Peroxisome Deficiency Impairs BDNF Signaling and Memory.

Peroxisome is an intracellular organelle that functions in essential metabolic pathways including ß-oxidation of very-long-chain fatty acids and biosynthesis of plasmalogens. Peroxisome biogenesis disorders (PBDs) manifest severe dysfunction in multiple organs including central nervous system (CNS), whilst the pathogenic mechanisms are largely unknown. We recently reported that peroxisome-deficient neural cells secrete an increased level of brain-derived neurotrophic factor (BDNF), resulting in the cerebellar malformation. Peroxisomal functions in adulthood brain have been little investigated. To induce the peroxisome deficiency in adulthood brain, we here established tamoxifen-inducible conditional Pex2-knockout mouse. Peroxisome deficiency in the conditional Pex2-knockout adult mouse brain induces the upregulated expression of BDNF and its inactive receptor TrkB-T1 in hippocampus, which notably results in memory disturbance. Our results suggest that peroxisome deficiency gives rise to the dysfunction of hippocampal circuit via the impaired BDNF signaling.

Anterograde and retrograde signaling by an Aplysia neurotrophin forms a transsynaptic functional unit.

Whereas short-term synaptic plasticity is often either pre- or postsynaptic, intermediate- and long-term plasticity generally require coordinated pre- and postsynaptic mechanisms. Thus, the transition from presynaptic short-term facilitation (STF) to intermediate-term facilitation (ITF) induced by 5HT at Aplysia sensory-to-motor neuron synapses requires the recruitment of postsynaptic mechanisms and activation of protein synthesis in both neurons. In the companion paper to this report, we found that presynaptic autocrine signaling by an Aplysia neurotrophin (ApNT) forms a positive feedback loop that drives the synapses from STF to ITF. Here we report that ApNT also acts through both anterograde and retrograde signaling to form a transsynaptic positive feedback loop that orchestrates cellular functions in both the presynaptic and postsynaptic neurons during the induction of ITF. These two feedback loops activate protein synthesis in each synaptic compartment, which in both cases depends on signaling from the other synaptic compartment. These results suggest that the pre- and postsynaptic compartments act as one functional unit during the consolidation of learning-related facilitation induced by 5HT.

Autocrine signaling by an Aplysia neurotrophin forms a presynaptic positive feedback loop.

Whereas short-term plasticity is often initiated on one side of the synapse, long-term plasticity involves coordinated changes on both sides, implying extracellular signaling. We have investigated the possible signaling role of an Aplysia neurotrophin (ApNT) in facilitation induced by serotonin (5HT) at sensory-to-motor neuron synapses in culture. ApNT is an ortholog of mammalian BDNF, which has been reported to act as either an anterograde, retrograde, or autocrine signal, so that its pre- and postsynaptic sources and targets remain unclear. We now report that ApNT acts as a presynaptic autocrine signal that forms part of a positive feedback loop with ApTrk and PKA. That loop stimulates spontaneous transmitter release, which recruits postsynaptic mechanisms, and presynaptic protein synthesis during the transition from short- to intermediate-term facilitation and may also initiate gene regulation to trigger the transition to long-term facilitation. These results suggest that a presynaptic ApNT feedback loop plays several key roles during consolidation of learning-related synaptic plasticity.

Early-life Photoperiod Influences Depression-like Behavior, Prepulse Inhibition of the Acoustic Startle Response, and Hippocampal Astrogenesis in Mice.

Environmental factors during early life stages affect behavioral and physiological phenotypes in adulthood. We examined the effect of photoperiods during development on neurogenesis and affective behaviors during adolescence/adulthood using C57BL/6J mice. Mice were born and raised until weaning under long-day conditions (LDs) or short-day conditions (SDs), followed by a 12L12D cycle until adulthood. Adult mice born under SD showed a shorter latency to first immobility in the forced swim test when compared with the mice born under LD. The mice born under SD also exhibited significantly lower prepulse inhibition, which is a characteristic of schizophrenia. However, the mice exposed to SD and LD during the prenatal period only did not show differences in prepulse inhibition. At 4 weeks of age, there were less 5-bromo-2'-deoxyuridine (BrdU)-positive cells in the dentate gyrus (DG) of the hippocampus of mice born under SD when compared with mice born under LD. Double immunostaining showed that the mice born under SD showed less BrdU/glial fibrillary acidic protein (GFAP, an astrocyte marker) cells when compared with mice born under LD. Furthermore, expression of the glucocorticoid receptor in the DG was higher in mice born under SD, and the photoperiod-dependent changes in the number of BrdU-positive cells in the DG were abolished by administration of RU486, a glucocorticoid receptor antagonist. These results suggest that the photoperiod in early life alters astrogenesis in the hippocampus via the hypothalamic-pituitary-adrenal axis and may relate to affective behaviors in adulthood.

