Published papers

Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-II-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells.

Two abnormal SOD 1 mRNAs, exon 2-skipping and exon 2 and 3-skipping species, were identified from occipital brain tissue of sporadic amyotrophic lateral sclerosis (ALS) patients carrying no mutations in the SOD 1 gene. Both transcripts were ubiquitously expressed in non-neuronal as well as neuronal tissues from a subject without neurological diseases. The expression pattern did not show disease specificity or lesional selectivity associated with ALS. Transient expression studies revealed weak expression of the proteins derived from the exon 2-skipping SOD 1 cDNA in a cell-free translation system but not in cells. The putative abnormal SOD 1 protein may accumulate and exert toxic effects on motor neurons in ALS when the proteolytic system is disturbed by aging or some causal factors. (C) 2000 Lippincott Williams and Wilkins.

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-α as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-α complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.

By reverse transcription-polymerase chain reaction, Southern blot analysis, direct sequencing, and immunohistochemistry, we studied the expression of cholinergic neuronal markers (choline acetyltransferase [ChAT], vesicular acetylcholine transporter [VAChT], and a high-affinity choline transporter [CHT1]), and gene regulatory molecules (repressor element-1 silencing transcription factor/neuron-restrictive silencer factor [REST/NRSF] and CoREST) in the human spinal cord and term placenta, both of which are well known to contain cells synthesizing acetylcholine. H-type, M-type, N2-type, and R-type ChAT mRNAs, VAChT mRNA, and CHT1 mRNA were detected in the spinal cord, but only H-type, M-type, and N2-type ChAT mRNAs, in the term placenta. REST/NRSF and CoREST were detected in the spinal cord and the placenta, but the amounts of both mRNAs were greater in the placenta than in the spinal cord. Further microdissection analyses revealed that the placental trophoblastic cells contained more REST/NRSF and CoREST transcripts than the spinal large motor neurons. Large motor neurons in the anterior horn of the spinal cord were immunohistochemically stained for ChAT and VAChT. In the placenta, stromal fibroblasts, endothelial cells, and trophoblastic cells of the chorionic villi were positively stained with anti-ChAT antibody but not with anti-VAChT antibody. These findings suggest that transcriptions of the R-type ChAT and VAChT mRNAs are coordinately suppressed in the human term placenta, which might be regulated in part by a REST/NRSF complex that binds to a consensus sequence of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) in the 5′ region upstream from exon R, whereas transcriptions of the H-type, M-type, and N2-type ChAT mRNAs might be independent of control by RE1/NRSE. It is possible that at least two separate regulatory mechanisms of gene expression are present for the human cholinergic gene locus, which might be selected by different combinations of DNA motifs and binding proteins to function in neuronal and non-neuronal cells.

Mutant Cu/Zn-superoxide dismutase (SOD1) protein aggregation has been suggested as responsible for amyotrophic lateral sclerosis (ALS), although the operative mediating factors are as yet unestablished. To evaluate the contribution of motoneuronal Ca2+ -permeable (GluR2 subunit-lacking) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors to SOD1-related motoneuronal death, we generated chat-GluR2 transgenic mice with significantly reduced Ca2+ -permeability of these receptors in spinal motoneurons. Crossbreeding of the hSOD1G93A transgenic mouse model of ALS with chat-GluR2 mice led to marked delay of disease onset (19.5%), mortality (14.3%) and the pathological hallmarks such as release of cytochrome c from mitochondria, induction of cox2 and astrogliosis. Subcellular fractionation analysis revealed that unusual SOD1 species first accumulated in two fractions dense with neurofilaments/ glial fibrillary acidic protein/nuclei and mitochondria long time before disease onset, and then concentrated into the former fraction by disease onset. All these processes for unusual SOD1 accumulation were considerably delayed by GluR2 overexpression. Ca2+ -influx through atypical motoneuronal AMPA receptors thus promotes a misfolding of mutant SOD1 protein and eventual death of these neurons.

