Tsutomu Hashikawa

CHIP Is Associated with Parkin, a Gene Responsible for Familial Parkinson's Disease, and Enhances Its Ubiquitin Ligase Activity

Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity.

Retrograde modulation of presynaptic release probability through signaling mediated by PSD-95–neuroligin

The structure and function of presynaptic and postsynaptic components of the synapse are highly coordinated. How such coordination is achieved and the molecules involved in this process have not been clarified. Several lines of evidence suggest that presynaptic functionalities are regulated by retrograde mechanisms from the postsynaptic side. We therefore sought postsynaptic mechanisms responsible for trans-synaptic regulation of presynaptic function at excitatory synapses in rat hippocampal CA1 pyramidal neurons. We show here that the postsynaptic complex of scaffolding protein PSD-95 and neuroligin can modulate the release probability of transmitter vesicles at synapse in a retrograde way, resulting in altered presynaptic short-term plasticity. Presynaptic β-neurexin serves as a likely presynaptic mediator of this effect. Our results indicate that trans-synaptic protein-protein interactions can link postsynaptic and presynaptic function.

Tau filament formation and associative memory deficit in aged mice expressing mutant (R406W) human tau

The R406W tau mutation found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) causes a hereditary tauopathy clinically resembling Alzheimers disease. Expression of modest levels of the longest human tau isoform with this mutation under the control of the α-calcium–calmodulin-dependent kinase-II promoter in transgenic (Tg) mice resulted in the development of congophilic hyperphosphorylated tau inclusions in forebrain neurons. These inclusions appeared as early as 18 months of age. As with human cases, tau inclusions were composed of both mutant and endogenous wild-type tau, and were associated with microtubule disruption and flame-shaped transformations of the affected neurons. Straight tau filaments were recovered from Sarkosyl-insoluble fractions from only the aged Tg brains. Behaviorally, aged Tg mice had associative memory impairment without obvious sensorimotor deficits. Therefore, these mice that exhibit a phenotype mimicking R406W FTDP-17 provide an animal model for investigating the adverse properties associated with this mutation, which might potentially recapitulate some etiological events in Alzheimers disease.

Magnetic Resonance Imaging of Neuronal Connections in the Macaque Monkey

Recently, an MRI-detectable, neuronal tract-tracing method in living animals was introduced that exploits the anterograde transport of manganese (Mn2+). We present the results of experiments simultaneously tracing manganese chloride and wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) to evaluate the specificity of the former by tracing the neuronal connections of the basal ganglia of the monkey. Mn2+ and WGA-HRP yielded remarkably similar and highly specific projection patterns. By showing the sequential transport of Mn2+ from striatum to pallidum-substantia nigra and then to thalamus, we demonstrated MRI visualization of transport across at least one synapse in the CNS of the primate. Transsynaptic tract tracing in living primates will allow chronic studies of development and plasticity and provide valuable anatomical information for fMRI and electrophysiological experiments in primates.

TONOTOPIC ORGANIZATION OF AUDITORY CORTICAL FIELDS DELINEATED BY PARVALBUMIN IMMUNOREACTIVITY IN MACAQUE MONKEYS

Tonotopic maps, obtained from single and multi‐unit recordings in the primary and surrounding areas of the auditory cortex, were related to chemoarchitecture of the supratemporal plane, as delineated by immunoreactivity for parvalbumin. Neurons in the central core were sharply tuned and formed two complete tonotopic representations corresponding to the primary auditory area (AI) and the rostral (R) area. High frequencies were represented posteriorly in AI and anteriorly in R, the representation reversing in the anterior part of the core. Neurons in regions of less dense immunostaining previously described as lateral (L) and posteromedial (P‐m) fields, showed broader frequency tuning. Two tonotopic representations were found in L: in an anterolateral (AL) field, corresponding to a field previously reported by others, high frequencies were represented anteriorly and low frequencies posteriorly; in a posterolateral field (PL) the trend reversed. There was a further reversal on entering P‐m from the high frequency representation in PL and progressively lower frequencies tended to be represented more medially in P‐m, but P‐m may contain two representations reported by others.

Distal Extension of Climbing Fiber Territory and Multiple Innervation Caused by Aberrant Wiring to Adjacent Spiny Branchlets in Cerebellar Purkinje Cells Lacking Glutamate Receptor δ2

Organized synapse formation on to Purkinje cell (PC) dendrites by parallel fibers (PFs) and climbing fibers (CFs) is crucial for cerebellar function. In PCs lacking glutamate receptor δ2 (GluRδ2), PF synapses are reduced in number, numerous free spines emerge, and multiple CF innervation persists to adulthood. In the present study, we conducted anterograde and immunohistochemical labelings to investigate how CFs innervate PC dendrites under weakened synaptogenesis by PFs. In the GluRδ2 knock-out mouse, CFs were distributed in the molecular layer more closely to the pial surface compared with the wild-type mouse. Serial electron microscopy demonstrated that CFs in the knock-out mouse innervated all spines protruding from proximal dendrites of PCs, as did those in the wild-type mouse. In the knock-out mouse, however, CF innervation extended distally to spiny branchlets, where nearly half of the spines were free of innervation in contrast to complete synapse formation by PFs in the wild-type mouse. Furthermore, from the end point of innervation, CFs aberrantly jumped to form ectopic synapses on adjacent spiny branchlets, whose proximal portions were often innervated by different CFs. Without GluRδ2, CFs are thus able to expand their territory along and beyond dendritic trees of the target PC, resulting in persistent surplus CFs by innervating the distal dendritic segment. We conclude that GluRδ2 is essential to restrict CF innervation to the proximal dendritic segment, by which territorized innervation by PFs and CFs is properly structured and the formation of excess CF wiring to adjacent PCs is suppressed.