An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination.

One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter). Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I-mediated cloning. The method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZα. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells.

VEGF-induced antidepressant effects involve modulation of norepinephrine and serotonin systems.

Throughout life, we are exposed to a variety of stresses, which may be inevitable and noxious sometimes. During evolution, animals must have acquired some physiological means to counteract stress. Vascular endothelial growth factor (VEGF) is an angiogenic and neurogenic factor, which has been shown to elicit antidepressant-like effects in response to different external stimuli, potentially functioning as an anti-stress molecule. However, it remains largely unknown how VEGF modulates mood-related behaviors. To investigate molecular correlates, we analyzed monoaminergic systems of VEGF transgenic mice that display antidepressant-like behavior. Immunostaining showed that overall morphologies of monoaminergic nuclei and their processes were normal. However, we found imbalances in brain monoamine contents, in which the levels of norepinephrine and serotonin, but not dopamine, were decreased exclusively in the regions where VEGF was expressed. The turnover of norepinephrine showed a marked increase and serotonin turnover showed a modest reduction, whereas dopamine turnover was not affected. The protein levels of tyrosine hydroxylase and tryptophan hydroxylase, the rate-limiting enzymes of catecholamine and serotonin synthesis, remained constant. The mRNA levels of monoamine receptors were generally similar but adrenergic receptors of ADRα1A and ADRß1 were down-regulated. Behavioral tests showed that serotonin- or norepinephrine-selective antidepressant drugs failed to additively enhance antidepressant-like behaviors, whereas monoamine depleting drugs attenuated VEGF-mediated antidepressant-like effect. These data suggest that VEGF-induced antidepressant-like effects involve modulation of norepinephrine and serotonin systems.

Behavioral analyses of visually impaired Crx knockout mice revealed sensory compensation in exploratory activities on elevated platforms.

Visual perception is important for acquiring spatial information in many animals, and loss of vision often causes devastating effects on their survival. However, it may be compensated to some extent by utilizing other intact sensory modalities. The cone-rod homeobox (Crx) gene plays a key role in development of photoreceptor cells, but behavioral consequences of the gene deletion have not been well characterized. In this study, we analyzed homozygous knockout (Crx(-/-)) mice by comparing with heterozygous knockout (Crx(+/-)) mice as controls. We first checked their vision with three different behavioral paradigms of the glass table visual recognition test, the light-dark transition test, and the Barnes maze test with a visual cue, all of which indicated that Crx(-/-) mice were blind while Crx(+/-) mice were sighted. In the fear conditioning test, Crx(-/-) mice were able to acquire both contextual and cued memory using non-visual information. Crx(-/-) mice showed normal thigmotaxis, but the exploratory activities were significantly increased. In the elevated plus maze test, it was unexpected that Crx(-/-) mice rarely fell down from the narrow platform. There was no reduction in their moving speeds and the moving distance was rather increased in Crx(-/-) mice. Such behaviors were not affected by trimming their whiskers. However, attachment of earplugs significantly reduced their exploratory activities. In summary, these data suggest that Crx(-/-) mice were behaviorally blind but were able to learn and recognize external environment utilizing non-visual information, as exemplified by sensory compensation in exploratory activities on elevated platforms.

Collagen peptides enhance hippocampal neurogenesis and reduce anxiety related behavior in mice.

The present study examined the effects of enzymatically hydrolyzed collagen peptides on the level of hippocampal neurogenesis and emotional behavior in adult mice. For this purpose, two kinds of enzymatically hydrolyzed collagen peptides, the lower or higher molecular weight peptides (LP: below 2,000, HP: about 30,000) were administered orally to C57BL/6 mice for 4 weeks. As a result, the density of proliferating cells in subgranular zone of hippocampus showed a 1.2-fold increase in LP mice as compared with HP mice. Additionally, LP mice spent less time in closed arms than HP mice in elevated plus maze test to examine anxiety-related behavior. These results suggest that oral administration of the lower molecular weight peptides derived from collagen enhanced the hippocampal neurogenesis and exerted emotional behavior in adult mice.