In cholinergic neurons, Na+- and Cl--dependent, hemicholinium-3-sensitive, high-affinity choline uptake system is thought to be the rate-limiting step in acetylcholine (ACh) synthesis. The system is highly regulated by neuronal activity; the choline uptake is increased by a condition in which ACh release is favored. Here we analyzed the ultrastructural localization of the high-affinity choline transporter (CHT) in the rat neuromuscular junctions with two separate antibodies. The majority (>90%) of immunogold labeling of CHT was observed on synaptic vesicles rather than the presynaptic plasma membrane. Less than 5% of the gold-silver particles were associated with the plasma membrane, and more than 70% of such particles were localized within or in close vicinity to presynaptic active zones. Our morphological data support the recent hypothesis that trafficking of CHT from synaptic vesicles to the plasma membrane couples neuronal activity and choline uptake.

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.

Basal forebrain cholinergic neurons (BFCNs) are involved in cognitive functions such as learning and memory, and are affected in several neurodegenerative diseases (e.g. Alzheimer's disease). Despite their importance, the molecular mechanisms of their development are not fully elucidated. A recent report demonstrated that some BFCNs in adult rat are positive for L3/Lhx8, a LIM homeobox transcription factor. To examine the function of L3/Lhx8 in the development of BFCNs, we generated L3/Lhx8 gene-disrupted mice. In these mice, cells expressing cholinergic neuron markers, such as choline acetyltransferase, vesicular acetylcholine transporter and p75 low-affinity NGF receptor, were markedly reduced in the basal forebrain, whereas other cholinergic neurons including brain stem and spinal motor neurons expressed the markers. Neurotransmitter phenotypes other than cholinergic in the basal forebrain appeared intact. From these results, we suggested that L3/Lhx8 has a pivotal and specific role in the development and/or maintenance of BFCNs.

Synapses on the motor neurons of patients with Duchenne muscular dystrophy (DMD) and myotonic dystrophy (MD) were studied immunohistochemically using antibodies against synaptobrevin and synaptophysin. Some motor neurons showed hyperproliferation of synapses on the soma and the proximal dendrites. Hyperproliferated synapses were non-cholinergic, because they were not identified with antibodies against vesicular acetylcholine transporter. Regeneration of motor nerve terminals in patients with muscular diseases may affect reorganization of the subsets of central synapses.

Mutant copper/zinc superoxide dismutase (SOD1)-overexpressing transgenic mice, a mouse model for familial amyotrophic lateral sclerosis (ALS), provides an excellent resource for developing novel therapies for ALS. Several observations suggest that mitochondria-dependent apoptotic signaling, including caspase-9 activation, may play an important role in mutant SOD1-related neurodegeneration. To elucidate the role of caspase-9 in ALS, we examined the effects of an inhibitor of X chromosome-linked inhibitor of apoptosis (XIAP), a mammalian inhibitor of caspase-3, -7 and -9, and p35, a baculoviral broad caspase inhibitor that does not inhibit caspase-9. When expressed in spinal motor neurons of mutant SOD1 mice using transgenic techniques, XIAP attenuated disease progression without delaying onset. In contrast, p35 delayed onset without slowing disease progression. Moreover, caspase-9 was activated in spinal motor neurons of human ALS subjects. These data strongly suggest that caspase-9 plays a crucial role in disease progression of ALS and constitutes a promising therapeutic target.

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in -40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre.Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.

Epilepsy is a debilitating disease with a strong genetic component. Positional cloning has identified a few genes for rare monogenic epilepsy syndromes; however, the genetics of common human epilepsies are too complex to be analyzed easily by current techniques. Mouse models of epilepsy can further this analysis by eliminating genetic background heterogeneity and enabling the production of sufficient numbers of offspring. Here, we report that Black Swiss mice have a heretofore unrecognized specific susceptibility to audiogenic seizures. These seizures are characterized by wild running, loss of righting reflex, and tonic flexion and extension, and are followed by a postictal period. The susceptibility to these seizures is developmentally regulated, peaking at 21 d of age and nearly disappearing by adulthood. Interestingly, both the susceptibility to seizures and their developmental regulation appear unrelated to hearing thresholds in the Black Swiss strain and backcrossed progeny. Genetic mapping and linkage analysis of hybrid mice localize the seizure gene, jams1 (juvenile audiogenic monogenic seizures), to a 1.6 ± 0.5 centimorgan (cM) region on mouse chromosome 10, delimited by the gene basigin (Bsg) and marker D10Mit140. Interestingly, the majority of the critical region is syntenic to a region on human chromosome 19p13.3 implicated in a familial form of juvenile febrile convulsions. Cloning the gene for audiogenic seizures in these mice may provide important insight into the fundamental mechanisms for developmentally regulated human epilepsy syndromes.