Mitochondrial protease Omi/HtrA2 enhances caspase activation through multiple pathways.

AbstractOmi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis and promotes cytochrome c (Cyt c)dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs) via its IAP-binding motif. The protease activity of Omi/HtrA2 also contributes to the progression of both apoptosis and caspase-independent cell death. In this study, we found that wild-type Omi/HtrA2 is more effective at caspase activation than a catalytically inactive mutant of Omi/HtrA2 in response to apoptotic stimuli, such as UV irradiation or tumor necrosis factor. Although similar levels of Omi/HtrA2 expression, XIAP-binding activity, and Omi/HtrA2 mitochondrial release were observed among cells transfected with catalytically inactive and wild-type Omi/HtrA2 protein, XIAP protein expression after UV irradiation was significantly reduced in cells transfected with wild-type Omi/HtrA2. Recombinant Omi/HtrA2 was observed to catalytically cleave IAPs and to inactivate XIAP in vitro, suggesting that the protease activity of Omi/HtrA2 might be responsible for its IAP-inhibiting activity. Extramitochondrial expression of Omi/HtrA2 indirectly induced permeabilization of the outer mitochondrial membrane and subsequent Cyt c-dependent caspase activation in HeLa cells. These results indicate that protease activity of Omi/HtrA2 promotes caspase activation through multiple pathways.

Differential expression of γ-aminobutyric acid type B receptor-1a and -1b mRNA variants in GABA and non-GABAergic neurons of the rat brain

To understand the heterogeneity of γ‐aminobutyric acid type B receptor (GABABR)‐ mediated events, we investigated expression of GABABR1a and 1b mRNA variants in GABA and non‐GABAergic neurons of the rat central nervous system (CNS), by using nonradioactive in situ hybridization histochemistry and, in combination with GABA immunocytochemistry, double labeling. In situ hybridization with a pan probe, which recognizes a common sequence of both GABABR1a and GABABR1b mRNA variants, demonstrated widespread expression of GABABR1 mRNA at various levels in the CNS. Both GABABR1a and GABABR1b were expressed in the neocortex, hippocampus, dorsal thalamus, habenula, and septum, but only GABABR1a was detected in cerebellar granule cells, in caudate putamen, and most hindbrain structures. A majority of GABA neurons in cerebral cortex showed hybridization signals for both GABABR1a and GABABR1b, whereas those in most subcortical structures expressed either or neither of the two. GABA neurons in thalamic reticular nucleus and caudate putamen hybridized primarily for GABABR1a. Purkinje cells in the cerebellar cortex expressed predominantly GABABR1b. GABA neurons in dorsal lateral geniculate nucleus did not display significant levels of either GABABR1a or GABABR1b mRNAs. These data suggested widespread availability of GABABR‐mediated inhibition in the CNS. The differential but overlapping expression of GABABR1 mRNA variants in different neurons and brain structures may contribute to the heterogeneity of GABABR‐mediated inhibition. Some GABA neurons possessed, but others might lack the molecular machinery for GABABR‐mediated disinhibition, autoinhibition, or both. J. Comp. Neurol. 416:475–495, 2000.

Formation of filamentous tau aggregations in transgenic mice expressing V337M human tau

Formation of neurofibrillary tangles (NFTs) is the most common feature in several neurodegenerative diseases, including Alzheimers disease (AD). Here we report the formation of filamentous tau aggregations having a beta-sheet structure in transgenic mice expressing mutant human tau. These mice contain a tau gene with a mutation of the frontotemporal dementia parkinsonism (FTDP-17) type, in which valine is substituted with methionine residue 337. The aggregation of tau in these transgenic mice satisfies all histological criteria used to identify NFTs common to human neurodegenerative diseases. These mice, therefore, provide a preclinical model for the testing of therapeutic drugs for the treatment of neurodegenerative disorders that exhibit NFTs.

Visualization of synaptic Ca2+ /calmodulin-dependent protein kinase II activity in living neurons.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the CNS and critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, because of a lack of specific methods. We tried to address this by optically detecting the conformational change in CaMKII during activation using fluorescence resonance energy transfer (FRET). The engineered FRET probe Camuiα detects calmodulin binding and autophosphorylation at threonine 286 that renders the enzyme constitutively active. In combination with two-photon microscopy, we demonstrate that Camuiα can be used to observe temporal and spatial regulation of CaMKII activity in living neurons.