The Possible Linkage Between Two Pathological Causes Of Neurodegenerative Disease

Neurodegenerative diseases place an increasing medical and social burden on developed societies,whose populations have ever-increasing numbers of elderly individuals.However,there is lack of effective treatment methods for these diseases.Therefore,in-depth exploration of the mechanism of neurodegenerative diseases and the search for new therapeutic targets have become the key to the development of neurodegenerative diseases treatment.Posttranslational modifications of tubulin are emerging regulators of microtubule functions.Tubulin glutamylation is the most abundant tubulin modification in the adult mammalian brain where it increases during postnatal neuronal maturation.Microtubules（MTs）are building blocks of the cytoskeletal framework of all eukaryotes and are formed by hetero-dimerization ofα-andβ-tubulin.When tubulin dimers polymerize into microtubules（MTs）,the C-terminal tails of tubulin become exposed to the outer surface of the tubules.The C-terminus of tubulin have a variety of protein post-translational modifications（PTMs）,including polyglutamylation etc.In polyglutamylation,a glutamate residue is enzymatically linked to theγ-carboxyl group of a glutamate in the primary sequence of protein substrates.Additional glutamates are then sequentially added viaα-carboxyl–linkages to the growing glutamate side chain.The formation of polyglutamate chains are catalyzed by TTLL（Tubulin Tyrosine Ligase Like,TTLL）family members.In contrast,CCP（cytosolic carboxy peptidase）family members catalyze the removal of glutamate side chains.Mutation of Agtpbp1,the gene encoding CCP1,is the cause of the recessive mutant Purkinje cell degeneration（pcd）mice.The pcd mice exhibit Purkinje cell degeneration leading to the ataxia phenotype.It was found that loss of CCP1 function in pcd mice caused a significant increase in cerebellar tubulin polyglutamylation.In Hela cells,it was found that the longer polyglutamylate side chains induced by TTLL6 activated the microtubule severing activity of spastin,therefore leading to the microtubule instability.Spastin protein is encoded by the Spast gene,which is the most common pathological cause of neurodegenerative disease-hereditary spastic paraplegia（HSP）.Although the level of cerebellar tubulin polyglutamylation increases significantly in pcd mice due to CCP1 loss-of-function,whether this tubulin hyperglutamylation is the cause of Purkinje cells degeneration in pcd mice remains unknown.Excessive tubulin polyglutamylation can activate the activity of microtubule-severing enzyme spastin and reduce the stability of microtubules in vitro.Is this mechanism the cause of neurodegeneration in pcd mice?If it is true,knocking out Spast in pcd mice should rescue the phenotypes.To address the issue,we generated double mutant Spast-KO/pcd（DKO）mice in this study.It was found that spastin knockout did not rescue the motor deficiency and Purkinje cell loss in pcd mice.Therefore,these findings demonstrated that stimulated spastin severing activity by hyperglutamylation of tubulin was not the cause of neurodegeneration in pcd mice.In addition to spastin,two other microtubule severing enzymes—katanin and fidgetin are also present in the nervous system.It is reported that katanin can also be regulated by microtubule polyglutamylation.To assess whether the unrescued Purkinje cell loss and motor deficiency in DKO mice may result from a functional compensation of katanin and fidgetin,the expression levels of katanin and fidgetin were detected by q RT-PCR.The results showed that compared with wild-type mice,the m RNA level of katanin and fidgetin in cerebellum and other brain tissues of Spast^-/-and DKO mice did not have significant change.Interestingly,the fidgetin m RNA level in the spinal cord of DKO mice was reduced.However,whether excessive polyglutamylation of the cerebellar microtubules in pcd mice leads to increased microtubule severing activity of spastin has not yet been proved.So,this study intends to test the severing activity of spastin towards microtubules purified from the cerebellum of pcd mice in vitro.The truncated mouse spastin（His-GST-Spastin-C389）fusion protein and its mutation（His-GST-Spastin-C389-E439A）have been expressed in E.coli cells and purified in vitro.The truncated spastin has been proved to have ATPase activity and mutated version were inactive by in vitro experiments.In addition,we have established the tubulin purification method from mouse brain using TOG affinity column.These works provide a practical basis for the future spastin microtube severing assays.