In cholinergic nerve terminals, Na+- and Cl--dependent, hemicholinium-3-sensitive, high-affinity choline uptake is thought to be the rate-limiting step in acetylcholine synthesis. The high-affinity choline transporter cDNA responsible for the activity was recently cloned. Here we report production of a highly specific antibody to the high-affinity choline transporter and distribution of the protein in the CNS of the rat. The antibody stained almost all known cholinergic neurons and their terminal fields. High-affinity choline transporter-immunoreactive cell bodies were demonstrated in the olfactory tubercle, basal forebrain complex, striatum, mesopontine complex, medial habenula, cranial nerve motor nuclei, and ventral horn and intermediate zone of the spinal cord. Noticeably, high densities of high-affinity choline transporter-positive axonal fibers and puncta were encountered in many brain regions such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem. Transection of the hypoglossal nerve resulted in a loss of high-affinity choline transporter immunoreactivity in neurons within the ipsilateral hypoglossal motor nucleus, which paralleled a loss of immunoreactivity to choline acetyltransferase. The antibody also stained brain sections from human and mouse, suggesting cross-reactivity. These results confirm that the high-affinity choline transporter is uniquely expressed in cholinergic neurons and is efficiently transported to axon terminals. The antibody will be useful to investigate possible changes in cholinergic cell bodies and axon terminals in human and rodents under various pathological conditions.

Previous studies have shown that NGFI-B mRNA is highly expressed in the adult striatum. In the present study we analyzed the anatomical distribution of NGFI-B mRNA within this brain region as well as the degree of co-existence of NGFI-B with different striatal markers in the adult brain. NGFI-B mRNA levels were found to be significantly higher within the dorsomedial portion of the striatum as compared to the ventrolateral striatum. This distribution pattern was maintained throughout the rostro-caudal axis of the striatum. Double in situ hybridization studies showed that striatal NGFI-B mRNA colocalized with a subset of preproenkephalin and prodynorphin positive spiny neurons within the dorsomedial striatum; 22-28% of all opiate-peptide positive cells co-expressed NGFI-B mRNA. NGFI-B did not colocalize with striatal aspiny interneurons expressing choline acetyl transferase mRNA or those containing the calcium-binding protein parvalbumin. The pattern of NGFI-B mRNA expression within different striatal spiny projecting neurons suggests that this transcription factor may have a direct effect on the function of different striatal efferent pathways.

Proteins containing PDZ (postsynaptic density-95, discs large, zonula occludens) domains play a general role in recruiting receptors and enzymes to specific synaptic sites. In Caenorhabditis elegans, a complex of three PDZ proteins, LIN-2/7/10, mediates basolateral targeting of a receptor tyrosine kinase. Homologs of these LIN proteins have also been identified in higher organisms, and here we analyze the MALS/Veli (mammalian LIN-7/vertebrate homolog of LIN-7) proteins in brain. Immunohistochemical staining and in situ hybridization show that MALS occur differentially in discrete populations of neurons throughout the brain. Most neurons express only one MALS protein, although some cells contain two or even all three MALS isoforms. At the subcellular level, MALS proteins are found in both dendritic and axonal locations, suggesting that they may regulate processes at both pre- and postsynaptic sites. Targeted disruption of MALS-1 and MALS-2 does not yield a detectable phenotype, and hippocampal synaptic function and plasticity are intact in the MALS-1/2 double knockouts. Interestingly, MALS-3 protein is dramatically induced in the MALS-1/2 double knockouts, implying that dynamic changes in protein expression may play an important regulatory role for this family of synaptic PDZ proteins.