Spontaneous transmitter release is critical for the induction of long-term and intermediate-term facilitation in Aplysia.

Long-term plasticity can differ from short-term in recruiting the growth of new synaptic connections, a process that requires the participation of both the presynaptic and postsynaptic components of the synapse. How does information about synaptic plasticity spread from its site of origin to recruit the other component? The answer to this question is not known in most systems. We have investigated the possible role of spontaneous transmitter release as such a transsynaptic signal. Until recently, relatively little has been known about the functions of spontaneous release. In this paper, we report that spontaneous release is critical for the induction of a learning-related form of synaptic plasticity, long-term facilitation in Aplysia. In addition, we have found that this signaling is engaged quite early, during an intermediate-term stage that is the first stage to involve postsynaptic as well as presynaptic molecular mechanisms. In a companion paper, we show that spontaneous release from the presynaptic neuron acts as an orthograde signal to recruit the postsynaptic mechanisms of intermediate-term facilitation and initiates a cascade that can culminate in synaptic growth with additional stimulation during long-term facilitation. Spontaneous release could make a similar contribution to learning-related synaptic plasticity in mammals.

Spontaneous transmitter release recruits postsynaptic mechanisms of long-term and intermediate-term facilitation in Aplysia.

Whereas short-term (minutes) facilitation at Aplysia sensory-motor neuron synapses is presynaptic, long-term (days) facilitation involves synaptic growth, which requires both presynaptic and postsynaptic mechanisms. How are the postsynaptic mechanisms recruited, and when does that process begin? We have been investigating the possible role of spontaneous transmitter release from the presynaptic neuron. In the previous paper, we found that spontaneous release is critical for the induction of long-term facilitation, and this process begins during an intermediate-term stage of facilitation that is the first stage to involve postsynaptic as well as presynaptic mechanisms. We now report that increased spontaneous release during the short-term stage acts as an orthograde signal to recruit postsynaptic mechanisms of intermediate-term facilitation including increased IP3, Ca(2+), and membrane insertion and recruitment of clusters of AMPA-like receptors, which may be first steps in synaptic growth during long-term facilitation. These results suggest that the different stages of facilitation involve a cascade of pre- and postsynaptic mechanisms, which is initiated by spontaneous release and may culminate in synaptic growth.

Rapid increase in clusters of synaptophysin at onset of homosynaptic potentiation in Aplysia.

Imaging studies have shown that even the earliest phases of long-term plasticity are accompanied by the rapid recruitment of synaptic components, which generally requires actin polymerization and may be one of the first steps in a program that can lead to the formation of new stable synapses during late-phase plasticity. However, most of those results come from studies of long-term potentiation in rodent hippocampus and might not generalize to other forms of synaptic plasticity or plasticity in other brain areas and species. For example, recruitment of presynaptic proteins during long-term facilitation by 5HT in Aplysia is delayed for several hours, suggesting that whereas activity-dependent forms of plasticity, such as long-term potentiation, involve rapid recruitment of presynaptic proteins, neuromodulatory forms of plasticity, such as facilitation by 5HT, involve more delayed recruitment. To begin to explore this hypothesis, we examined an activity-dependent form of plasticity, homosynaptic potentiation produced by tetanic stimulation of the presynaptic neuron in Aplysia. We found that homosynaptic potentiation involves presynaptic but not postsynaptic actin and a rapid (under 10 min) increase in the number of clusters of the presynaptic vesicle-associated protein synaptophysin. These results indicate that rapid recruitment of synaptic components is not limited to hippocampal potentiation and support the hypothesis that activity-dependent types of plasticity involve rapid recruitment of presynaptic proteins, whereas neuromodulatory types of plasticity involve more delayed recruitment.

Rapid and long-lasting increase in sites for synapse assembly during late-phase potentiation in rat hippocampal neurons.

