β-Secretase, BACE1 is a neuron-specific membrane-associated protease that cleaves amyloid precursor protein (APP) to generate β-amyloid protein (Aβ). BACE1 is partially localized in lipid rafts. We investigated whether lipid raft localization of BACE1 affects Aβ production in neurons using a palmitoylation-deficient mutant and further analyzed the relationship between palmitoylation of BACE1 and its shedding and dimerization. We initially confirmed that BACE1 is mainly palmitoylated at four C-terminal cysteine residues in stably transfected neuroblastoma cells. We found that raft localization of mutant BACE1 lacking the palmitoylation modification was markedly reduced in comparison to wild-type BACE1 in neuroblastoma cells as well as rat primary cortical neurons expressing BACE1 via recombinant adenoviruses. In primary neurons, expression of wild-type and mutant BACE1 enhanced production of Aβ from endogenous or overexpressed APP to similar extents with the β-C-terminal fragment (β-CTF) of APP mainly distributed in nonraft fractions. Similarly, β-CTF was recovered mainly in nonraft fractions of neurons expressing Swedish mutant APP only. These results show that raft association of BACE1 does not influence β-cleavage of APP and Aβ production in neurons, and support the view that BACE1 cleaves APP mainly in nonraft domains. Thus, we propose a model of neuronal Aβ generation involving mobilization of β-CTF from nonraft to raft domains. Additionally, we obtained data indicating that palmitoylation plays a role in BACE1 shedding but not dimerization.
Guanosine triphosphate cyclohydrolase I (GCH1) mutations are associated with increased risk for dopa-responsive dystonia (DRD) and Parkinson's disease (PD). Herein, we investigated the frequency of GCH1 mutations and clinical symptoms in patients with clinically diagnosed PD and DRD. We used the Sanger method to screen entire exons in 268 patients with PD and 26 patients with DRD, with the examinations of brain magnetic resonance imaging scans, striatal dopamine transporter scans, and [I] metaiodobenzylguanidine (MIBG) myocardiac scintigraphy scans. We identified 15 patients with heterozygous GCH1 mutations from seven probands and five sporadic cases. The prevalence of GCH1 mutations in probands was different between PD [1.9% (5/268)] and DRD [26.9% (7/26)] (p value < 0.0001). The onset age tends to be different between PD and DRD patients: 35.4 ± 25.3 and 16.5 ± 13.6, respectively (average ± SD; p = 0.08). Most of the patients were women (14/15). Dystonia was common symptom, and dysautonomia and cognitive decline were uncommon in our PD and DRD. All patients presented mild parkinsonism or dystonia with excellent response to levodopa. Seven of seven DRD and three of five PD presented normal heart-to-mediastinum ratio on MIBG myocardial scintigraphy. Five of six DRD and three of four PD demonstrated normal densities of dopamine transporter. Our findings elucidated the clinical characteristics of PD and DRD patients due to GCH1 mutations. PD patients with GCH1 mutations also had different symptoms from those seen in typical PD. The patients with GCH1 mutations had heterogeneous clinical symptoms.
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