Haruka Sudo

A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ

Through functional expression screening, we identified a gene, designated Humanin (HN) cDNA, which encodes a short polypeptide and abolishes death of neuronal cells caused by multiple different types of familial Alzheimers disease genes and by Aβ amyloid, without effect on death by Q79 or superoxide dismutase-1 mutants. Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. The rescue action clearly depended on the primary structure of HN. This polypeptide would serve as a molecular clue for the development of new therapeutics for Alzheimers disease targeting neuroprotection.

Acetylation of Microtubules Influences Their Sensitivity to Severing by Katanin in Neurons and Fibroblasts

Here we investigated whether the sensitivity of microtubules to severing by katanin is regulated by acetylation of the microtubules. During interphase, fibroblasts display long microtubules with discrete regions rich in acetylated tubulin. Overexpression of katanin for short periods of time produced breaks preferentially in these regions. In fibroblasts with experimentally enhanced or diminished microtubule acetylation, the sensitivity of the microtubules to severing by katanin was increased or decreased, respectively. In neurons, microtubules are notably more acetylated in axons than in dendrites. Experimental manipulation of microtubule acetylation in neurons yielded similar results on dendrites as observed on fibroblasts. However, under these experimental conditions, axonal microtubules were not appreciably altered with regard to their sensitivity to katanin. We hypothesized that this may be attributable to the effects of tau on the axonal microtubules, and this was validated by studies in which overexpression of tau caused microtubules in dendrites and fibroblasts to be more resistant to severing by katanin in a manner that was not dependent on the acetylation state of the microtubules. Interestingly, none of these various findings apply to spastin, because the severing of microtubules by spastin does not appear to be strongly influenced by either the acetylation state of the microtubules or tau. We conclude that sensitivity to microtubule severing by katanin is regulated by a balance of factors, including the acetylation state of the microtubules and the binding of tau to the microtubules. In the neuron, this contributes to regional differences in the microtubule arrays of axons and dendrites.

Antibody-regulated neurotoxic function of cell-surface β-amyloid precursor protein

APP is a transmembrane precursor of beta-amyloid, and its mutations cause early-onset familial Alzheimers disease. We report a toxic function of normal wild-type APP (wtAPP). Treatment of neuronal F11 cells, immortalized embryonic day 13 neurons, overexpressing wtAPP with anti-APP antibodies caused death. Death was not induced by antibody in parental F11 cells. Death by antibody occurred through cell-surface APP, not through secreted APP, in a pertussis toxin-sensitive manner and was typical apoptosis, not observed in primary astrocytes or glioma cells overexpressing wtAPP, but observed in primary cortical neurons. Cell-surface APP thus performs a toxic function as an extracellularly controllable regulator of neuronal death. This study provides a novel insight into the normal and pathological functions of cell-surface wtAPP.

Strategies for diminishing katanin-based loss of microtubules in tauopathic neurodegenerative diseases

It is commonly stated that microtubules gradually disintegrate as tau becomes dissociated from them in tauopathies such as Alzheimers disease. However, there has been no compelling evidence to date that such disintegration is due to depolymerization of microtubules from their ends. In recent studies, we have shown that neurons contain sufficient levels of the microtubule-severing protein termed katanin to completely break down the axonal microtubule array if not somehow attenuated. The presence of tau on axonal microtubules renders them notably less sensitive to katanin, prompting us to posit that microtubule disintegration in tauopathies may result from elevated severing of the microtubules as they lose tau. In support of this hypothesis, we demonstrate here that pathogenic tau mutants that bind less strongly to microtubules than wild-type tau provide correspondingly less protection against katanin-based severing. Using cultured rat hippocampal neurons, we pursued two potential therapies for fortifying axonal microtubules against excess severing by katanin, under conditions of tau depletion. We found that either deacetylating the microtubules via overexpression of HDAC6 or treating the neurons with NAP, a microtubule-interacting neuroprotective peptide, resulted in notable protection of the microtubules against katanin-based loss. In both cases, we found that these treatments also diminished the characteristic increase in axonal branching that normally accompanies tau depletion, an effect that is also known to be directly related to the severing of microtubules. These observations may be useful in developing therapeutic regimes for preserving microtubules against loss in the axons of patients suffering from tauopathies.

p53-independent apoptosis is induced by the p19ARF tumor suppressor.

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.

LAPSER1/LZTS2: a pluripotent tumor suppressor linked to the inhibition of katanin-mediated microtubule severing

Human chromosome region 10q23-24 is one of the most frequently found regions that show loss of heterozygosity in prostate cancers. A candidate tumor suppressor LAPSER1/LZTS2 (LAPSER1) is located in 10q24.3 that has been reported to be deleted as frequently as the neighboring PTEN locus. We previously reported that LAPSER1 binds p80 katanin, a subunit of the katanin heterodimer. In this report, we show that the LAPSER1 C terminal domain inhibits katanin-mediated microtubule severing in vitro and we detected this inhibition at centrosomes by tracing the nucleated de novo, severed, and transported microtubules in cells. This functional association is also supported by the intracellular localization. Centrosomal localization of LAPSER1 was independent of microtubules and was preferential to mother centrioles. In primary cultured neurons, LAPSER1 also colocalizes with p80 katanin. LAPSER1 alters cell proliferation by regulating cytokinesis. As subcellular mechanisms that underlie the tumor suppressive activity, exogenous LAPSER1 expression inhibited central spindle formation by abrogating microtubule transportation and a similar mode of inhibition was found in axogenesis. Katanin knockdown and dominant negative inhibitor of katanin provided similar phenotypes. Prophase LAPSER1 inhibited centrosomal gamma-tubulin accumulation, which resulted in retardation of mitotic entry. Furthermore, interphase inhibition of katanin by LAPSER1 expression resulted in prevention of cell motility that was accompanied by the increased acetylated microtubules. LAPSER1 knockdown increased cell migration that was inhibited by the expression of ninein, a microtubule release inhibitor. These results indicate that microtubule severing at centrosomes is a novel tumor-associated molecular subcircuit in cells, in which LAPSER1 is a regulator.

Multiple domains of the mouse p19ARF tumor suppressor are involved in p53-independent apoptosis.

The ARF (p19ARF for the mouse ARF consisting of 169 amino acids and p14ARF for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19ARF. We constructed various deletion mutants of p19ARF with a cre/loxP-regulated adenoviral vector to determine the regions of p19ARF which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19ARF whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19ARF1-129, a p19ARF deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14ARF did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution.

More microtubule severing proteins: more microtubules.

Comment on: Sonbuchner TM, et al. Cell Cycle 2010; 9:In this issue.