ABSTRACT
Delayed cerebellar development is a hallmark of Zellweger syndrome (ZS), a severe neonatal neurodegenerative disorder. ZS is caused by mutations in PEX genes, such as PEX13, which encodes a protein required for import of proteins into the peroxisome. The molecular basis of ZS pathogenesis is not known. We have created a conditional mouse mutant with brain-restricted deficiency of PEX13 that exhibits cerebellar morphological defects. PEX13 brain mutants survive into the postnatal period, with the majority dying by 35 days, and with survival inversely related to litter size and weaning body weight. The impact on peroxisomal metabolism in the mutant brain is mixed: plasmalogen content is reduced, but very-long-chain fatty acids are normal. PEX13 brain mutants exhibit defects in reflex and motor development that correlate with impaired cerebellar fissure and cortical layer formation, granule cell migration and Purkinje cell layer development. Astrogliosis and microgliosis are prominent features of the mutant cerebellum. At the molecular level, cultured cerebellar neurons from E19 PEX13-null mice exhibit elevated levels of reactive oxygen species and mitochondrial superoxide dismutase-2 (MnSOD), and show enhanced apoptosis together with mitochondrial dysfunction. PEX13 brain mutants show increased levels of MnSOD in cerebellum. Our findings suggest that PEX13 deficiency leads to mitochondria-mediated oxidative stress, neuronal cell death and impairment of cerebellar development. Thus, PEX13-deficient mice provide a valuable animal model for investigating the molecular basis and treatment of ZS cerebellar pathology.
Subject(s)
Cerebellum/abnormalities , Cerebellum/embryology , Gliosis/pathology , Membrane Proteins/deficiency , Organogenesis , Oxidative Stress , Zellweger Syndrome/pathology , Animals , Apoptosis , Cell Differentiation , Cell Movement , Cells, Cultured , Cerebellum/pathology , Cerebellum/physiopathology , Disease Models, Animal , Gliosis/complications , Gliosis/physiopathology , Membrane Proteins/metabolism , Mice , Mice, Neurologic Mutants , Motor Activity , Mutation/genetics , Neurons/metabolism , Neurons/pathology , Organ Specificity , Purkinje Cells/metabolism , Purkinje Cells/pathology , Reflex , Survival Analysis , Zellweger Syndrome/complications , Zellweger Syndrome/physiopathologyABSTRACT
(+/-)-4,5-Bis-epi-neovibsanin A and B were synthesized in 12 steps. The acid-catalyzed, one-pot, five-step cascade reaction was central toward the formation of the tricyclic core. The two diastereomers of natural neovibsanin A and B acted as desirable derivatives for structure-activity relationship studies to probe neurotrophic activity. Both (+/-)-4,5-bis-epi-neovibsanin A and B strongly potentiate neurite outgrowth in NGF-stimulated PC12 cells. Furthermore, the tricyclic core appears to be largely responsible for promoting a biological response.
Subject(s)
Diterpenes/chemical synthesis , Diterpenes/pharmacology , Nerve Growth Factor/chemical synthesis , Nerve Growth Factor/pharmacology , Neurites/physiology , Animals , Diterpenes/chemistry , Nerve Growth Factor/chemistry , PC12 Cells , Rats , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The Cre/loxP-system has become an invaluable tool for the generation of tissue-specific gene disruption in mice. However, because Cre recombinase excision of individual genes can be variable, an accurate and sensitive method is necessary to determine the ultimate level of gene disruption. The analysis of gene disruption is particularly difficult for tissue that has been fixed for (immuno)histochemical analysis with paraformaldehyde. Here, we describe a simple, rapid and cost effective method for measurement of gene disruption using quantitative real-time PCR, through application to the analysis of PEX13 gene disruption in a brain-specific PEX13 mouse mutant. We show that this general protocol is suitable for both normal and paraformaldehyde-fixed tissue.
