Hideaki Kume

Reticulons RTN3 and RTN4‐B/C interact with BACE1 and inhibit its ability to produce amyloid β‐protein

β‐Secretase β‐site APP cleaving enzyme 1 (BACE1), is a membrane‐bound aspartyl protease necessary for the generation of amyloid β‐protein (Aβ), which accumulates in the brains of individuals with Alzheimers disease (AD). To gain insight into the mechanisms by which BACE1 activity is regulated, we used proteomic methods to search for BACE1‐interacting proteins in human neuroblastoma SH‐SY5Y cells, which overexpress BACE1. We identified reticulon 4‐B (RTN4‐B; Nogo‐B) as a BACE1‐associated membrane protein. Co‐immunoprecipitation experiments confirmed a physical association between BACE1 and RTN4‐B, RTN4‐C (the shortest isoform of RTN‐4), and their homologue reticulon 3 (RTN3), both in SH‐SY5Y cells and in transfected human embryonic kidney (HEK) 293 cells. Overexpression of these reticulons (RTNs) resulted in a 30–50% reduction in the secretion of both Aβ40 and Aβ42 from HEK293 cells expressing the AD‐associated Swedish mutant amyloid precursor protein (APP), but did not affect Aβ secretion from cells expressing the APP β‐C‐terminal fragment (β‐CTF), indicating that these RTNs can inhibit BACE1 activity. Furthermore, a BACE1 mutant lacking most of the N‐terminal ectodomain also interacted with these RTNs, suggesting that the transmembrane region of BACE1 is critical for the interaction. We also observed a similar interaction between these RTNs and the BACE1 homologue BACE2. Because RTN3 and RTN4‐B/C are substantially expressed in neural tissues, our findings suggest that they play important roles in the regulation of BACE1 function and Aβ production in the brain.

Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family.

By DNA cloning, we have identified the BSRP (brain‐specific receptor‐like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple‐knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase Cα (PKCα) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCα activation in PCs.

Norbin, a neurite-outgrowth-related protein, is a cytosolic protein localized in the somatodendritic region of neurons and distributed prominently in dendritic outgrowth in Purkinje cells

Distribution of norbin protein in rats was characterized by immunohistochemical study. It was distributed not only in whole brain but also in peripheral nervous system. The protein was localized in the somata, except for nuclei, and dendrites of neurons. Subcellular fractionation revealed that norbin is a cytosolic protein. Prominent distribution was observed in dendrites of dendritic outgrowth in Purkinje cells. Norbin should play a role in somatodendritic functions of neurons.

IGF-1 promotes β-amyloid production by a secretase-independent mechanism

Beta-amyloid peptide (Abeta) is generated via the sequential proteolysis of beta-amyloid precursor protein (APP) by beta- and gamma-secretases, and plays a crucial role in the pathogenesis of Alzheimers disease (AD). Here, we sought to clarify the role of insulin-like growth factor-1 (IGF-1), implicated in the AD pathomechanism, in the generation of Abeta. Treatment of neuroblastoma SH-SY5Y cells expressing AD-associated Swedish mutant APP with IGF-1 did not alter cellular levels of APP, but significantly increased those of beta-C-terminal fragment (beta-CTF) and secreted Abeta. IGF-1 also enhanced APP phosphorylation at Thr668. Treatment of beta-CTF-expressing cells with IGF-1 increased the levels of beta-CTF and secreted Abeta. The IGF-1-induced augmentation of beta-CTF was observed in the presence of gamma-secretase inhibitors, but not in cells expressing beta-CTF with a Thr668 to alanine substitution. These results suggest that IGF-1 promotes Abeta production through a secretase-independent mechanism involving APP phosphorylation.

Mutant presenilin (A260V) affects Rab8 in PC12D cell

Most familial early-onset Alzheimers disease (FAD) is caused by mutations in the presenilin-1 (PS1) gene. Abeta is derived from amyloid precursor protein (APP) and an increased concentration of Abeta 42 is widely believed to be a pathological hallmark of abnormal PS function. Therefore, the interaction between PS1 and APP is a central theme in attempts to clarify the molecular mechanism of AD. To examine the effect of PS1 mutations on APP metabolism, we made PC12D cell lines that express human PS1 or mutant PS1 (A260V). In PC12D cells expressing the PS1A260V mutant, we found that Rab8, a GTPase involved in transport from the trans-Golgi network (TGN) to the plasma membrane (PM), was significantly reduced in PC12D cells expressing the A260V mutant and that APP C-terminal fragment (CTF), the direct precursor of Abeta, accumulated in the heavy membrane fraction including membrane vesicles involved in TGN-to-PM transport. Furthermore, the total intracellular Abeta production was reduced in these cells. Combined together, we have observed that PS1 mutation disturbs membrane vesicle transport, resulting in prolonged residence of APP CTF during TGN-to-PM transport pathway. Therefore, it is highly likely that reduction of Abeta is closely related to the retention of APP CTF during TGN-to-PM transport.

Neuronal β-amyloid generation is independent of lipid raft association of β-secretase BACE1: analysis with a palmitoylation-deficient mutant

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.

Expression of reticulon 3 in Alzheimer's disease brain.

