic version of Nudt7 (scAAV9-Syn-Nudt7cyt) exhibit a significant decrease in brain CoA levels in conjunction with a reduction in motor coordination. These results strongly support the existence of a link between CoA levels and neuronal function and show that scAAV9-Syn-Nudt7cyt mice can be used to model PKAN.

Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform

“…We confirmed these findings by measuring the activity of recombinant Nudt19 against several acyl-CoAs and comparing it to the better characterized Nudt7 (17,25) (Fig. 1A and 1B).…”

“…Independent studies and approaches have demonstrated the localization of Nudt7 in the peroxisomes (17,25). Nudt7 mRNA and protein levels in the liver are regulated by the nutritional state and are inversely correlated with the tissue concentration of CoA (9,26).…”

“…Additionally, overexpression of a catalytically active, cytosolic form of Nudt7 has also recently been shown to cause a decrease in CoA levels in vivo (25).…”

“…The concentration of total CoA (free CoA plus CoA thioesters) in kidney homogenates was determined after conversion of the cofactor to the monobromobimane derivative, as previously described (25). For western blot analysis, flash frozen tissues were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Biotool), and centrifuged at 10,000 x g for 10 min at 4°C.…”

“…For enzymatic assay, the enzymes were purified as previously described (25). Fractions containing ∼90% pure enzymes were combined and dialyzed overnight at 4°C in buffer containing 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol.…”