Naohiko Matsumoto

Regulation of Notch Signaling by Dynamic Changes in the Precision of S3 Cleavage of Notch-1†

ABSTRACT Intramembrane proteolysis by presenilin-dependent γ-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-β. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val→Gly and the Lys→Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.

Major phosphorylation site (Ser55) of neurofilament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture.

Abstract: Ser55 of neurofilament L (NF‐L) is reported to be partly phosphorylated in neurons and to be phosphorylated by cyclic AMP‐dependent protein kinase (PKA). Bovine NF‐L was phosphorylated by PKA in a low concentration of MgCl2 (0.3 mM) and digested by trypsin. Trypsin‐digested fragments were assigned by MALDI/TOF (matrix‐assisted laser desorption and ionization/time‐of‐flight) mass spectrometry. Phosphorylation sites were found at Ser41, Ser55, and Ser62 in the head region, with Ser55 considered the preferred site. A site‐specific phosphorylation‐dependent antibody against Ser55 rendered NF‐L phosphorylated at Ser55 detectable in primary cultured rat neurons. One‐hour treatment with 20 nM okadaic acid increased the phosphorylation level of Ser55, and co‐treatment with 10 μM forskolin enhanced it. However, forskolin alone did not elevate the phosphorylation level. As a consequence, NF‐L may be phosphorylated at Ser55 by PKA or by a PKA‐like kinase in vivo; however, the phosphorylation level of Ser55 may be modulated by certain phosphatases sensitive to okadaic acid.

FAD-linked presenilin-1 mutants impede translation regulation under ER stress.

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.

Localized phosphorylation of vimentin by Rho-kinase in neuroblastoma N2a cells

Vimentin, which is one of the intermediate filaments, is the major cytoskeletal component in developing neurones or neuroblastoma cells. Rho‐associated kinase (Rho‐kinase), is rich in neurones and is found downstream of Rho. It is involved in the agonist‐induced neurite retraction of neuronal cells, and phosphorylates vimentin at Ser‐38 and Ser‐71 resulting in in vitro disassembly of the filaments.

Phosphorylation of neurofilament-L during LTD

We recently reported that CaMKII-dependent phosphorylation of the neurofilament-L (NF-L) head domain was induced in the apical dendrites during long-term potentiation. Long-term depression (LTD) is another cellular model for neuronal plasticity. In the present study, we examined the phosphorylation of NF-L during hippocampal LTD using a series of phospho-specific antibodies against the NF-L head domain. During LTD, these antibodies visualized NF-L phosphorylation at Ser57 in the apical dendrites of the hippocampal pyramidal neurons. The assembly and disassembly of NF-L filaments are regulated by phosphorylation of its head domain. Thus, our results indicated that NF-L phosphorylation might be associated with alterations of the neuronal structure during LTD.

Visual stimulation-induced phosphorylation of neurofilament-L in the visual cortex of dark-reared rats.

In dark‐reared animals, visual exposure is expected to induce drastic changes in both the physiology and anatomy of the cortical neurons, including the rearrangement of their cytoskeletal structures. Phosphorylation of neurofilament‐L (NF‐L) is probably associated with relatively short‐term structural plasticity in vivo, because the assembly and disassembly of the filaments are regulated by phosphorylation of the head domain of NF‐L. Thus, by using a series of site‐ and phosphorylation state‐specific antibodies against NF‐L, we examined how visual activation induces the phosphorylation of NF‐L in the rat brain. We found no specific immunoreactivity for phosphorylated NF‐L in the brain of naive rats, whereas one‐hour ambient light exposure after dark rearing for ten weeks from birth induced marked phosphorylation of NF‐L selectively. Also, the NF‐L phosphorylation was found to be localized in the primary and secondary visual cortical areas. These findings suggest that the selective phosphorylation of NF‐L plays an important role in the structural plasticity related to the visual experience.

Effect of Presenilin 1 Missense Mutation and Aluminum on Early Neuronal Development of the Mouse Brain

Background: The pathology of Alzheimers disease (AD) has not been fully elucidated. Presenilin 1 (PS1) mutation is one of the major genetic factors of AD, while aluminum is a candidate for the environmental risk factor.

Aluminum alters cell viability and axonal transport system in Alzheimer's disease pathogenic mutation‐bearing cells

Background:  We previously reported that pulse exposure of cultured rat neurons to aluminum‐maltol resulted in an abnormal distribution of both neurofilament‐L (NF‐L) and fast axonal transported proteins. It was also found that the presenilin 1 (PS1) missense mutation and aluminum affected early neuronal development of the mouse brain. It has been reported that the presenilin (PS) mutation alters neurite outgrowth, and the axonal transport of the amyloid precursor protein (APP)/PS complex is mediated by kinesin. The present study hypothesizes that aluminum might modulate axonal transport and neurite outgrowth in APP/PS mutant‐bearing cells.

Involvement of Rho‐associated kinase in neurite sprouting and amyloid beta production of rat cortical neurons cultured in insulin‐free medium

Background: Abnormal sprouting of neurites is one of the neuropathological features of Alzheimers disease. The mechanism of neurite sprouting of cultured neurons remains unknown. Among small guanosine triphosphate binding proteins, participation of the Rho family has been speculated.