The κ-opioid receptor agonists including U-50,488H and dynorphin A (1-17) in ranges of 0.1-100 nM inhibited the hydrolysis of GTP to GDP (Pi release) inherent in GTP-binding proteins (G proteins) in guinea pig cerebellar membranes. U-50,488H inhibited only high-affinity GTPase activity, not low-affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations >1 μM. The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations >1 μg/ml but not with preactivated cholera toxin (30 μg/ml). Similar blockade of κ-receptor-mediated inhibition was also observed when membranes were pretreated with a low concentration (8 μM) of N-ethylmaleimide (NEM) at low temperature (4°C), which alkylates the cysteine residue to be ADP-ribosylated by PTX; but this treatment caused no significant change in κ-agonist binding. When purified Gi1, but not Go, was reconstituted into membranes pretreated with NEM, the κ-receptor-mediated inhibition was recovered. These findings suggest that a subtype of κ-opioid receptor is coupled to inhibition of intrinsic activity of Gi1.

Secretion of acetylcholine (ACh) in neuroblastoma cells overexpressing choline acetyltransferase (ChAT) was examined. With transient transfection of ChAT cDNA, neuroblastoma cells, which have no endogenous ChAT and either adhere to myotubes or not, failed to form functional synapses, and thus no evidence for release of ACh was detected. Stable neuroblastoma cell lines overexpressing ChAT accumulated ACh inside the cell, and slowly released ACh to the outside of the cell in a calcium-independent fashion. However, after co-culturing them with rat muscle cells, these transformed cells adhered to myotubes and ACh was secreted in a discrete fashion into the synaptic cleft efficiently in some neuroblastoma cell lines but rather inefficiently in another cell line. The results show that the latent secretion machinery of ChAT overexpressing neuroblastoma cells either is competent or possess defect(s) in ACh release.

Neuroblastoma (clones NS-20Y, N1E-115, and Neuro2A) and neuroblastoma × glioma hybrid (NG108-15) cells were transfected with mouse choline acetyltransferase (ChAT) complementary DNA (cDNA) or vector DNA alone and stably transformed cell lines were established to examine their ability to secrete acetylcholine (ACh). Membrane potentials were recorded from either presynaptic neuroblastoma and hybrid cells or postsynaptic myotubes in co-culture. After transformation with ChAT, synapses were formed and miniature end-plate potentials (MEPPs) were recorded in myotubes co-cultured with Neuro2A and N1E-115 cells, while parental and mock-transfected control cells totally lacked this ability. The rate of synapse formation and/or MEPP frequency was higher in transformed NG108-15 hybrid and NS-20Y cells than that in the control cells. Action potentials of NS-20Y, Neuro2A or NG108-15 cells overexpressing ChAT were able to evoke end-plate potentials in myotubes, though the average quantum content of these cells was 0.04-0.14, which is as low as the control value. The results show that increased concentrations of ACh by ChAT cDNA transfection reveal a masked property in vesicular ACh release from Neuro2A and N1E-115 cells with no endogenous ChAT activity, or modify their secretory capacity upwardly from NG108-15 and NS-20Y cells with endogenous activity.

The neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT) and transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Recently it has been reported that the entire coding region of VAChT mRNA is located in the first intron of the ChAT gene. In this study, ChAT and VAChT mRNAs were analysed in cultured sympathetic neurones. Cholinergic differentiation factor/leukaemia inhibitory factor and ciliary neurotrophic factor induced strong expression of ChAT and VAChT mRNAs in parallel. RT-PCR analysis of ChAT mRNAs revealed that five types of ChAT transcripts which differed in the 5' non coding regions were increased. RT-PCR analysis of VAChT mRNA indicated that the cytokines induced only VAChT mRNA species which did not contain the R-exon, and not those containing the R-exon. The results indicate that ChAT and VAChT expressions are coordinately but differentially regulated in cultured sympathetic neurones.

PLC activity was stimulated either by 1-100 μM of GTP or by 100-3,000 μM Ca2+ in lysed synaptosomal membranes of the guinea pig cerebellum. The κ-opioid receptor agonist selectively inhibited the PLC activity stimulated by 100 μM GTP, but not by 100-3,000 μM Ca2+. Pretreatment of membranes with PTX abolished such a κ-agonist-induced inhibition of PLC activity. The reconstitution of Gi1, but not of Go purified from porcine brains with PTX-treated membranes showed a complete recovery of the κ-agonist-inhibition of PLC activity. These findings suggest that a novel subtype κ-receptor mediates inhibition of PLC through inhibiting the intrinsic activity of PTX-substrate G-proteins.