Tatsuro Koike

Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death.

Typical programmed cell death requires de novo macromolecular synthesis and shares common morphological changes referred to as apoptosis. To elucidate the molecular mechanism of apoptosis, we isolated cDNA clones that are induced in various types of apoptosis by the differential display method. Among such clones, the MA-3 mRNA was induced in all apoptosis-inducible cell lines tested so far, including thymocytes, T cells, B cells and pheochromocytoma. The nucleotide sequence of the MA-3 cDNA predicted an amino acid (aa) sequence of 469 aa, which did not reveal significant similarity to any known proteins and functional aa motifs in databases. The MA-3 mRNA was strongly expressed in the thymus although small amounts of the MA-3 mRNA were ubiquitously expressed in mouse adult tissues. The MA-3 gene was highly conserved during evolution and cross-hybridization bands were found not only in vertebrates but also in Drosophila melanogaster.

Induction of autophagy in neurite degeneration of mouse superior cervical ganglion neurons

Emerging lines of evidence show that the mechanisms of neurite degeneration are convergent, with poor neuritic transport, mitochondrial dysfunction and an increase in intra‐axonal calcium being the principal convergence points. Nevertheless, the details are unclear. Here, we revealed the induction of autophagy in degenerating neurites of sympathetic neuron initiated by three different experimental paradigms. Autophagosomes were colocalized with collapsed cytoskeletal proteins in neuritic beadings during degeneration. Accumulation of microtubule‐associated protein light chain 3‐II, which is the most reliable marker for autophagy, was observed in the early stage of neurite degeneration. The autophagy inhibitor 3‐methyladenine efficiently suppressed neurite degeneration by protecting neurites from the loss of viability and mitochondrial function. Furthermore, knocking down the key autophagy‐related genes Atg7 and Beclin1 significantly delayed axonal and dendritic degeneration after nerve growth factor deprivation. Reduced expression of Atg7 also suppressed neurite fragmentation after transection. Therefore, our present data suggest the critical role of autophagy in neurite degeneration and may provide a valuable clue in understanding the mechanism of axonal and dendritic degeneration.

Non-apoptotic neurite degeneration in apoptotic neuronal death: pivotal role of mitochondrial function in neurites.

The length and thinness of neurites render them greatly susceptible to a variety of insults. Accumulating evidence suggests that neurite degeneration is not a passive, but an active and causative, event in some neurodegenerative diseases. Nonetheless, the mechanisms underlying neurite degeneration remain largely unknown. To elucidate the relevant mechanisms, we employed a mutant C57BL/Wld mouse with a unique phenotype of resistance to Wallerian degeneration, and separately analyzed the destruction of cell soma and neurites following treatment with vinblastine, a microtubule-disrupting agent, in superior cervical ganglion neurons. Vinblastine induced macromolecular synthesis-dependent cell death, which was indistinguishable between the wild-type and mutant mice. Evidence for a loss of mitochondrial cytochrome c, caspase activation, and nuclear fragmentation, has indicated that this type of cell death is entirely apoptotic. Consistent with this, the ATP level in the cell soma was well maintained and indistinguishable between wild-type and mutant mice. In neurites of wild-type neurons, vinblastine induced an early loss of mitochondrial membrane potential (MMP) and ATP depletion preceding caspase-independent degeneration, suggesting that this type of neurite degeneration is principally non-apoptotic. In contrast, neurites of mutant neurons were markedly resistant to vinblastine-induced degeneration, and both the MMP and the ATP content in the neurites were well maintained. Exposure of mutant neurons to carbonyl cyanide m-chlorophenyl-hydrazone, an uncoupler, caused extreme neurite degeneration following rapid MMP loss. Collectively, our findings suggest that: 1) neurite degeneration is regulated through a non-apoptotic process achieved by mitochondrial dysfunction in neurites; 2) the mitochondrial functional status is controlled separately in neurites and in the neuronal soma.

Mammalian Sir2-related protein (SIRT) 2-mediated modulation of resistance to axonal degeneration in slow Wallerian degeneration mice: a crucial role of tubulin deacetylation.