Long-term potentiation in hippocampal neurons has stages that correspond to the stages of learning and memory. Early-phase (10-30 min) potentiation is accompanied by rapid increases in clusters or puncta of presynaptic and postsynaptic proteins, which depend on actin polymerization but not on protein synthesis. We have now examined changes in pre- and postsynaptic puncta and structures during glutamate-induced late-phase (3 hr) potentiation in cultured hippocampal neurons. We find that (1) the potentiation is accompanied by long-lasting maintenance of the increases in puncta, which depends on protein synthesis, (2) most of the puncta and synaptic structures are very dynamic, continually assembling and disassembling at sites that are more stable than the puncta or structures themselves, (3) the increase in presynaptic puncta appears to be due to both rapid and more gradual increases in the number of sites where the puncta may form, and also to the stabilization of existing puncta, (4) under control conditions, puncta of postsynaptic proteins behave similarly to puncta of presynaptic proteins and share sites with them, and (5) the increase in presynaptic puncta is accompanied by a similar increase in presumably presynaptic structures, which may form at distinct as well as shared sites. The new sites could contribute to the transition between the early and late phase mechanisms of plasticity by serving as seeds for the formation and maintenance of new synapses, thus acting as local "tags" for protein synthesis-dependent synaptic growth during late-phase plasticity.

Postural support by a standing aid alleviating subjective discomfort among cooks in a forward-bent posture during food preparation.

In this study, we evaluated the effects on subjective discomfort among cooks during food preparation through use of a standing aid that we developed to alleviate the workload on the low back in the forward-bent posture. Twelve female cooks who worked in a kitchen in a nursing home were asked to prepare foods in 2 working postures: (a) supported by the standing aid (Aid) and (b) without the aid (No aid). They were instructed to evaluate discomfort in 13-body regions during food preparation and the degree of fatigue at the day's end and to enter their ratings after the end of the workday. Since a significant correlation was observed between body height and the improvement effect of discomfort through use of the standing aid, cooks were divided into two groups according to the height, and ratings were analyzed in each group. Among the tall cooks, subjective discomfort in the low back and the front and back of thighs was significantly less with the Aid posture than with the No aid posture. However, in short cooks these values tended to increase in the Aid posture compared with the No aid posture. The results suggest that the standing aid was effective in alleviating tall cooks' workload on the low back in the forward-bent posture.

Enhanced adult neurogenesis and angiogenesis and altered affective behaviors in mice overexpressing vascular endothelial growth factor 120.

Vascular endothelial growth factor (VEGF) is implicated as a molecular mediator for adult neurogenesis and behavioral effects of antidepressant drugs. However, these potential roles of VEGF in the CNS have not been clarified in model animals. Here we have created transgenic mice overexpressing a short active variant of VEGF-A (VEGF120) in forebrain. Expression of VEGF120 significantly enhanced cell proliferation and angiogenesis, as exemplified by the formation of an enlarged reddish brain. Adult neurogenesis in hippocampus was markedly stimulated without affecting cell differentiation of neural progenitor cells. Hippocampal neurogenesis was particularly robust in young adult animals, but it declined with age and reduced to control levels by 20 weeks under continuous expression of VEGF120. Thus, VEGF alone is not sufficient to support the long-term enhancement of adult neurogenesis, and VEGF-induced vascularization per se does not necessarily predict increased neurogenesis. In transgenic mice, we observed significant changes in affective behaviors. VEGF was found to have not only antidepressant effects but also anxiolytic effects. In addition, we found that VEGF significantly reduced fear and aggression. In contrast, basal activities under natural conditions were not affected much. Unexpectedly, these characteristic behaviors were maintained in older transgenic mice undergoing a reduced level of cell proliferation in hippocampus, suggesting that there is potential dissociation between adult neurogenesis and mood regulation. Our data indicate that VEGF exerts strong neurogenic and angiogenic effects in postnatal brain and influences different forms of affective behaviors.

Participatory ergonomic improvement in nursing home.

The number of nursing home has increased largely in Japan since 1990. The Long-term Care Insurance in 2000 has accelerated the increase of nursing homes. The care giving and cooking in nursing homes have high risk factors of muscle-skeletal diseases (MSDs). However, the working conditions have not yet been improved. Thus, the incidence of low back pain and cervico-brachial disorder is very high among the care workers and cooks. Therefore, it is important to prevent the MSDs among the care workers and cooks. This study has been conducted to make a model of the participatory improvement focusing on low back pain in a nursing home for three years. As a result of the study, many improvements have been implemented and the incidence of low back pain has been reduced.

Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons.

