Analysis of microtubule-severing enzymes in the nervous system of Mus musculus (Linnaeus, 1754)

Abstract

Microtubules are dynamic, polymeric structures of the eukaryotic cytoskeleton with diverse physiological functions. In recent years there has been increasing evidence of their essential role in the synaptic plasticity of the nervous system. Severing by microtubule-severing enzymes is a mechanism to cleave microtubules internally, thus contributing to their dynamics. However, the contribution of severing to synaptic plasticity is still mainly hypothetical, which is also due to the lack of suitable in vivo models. In the present work general and conditional "loss-of-function" mutants of the genes for the two microtubule-cutting enzymes spastin and katanin were generated in the mouse model. Using homologous recombination and by applying the "knockout-first" strategy, critical exons were flanked by loxP sites, so that by using Cre-recombinase a frameshift could arise. Using the gene-trap LacZ reporter in heterozygous knockout animals, the endogenous expression of the two genes spastin and katanin has been studied during the development of the mouse in different tissues. A strikingly weak promoter activity was observed for both genes, which further decreased with increasing age. The expression of the two genes was also analyzed by Western blotting in different tissues. For spastin, both brain-specific, as well as general knockouts could be produced. A general homozygous katanin knockout was embryonic lethal and only a conditional knockout could be achieved with the use of a tissue-specific promoter in the brain. An initial characterization of the general mutants for spastin showed evidence of a potential role of the gene in the CA1 region of the hippocampus. Furthermore, the animals displayed a reduced body weight, an impaired motor function and were sterile. In the first instance, no affectation of microtubule spine entry could be observed using EB3 live-cell imaging in cultured hippocampal neurons. Using the CamKIIα promoter, postnatal and forebrain-specific katanin knockouts could be generated and initially analyzed biochemically, and in the biology of behavior. Here, a possible role for katanin on long-term memory was found.