It has been shown that Wallerian degeneration, an anterograde degeneration of transected axons, is markedly delayed in a mutant mouse called slow Wallerian degeneration (Wld(S)). These mice also show resistance to axonal degeneration caused by microtubule depolymerizing drugs, suggesting that axonal microtubules are stabilized. Here, we have focused on tubulin acetylation, a post-translational modification associated with microtubule stability. We found that the basal level of microtubule acetylation was increased in cultured cerebellar granule cells from Wld(S) mice. Nicotinamide but not 3-aminobenzamide, an inhibitor for poly(ADP)ribose polymerase, enhanced tubulin acetylation and resistance to axonal degeneration in cultured cerebellar granule cells from wild-type (WT) mice, suggesting that mammalian Sir2-related protein (SIRT) 2, a nicotinamide adenine dinucleotide (NAD)--dependent tubulin deacetylase, could modulate resistance to axonal degeneration. Indeed, the levels of NAD and SIRT2 were decreased in the cytoplasm from Wld(S) granule cells. Moreover, SIRT2 overexpression abrogated microtubule hyperacetylation and resistance to axonal degeneration in these cells. Conversely, SIRT2 knockdown by using a lentiviral vector expressing small interfering RNA, enhanced microtubule acetylation and resistance to axonal degeneration in WT granule cells. Taken together, these results suggest that SIRT2-mediated tubulin deacetylation is involved in both microtubule hyperacetylation and resistance to axonal degeneration in Wld(S) granule cells.

Upregulation of a New Microglial Gene, mrf-1, in Response to Programmed Neuronal Cell Death and Degeneration

Cerebellar granule neurons isolated from postnatal day 7 (P7) rats and grown in normal K+ medium begin to degenerate at approximately 4 d in vitro (DIV) and die. To search for genes upregulated in the process of neuronal cell death, differential hybridization was performed with subtracted cDNA probes and a cDNA library from 5 DIV. One of the genes isolated was microglial response factor-1 (mrf-1), which encoded a sequence of 177 amino acids with a single EF-hand calcium-binding motif. By Northern blots, the transcript was upregulated in cerebellar culture at 4 DIV, peaked at 6 DIV, and decreased at 7 DIV. Upregulation was also found when the apoptosis of granule cells was induced by replacing high K+ medium with normal K+ medium. However, when non-neuronal cells were thoroughly eliminated with aphidicolin, an antimitotic agent, the upregulation at 4–7 DIV did not occur. By immunocytochemistry, MRF-1 was detected at 5 DIV in OX-42-positive cells (microglia), and it exhibited an increase in response to granule cell death. MRF-1 levels in microglia purified from cerebral cortex also upregulated in the presence of 5 DIV granule cells. In the developing cerebellum in vivo, levels ofmrf-1 mRNA transiently increased in the early postnatal stages, reaching a peak at P7 when cerebellar neurons and astrocytes undergo extensive apoptosis. In adult brain sections, MRF-1 was detected in the perikarya and processes of ramified/resting microglia, and peripheral motor nerve dissection prominently increased the expression in activated microglia surrounding injured central motoneurons. Therefore, mrf-1 appears to be one of the microglial genes that respond to neuronal cell death and degeneration.

Calpain‐mediated cleavage of collapsin response mediator protein(CRMP)‐2 during neurite degeneration in mice

Axon or dendrite degeneration involves activation of the ubiquitin–proteasome system, failure to maintain neuritic ATP levels, microtubule fragmentation and a mitochondrial permeability transition that occur independently of the somal death programs. To gain further insight into the neurite degeneration mechanims we have compared two‐dimensional gel electrophoresis patterns of neurite proteins from suprior cervical ganglia during degeneration caused by nerve growth factor (NGF) deprivation. We show here that collapsin response mediator protein (CRMP)‐2 and CMRP‐4 protein patterns were altered during beading formation, an early hallmark of neurite degeneration, prior to neurite fragmentation, the final stage of degeneration. Western blotting using a monoclonal antibody against CRMP‐2 shows that the native form (64 kDa) was cleaved to generate a truncated form (58 kDa). No cleavage of CRMP‐2 or ‐4 occurred in NGF‐deprived neurites from Wld s (Wallerian degeneration slow) mutant mice in which neurite degeneration is markedly delayed. Using different protease inhibitors, purified calpain 1 protein and calpain 1‐specific siRNA, we have demonstrated that CRMP‐2 is a substrate for calpain 1. Indeed, caplain activity was activated at an early phase of neuronal degeneration in cerebellar granule neurons, and down‐regulation of caplain 1 expression suppressed CRMP‐2 cleavage. Furthermore, this cleavage occurred after vinblastine treatment or in vitro Wallerian degeneration, suggesting that it represents a common step in the process of dying neurites. CRMP‐2 and ‐4 play a pivotal role in axonal growth and transport, and the C‐terminus region of CRMP‐2 is essential for its binding to kinesin‐1. Hence, this cleavage will render them dysfunctional and subject to autophagic processing associated with beading formation, as evidenced by the finding that the truncated form was localized in the beadings.