Rho GTPases are key transducers of integrin/extracellular matrix and growth factor signaling. Although integrin-mediated adhesion and trophic support suppress neuronal apoptosis, the role of Rho GTPases in neuronal survival is unclear. Here, we have identified Rac as a critical pro-survival GTPase in cerebellar granule neurons (CGNs) and elucidated a death pathway triggered by its inactivation. GTP-loading of Rac1 was maintained in CGNs by integrin-mediated (RGD-dependent) cell attachment and trophic support. Clostridium difficile toxin B (ToxB), a specific Rho family inhibitor, induced a selective caspase-mediated degradation of Rac1 without affecting RhoA or Cdc42 protein levels. Both ToxB and dominant-negative N17Rac1 elicited CGN apoptosis, characterized by cytochrome c release and activation of caspase-9 and -3, whereas dominant-negative N19RhoA or N17Cdc42 did not cause significant cell death. ToxB stimulated mitochondrial translocation and conformational activation of Bax, c-Jun activation, and induction of the BH3-only protein Bim. Similarly, c-Jun activation and Bim induction were observed with N17Rac1. A c-jun N-terminal protein kinase (JNK)/p38 inhibitor, SB203580, and a JNK-specific inhibitor, SP600125, significantly decreased ToxB-induced Bim expression and blunted each subsequent step of the apoptotic cascade. These results indicate that Rac acts downstream of integrins and growth factors to promote neuronal survival by repressing c-Jun/Bim-mediated mitochondrial apoptosis.

A novel protein specifically interacting with Homer2 regulates ubiquitin-proteasome systems.

Homer family proteins are encoded by three genes, homer1, 2 and 3. Most of these proteins are expressed constitutively in nervous systems and accumulated in postsynaptic regions. However, the functional significance of these proteins, especially the significance of the distinction among the proteins encoded by homer1, 2 and 3, is still obscure. In the present study, we isolated a cDNA clone encoding a novel protein by two-hybrid system screening using the C-terminal half of Homer2b as the bait. This protein, termed 2B28, has 297 amino acid residues and contains three major domains: a UBA domain, a coiled-coil region, and a UBX domain. When expressed in HEK293T cells, 2B28 showed colocalization with uniquitin and enhanced the expression levels of IkappaB or Homer1a proteins, which are known to be degraded by proteasomes, indicating that 2B28 is involved in ubiquitin-proteasome functions. 2B28 specifically interacted and colocalized with Homer2 proteins, but not with Homer1 proteins. So far, we have identified no counterpart of 2B28 for Homer1 experimentally or in the protein databases. These results suggest that the specific interaction of 2B28 with Homer2 may play a role in regulation of protein degradation by ubiquitin-proteasome systems and that this function may be specific to Homer2 proteins among Homer family proteins.

Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons.

Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). We have isolated cDNAs encoding Aplysia Rho, Rac, and Cdc42 and found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities.

Presynaptic and postsynaptic roles of NO, cGK, and RhoA in long-lasting potentiation and aggregation of synaptic proteins.

Recent results suggest that long-lasting potentiation at hippocampal synapses involves the rapid formation of clusters or puncta of presynaptic as well as postsynaptic proteins, both of which are blocked by antagonists of NMDA receptors and an inhibitor of actin polymerization. We have investigated whether the increase in puncta involves retrograde signaling through the NO-cGMP-cGK pathway and also examined the possible roles of two classes of molecules that regulate the actin cytoskeleton: Ena/VASP proteins and Rho GTPases. Our results suggest that NO, cGMP, cGK, actin, and Rho GTPases including RhoA play important roles in the potentiation and act directly in both the presynaptic and postsynaptic neurons, where they contribute to the increase in puncta of synaptic proteins. cGK phosphorylates synaptic VASP during the potentiation, whereas Rho GTPases act both in parallel and upstream of cGMP, in part by maintaining the synaptic localization of soluble guanylyl cyclase.

Activity-inducible protein Homer1a/Vesl-1S promotes redistribution of postsynaptic protein Homer1c/Vesl-1L in cultured rat hippocampal neurons.

In cultured rat hippocampal neurons, overexpression of Homer1a/Vesl-1S, an inducible protein upregulated by seizure or long-term potentiation, caused a reduction of punctate distribution of a postsynaptic protein Homer1c/Vesl-1L, without significant decrease in its total amount. Clusters of F-actin were also decreased. Treatments of cells with BDNF or a proteasome inhibitor, which cause increase in the expression level of endogenous Homer1a, also resulted in the reduction of Homer1c puncta. These results indicate that the accumulation of Homer1a, either exogenously expressed or endogenously induced, caused redistribution and dispersion of postsynaptic clusters of Homer1c and F-actin, suggesting an important role of Homer1a in synaptic remodeling.