Aims: Reticulon 3 (RTN3), a member of the reticulon family of proteins, interacts with the β‐secretase, β‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), and inhibits its activity to produce β‐amyloid protein. The aim of the present study was to clarify the biological role of RTN3 in the brain and its potential involvement in the neuropathology of Alzheimers disease (AD). Methods: We performed immunohistochemical and biochemical analyses using a specific antibody against RTN3 to investigate the expression and subcellular localization of RTN3 in control and AD brain tissue samples. Results: Western blot analysis revealed no significant differences in the RTN3 levels between control and AD brains. Immunohistochemical staining showed that RTN3 immunoreactivity was predominantly localized in pyramidal neurones of the cerebral cortex. The patterns of RTN3 immunostaining were similar in control and AD cerebral cortices, and senile plaques were generally negative for RTN3. Biochemical subcellular fractionation disclosed that RTN3 colocalized with BACE1 in various fractions, including the endoplasmic reticulum and the Golgi apparatus. Double‐immunofluorescence staining additionally indicated that RTN3 was localized in both endoplasmic reticulum and Golgi compartments in neurones. Conclusions: These results show that RTN3 is primarily expressed in pyramidal neurones of the human cerebral cortex and that no clear difference of RTN3 immunoreactivity is observable between control and AD brains. Our data also suggest that there is considerable colocalization of RTN3 with BACE1 at a subcellular level.

The two‐hydrophobic domain tertiary structure of reticulon proteins is critical for modulation of β‐secretase BACE1

β‐Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is a membrane‐bound protease that is essential for the production of β‐amyloid protein (Aβ). Given the crucial role of Aβ accumulation in Alzheimers disease (AD), inhibition of BACE1 activity may represent a feasible therapeutic strategy in the treatment of AD. Recently, we and others identified reticulon 3 (RTN3) and reticulon 4‐B/C (RTN4‐B/C or Nogo‐B/C) as membrane proteins that interact with BACE1 and inhibit its ability to produce Aβ. In this study, we employed various mutants of RTN3 and RTN4‐C and C. elegans RTN to investigate the molecular mechanisms by which RTNs regulate BACE1. We found that RTN3 mutants lacking the N‐terminal or C‐terminal or loop domain as well as a RTN4‐C mutant lacking the C‐terminal domain bound to BACE1 comparably to wild‐type RTN3 and RTN4‐C. Furthermore, overexpression of wild‐type RTN3, RTN4‐C, and these RTN mutants similarly reduced Aβ40 and Aβ42 secretion by cells expressing Swedish mutant APP. C. elegans RTN, which has low homology to human RTNs, also interacted with BACE1 and inhibited Aβ secretion. In contrast, two RTN3 mutants containing deletions of the first or second potential transmembrane domains and an RTN3 swap mutant of the second transmembrane domain bound BACE1 but failed to inhibit Aβ secretion. Collectively, these results suggest that the two‐transmembrane‐domain tertiary structure of RTN proteins is critical for the ability of RTNs to modulate BACE1 activity, whereas N‐terminal, C‐terminal and loop regions are not essential for this function.

Disease-Associated Mutations of TDP-43 Promote Turnover of the Protein Through the Proteasomal Pathway

TAR DNA-binding protein (TDP-43) is a major component of most ubiquitin-positive neuronal and glial inclusions of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A number of missense mutations in the TARDBP gene have been identified in patients with familial and sporadic ALS, as well as familial FTLD with ALS. In the diseased states, TDP-43 proteins exhibit characteristic alterations, including truncation, abnormal phosphorylation, and altered subcellular distribution. However, the mechanisms by which TDP-43 mutations induce neurodegeneration remain unclear at present. In the current study, we analyzed protein turnover and subcellular distribution of wild-type TDP-43 and two disease-associated mutants (G298S and A382T) in human neuroblastoma SH-SY5Y cells stably expressing TDP-43 with a C-terminal tag. Cycloheximide chase experiments revealed more rapid turnover of TDP-43 mutant proteins than their wild-type counterpart. The decrease in the TDP-43 level after cycloheximide treatment was partially recovered upon co-treatment with the proteasome inhibitor, epoxomicin, but not the lysosomotropic agent, chloroquine, suggesting involvement of the proteasomal pathway in TDP-43 degradation. Analysis of the subcellular distribution of TDP-43 revealed predominant localization in the nuclear fraction, whereas the relative level in the cytoplasm remained unaltered in cells expressing either mutant protein, compared with wild-type protein. Our results suggest that higher turnover of disease-associated mutant TDP-43 proteins through the ubiquitin proteasome system is pathogenetically relevant and highlight the significance of proteolysis in the pathogenetic mechanism of TDP-43 proteinopathy.

Molecular Cloning of a Partial cDNA Clone Encoding the C Terminal Region of Chicken Breast Muscle Connectin

Abstract The cDNA sequence encoding the C terminal region of chicken skeletal muscle connectin was described. Its predicted amino acid sequence had 1,021 amino acids comprising six motif lls (immunoglobulin C2 domain) and five interdomains. The sequence showed 70–75% homology with that of human cardiac connectin, but 168 amino acids including one motif II were missing in chicken skeletal muscle connectin. The C terminal sequence of chicken skeletal muscle connectin reported by the previous work (Maruyama et al., 1994) was erroneous due to the accidental ligation of the cDNA clone encoding a N terminal region of connectin with a partial porin cDNA clone.