Fukutin regulates tau phosphorylation and synaptic function: Novel properties of fukutin in neurons

Fukutin, a product of the causative gene of Fukuyama congenital muscular dystrophy (FCMD), is known to be responsible for basement membrane formation. Patients with FCMD exhibit not only muscular dystrophy but also central nervous system abnormalities, including polymicrogyria and neurofibrillary tangles (NFTs) in the cerebral cortex. The formation of NFTs cannot be explained by basement membrane disorganization. To determine the involvement of fukutin in the NFT formation, we performed molecular pathological investigations using autopsied human brains and cultured neurons of a cell line (SH-SYSY). In human brains, NFTs, identified with an antibody against phosphorylated tau (p-tau), were observed in FCMD patients but not age-matched control subjects and were localized in cortical neurons lacking somatic immunoreactivity for glutamic acid decarboxylase (GAD), a marker of inhibitory neurons. In FCMD brains, NFTs were mainly distributed in lesions of polymicrogyria. Immunofluorescence staining revealed the colocalization of immunoreactivities for p-tau and phosphorylated glycogen synthase kinase-3 beta (GSK-3 beta), a potential tau kinase, in the somatic cytoplasm of SH-SYSY cells; both the immunoreactivities were increased by fukutin knockdown and reduced by fukutin overexpression. Western blot analysis using SH-SYSY cells revealed consistent results. Enzyme-linked immunosorbent assay (ELISA) confirmed the binding affinity of fukutin to tau and GSK-3 beta in SH-SYSY cells. In the human brains, the density of GAD-immunoreactive neurons in the frontal cortex was significantly higher in the FCMD group than in the control group. GAD immunoreactivity on Western blots of SH-SYSY cells was significantly increased by fukutin knockdown. On immunofluorescence staining, immunoreactivities for fukutin and GAD were colocalized in the somatic cytoplasm of the human brains and SH-SYSY cells, whereas those for fukutin and synaptophysin were colocalized in the neuropil of the human brains and the cytoplasm of SH-SYSY cells. ELISA confirmed the binding affinity of fukutin to GAD and synaptophysin in SH-SYSY cells. The present results provide in vivo and in vitro evidence for novel properties of fukutin as follows: (i) there is an inverse relationship between fukutin expression and GSK-3 beta/tau phosphorylation in neurons; (ii) fukutin binds to GSK-3 beta and tau; (iii) tau phosphorylation occurs in non-GAD-immunoreactive neurons in FCMD brains; (iv) neuronal GAD expression is upregulated in the absence of fukutin; and (v) fukutin binds to GAD and synaptophysin in presynaptic vesicles of neurons.