Rapid induction and Ca2+ influx-mediated suppression of vitamin D3 up-regulated protein 1 (VDUP1) mRNA in cerebellar granule neurons undergoing apoptosis

Cerebellar granule neurons (CGNs) grown under depolarizing conditions with high K+ (HK; 30 mm) undergo apoptosis following replacement of HK by physiological K+ (5.4 mm). Differential display analysis identified eight genes up‐regulated in this paradigm of apoptosis. Vitamin D3 up‐regulated protein 1 (VDUP1) mRNA was markedly up‐regulated as early as 2 h following HK withdrawal. VDUP1 mRNA was up‐regulated in other paradigms of neuronal apoptosis as well both in vitro and in vivo. HK effectively suppressed the up‐regulation of VDUP1 mRNA in CGNs undergoing apoptosis via Ca2+ influx through voltage‐dependent L‐type Ca2+ channels, which did not require de novo protein synthesis. The up‐regulation occurred in parallel with that of the c‐jun transcript and c‐jun protein phosphorylation. Moreover, SB203580, p38 mitogen‐activated protein kinase inhibitor, suppressed up‐regulation of both c‐jun and VDUP1 mRNAs, and c‐jun phosphorylation in CGNs undergoing apoptosis. IGF‐1, one of the neuroprotective agents for CGNs, also inhibited VDUP1 mRNA up‐regulation through a phosphoinositide 3 kinase‐dependent pathway. These results suggest that the VDUP1 gene is a novel member of early response genes in neuronal apoptosis whose expression is directly regulated by Ca2+ influx and coordinately regulated with the transcription factor c‐jun in CGNs.

BRAIN-DERIVED NEUROTROPHIC FACTOR SUPPRESSES PROGRAMMED DEATH OF CEREBELLAR GRANULE CELLS THROUGH A POSTTRANSLATIONAL MECHANISM

Cerebellar granule cells isolated from 7-d-old rats have been shown to die in vitro unless they are continuously exposed to elevated K+ (25 mM). Here we have characterized this neuronal death, and examined whether its major features are shared with those of sympathetic neurons following nerve growth factor (NGF) deprivation. Granule cells underwent active cell death accompanied by morphological features of apoptosis. Brain-derived neurotrophic factor (BDNF), but not NGF, was capable of preventing this neuronal death by acting posttranslationally. Moreover, semiquantitative RT-PCR, Northern blot, and immunoblot analyses showed that trkB, the signal-transducing receptor for BDNF, was upregulated during neuronal death of granule cells in vitro. These results extend recent findings for the role of BDNF in granule cell development, and suggest that BDNF plays a pivotal role on the regulation of the neuronal death/survival of granule cells.

Axon & dendrite degeneration: its mechanisms and protective experimental paradigms.

Accumulating evidence suggests that axon and dendrite (or neurite) degeneration both in vivo and in vitro requires self-destructive programs independent of cell death programs to segregate neurite degeneration from cell soma demise. This review will deal with the mechanisms of neurite degeneration caused by several experimental paradigms including trophic factor deprivation and Wallerian degeneration as well as those under pathological conditions. The involvement of autophagy and mitochondrial dysfunction is emphasized in these mechanisms. The mechanisms through which protective agents including the Wld(s) protein rescue neurites from degeneration or fail to do so will be discussed.

Expression of microglial response factor-1 in microglia and macrophages following cerebral ischemia in the rat

Microglial response factor-1 is a newly isolated microglial gene, which encodes a Ca(2+) binding protein MRF-1 expressed in microglia and macrophages. We induced 1 h of focal cerebral ischemia or 10 min of global cerebral ischemia in the rat, and investigated the expression of MRF-1 immunoreactivity following ischemia. MRF-1 was present in resting microglia and was upregulated in response to microglial activation. MRF-1 was localized to all the cells of the mononuclear phagocyte system (microglia, monocytes, and perivascular cells) that appeared in the ischemic brain.