Research On Sodium Channel β4Subunit In Parkinson's Disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder afterAlzheimer's disease (AD). Age is the main risk factor, with onset most frequent in patientsaged more than60years. PD is clinically characterized by bradykinesia, resting tremor,and postural rigidity. As the disease progresses, patients frequently develop cognitiveimpairment and depression. PD results from a relatively selective loss of dopaminergicneurons in the substantia nigra pars compacta, with subsequent loss of striatal dopamineand accompanied by the formation of intraneuronal inclusions called Lewy bodies thatcontain α-synuclein as the major pathological changes.With the rapid development of proteomics and related technologies, many proteinshave been studied, such as parkin and DJ-1, the pathogenesis of PD is still unknown.Protein glycosylation represents the most common and complicated form, it regulatesmany properties of membrane protein, such as targeting, folding, affinity, and cell surfaceexpression. Furthermore, aberrant protein glycosylations have been recognized to playroles in human disorders, including PD. We have identified many glycoproteins isolatedfrom brain tissue and CSF of normal and different stages of PD patients usingglycoproteomics, a branch of proteomics that catalogs and quantifies glycoproteins. Usinga robust shotgun proteomic approach in conjunction with an isotope labeling technique,isobaric tagging for relative and absolute quantification (iTRAQ), we generated a list ofproteins from top-ranked GO categories which was identified from four independentexperiments.Among the top-ranked proteins, the expression of β4subunit was significant increasedin PD patient brain tissues. Herein, we quantitatively compared the protein profiles of braintissues with PD at different pathological stages of disease with age-matched controls. Inthis study, we mainly concentrate on the valdation of sodium channel β4subunit and the biological function of N-linked glycosylation of β4subunit in cultured cells.1. The validation of β4subunit in PD transgenic mice; The total RNA and cDNA wereprepared according to protocol from PD transgentic and wild-type mice, and RT-PCR wasperformed on a real-time PCR machine using a standard SYBR Green PCR kit. Therelative expression of sodium channel β4subunit compared with GAPDH was calculatedusing the2-ΔΔCtmethod. The membrane fractions of brain tissue were assayed usingwestern-blot, the expression of β4was calculated compared with Actin.2. The biological function of glycosylation of sodium channel β4subunit; Themembrane fractions of the brain from different postnatal ages were treated with PNGase F,which cleaves the glycosidic band between the N-acetyglucosamine group and theasparagines resides of N–linked glycoprotein. We also generated the β4plasmid anddeglycosylated plasmid β4-MUT, and then transfect in Neuro2A cells.The neuriteoutgrowth activities induced by β4-WT and β4-MUT in cultured cells were quantifiedusing a confocal microscope.The results from RT-PCR and western blot showed that the expression of sodiumchannel β4subunit was significant higher in PD transgenic mice. Immunoblot analysis ofthe membrane fractions showed that wild-type mice contain only~35kDa band untilpostnatal day7. The β4subunit heavily glycosylated and converted to a faster migratingband after PNGase F treatment. Overexpression of β4-MUT extended neurite length andincreased the number of filopodia-like protrusions than β4-WT and vector in cultured cells.Therefore, we concluded that the expression of sodium channel β4subunit wassignificant higher in PD transgenic mice. The expression of β4increased in neonatal PDtransgenic mice may imply that N-linked oligosaccharides play roles in neuriticdegeneration. Its sialic acid component may regulate cell adhesion and migration as well assodium channel activities.