Increased expression of alpha 1A Ca2+ channel currents arising from expanded trinucleotide repeats in spinocerebellar ataxia type 6.

The expansion of polyglutamine tracts encoded by CAG trinucleotide repeats is a common mutational mechanism in inherited neurodegenerative diseases. Spinocerebellar ataxia type 6 (SCA6), an autosomal dominant, progressive disease, arises from trinucleotide repeat expansions present in the coding region of CACNA1A (chromosome 19p13). This gene encodes alpha(1A), the principal subunit of P/Q-type Ca(2+) channels, which are abundant in the CNS, particularly in cerebellar Purkinje and granule neurons. We assayed ion channel function by introduction of human alpha(1A) cDNAs in human embryonic kidney 293 cells that stably coexpressed beta(1) and alpha(2)delta subunits. Immunocytochemical analysis showed a rise in intracellular and surface expression of alpha(1A) protein when CAG repeat lengths reached or exceeded the pathogenic range for SCA6. This gain at the protein level was not a consequence of changes in RNA stability, as indicated by Northern blot analysis. The electrophysiological behavior of alpha(1A) subunits containing expanded (EXP) numbers of CAG repeats (23, 27, and 72) was compared against that of wild-type subunits (WT) (4 and 11 repeats) using standard whole-cell patch-clamp recording conditions. The EXP alpha(1A) subunits yielded functional ion channels that supported inward Ca(2+) channel currents, with a sharp increase in P/Q Ca(2+) channel current density relative to WT. Our results showed that Ca(2+) channels from SCA6 patients display near-normal biophysical properties but increased current density attributable to elevated protein expression at the cell surface.

Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10; MIM 603516; refs 1,2) is an autosomal dominant disorder characterized by cerebellar ataxia and seizures. The gene SCA10 maps to a 3.8-cM interval on human chromosome 22q13-qter (refs 1,2). Because several other SCA subtypes show trinucleotide repeat expansions, we examined microsatellites in this region. We found an expansion of a pentanucleotide (ATTCT) repeat in intron 9 of SCA10 in all patients in five Mexican SCA10 families. There was an inverse correlation between the expansion size, up to 22.5 kb larger than the normal allele, and the age of onset (r2=0.34, P=0.018). Analysis of 562 chromosomes from unaffected individuals of various ethnic origins (including 242 chromosomes from Mexican persons) showed a range of 10 to 22 ATTCT repeats with no evidence of expansions. Our data indicate that the new SCA10 intronic ATTCT pentanucleotide repeat in SCA10 patients is unstable and represents the largest microsatellite expansion found so far in the human genome.

Repeat instability and motor incoordination in mice with a targeted expanded CAG repeat in the Sca1 locus.

To elucidate the pathophysiology of spinocerebellar ataxia type 1 (SCA1) and to evaluate repeat length instability in the context of the mouse Sca1 gene, we generated knock-in mice by inserting an expanded tract of 78 CAG repeats into the mouse Sca1 locus. Mice heterozygous for the CAG expansion show intergenerational repeat instability (+2 to -6) at a much higher frequency in maternal transmission than in paternal transmission. The majority of changes transmitted through the female germline were small contractions, as in humans, whereas small expansions occurred more frequently in paternal transmission. The frequency of intergenerational changes was age dependent for both paternal and maternal transmissions. Mice homozygous for mutant ataxin-1 on a C57BL/6J-129/SvEv mixed background performed significantly less well on the rotating rod than did wild-type littermates at 9 months of age, although they were not ataxic by cage behavior. Histological examination of brain tissue from mutant mice up to 18 months of age revealed none of the neuropathological changes observed in other transgenic models overexpressing expanded polyglutamine tracts. These data suggest that, even with 78 glutamines, prolonged exposure to mutant ataxin-1 at endogenous levels is necessary to produce a neurological phenotype reminiscent of human SCA1. Pathogenesis is thus a function of polyglutamine length, protein levels and duration of neuronal exposure to the mutant protein.

Amygdala-kindled and pentylenetetrazole-induced seizures in glutamate transporter GLAST-deficient mice.

The glutamatergic system has been shown to be important for the induction of epileptiform activity and the development of epileptogenesis. To investigate the role of the astroglial glutamate transporter GLAST in epileptogenesis, we examined amygdala (AM)-kindled and pentylenetetrazole (PTZ)-induced seizures in GLAST-deficient mice (GLAST(-/-)) and compared them to those observed in wild-type mice (GLAST(+/+)) and maternal C57Black6/J (C57) mice. AM-kindling resulted in no significant differences in afterdischarge threshold or in the seizure responses induced by first stimulation between these groups. In addition, although no significant differences were seen in kindled seizure development, the generalized seizure duration of AM-kindled seizures in GLAST(-/-) mice was significantly prolonged (approximately 35%) compared with that of C57 mice. Furthermore, GLAST(-/-) mice showed more severe stages of PTZ-induced seizures than GLAST(+/+) mice, and the latency to the onset of seizures was significantly shorter for the mutant mice. These results indicate that GLAST is one of factors determining seizure susceptibility.

Optical detection of synaptically induced glutamate transport in hippocampal slices.

Although it has long been believed that glial cells play a major role in transmitter uptake at synapses in the CNS, the relative contribution of glial and neuronal cells to reuptake of synaptically released glutamate has been unclear. Recent identification of the diverse glutamate transporter subtypes provides an opportunity to examine this issue. To monitor glutamate transporter activity, we optically detected synaptically induced changes of membrane potential from hippocampal CA1 field in slice preparations using a voltage-sensitive dye, RH155. In the presence of ionotropic glutamate-receptor blockers, synaptic inputs gave rise to a slow depolarizing response (SDR) in the dendritic field. The amplitude of SDR correlated well with presynaptic activities, suggesting that it was related to transmitter release. The SDR was found to be caused by the activities of glutamate transporters because it was not affected by blockers for GABAA, nACh, 5-HT3, P2X, or metabotropic glutamate receptors but was greatly reduced by dihydrokainate (DHK), a specific blocker for GLT-1 transporter, and by D, L-threo-beta-hydroxyaspartate (THA), a blocker for EAAC, GLAST, and GLT-1 transporters. When SDR was detected with RH482 dye, which stains both glial and neuronal cells, 1 mM DHK and 1 mM THA were equally effective in suppressing SDR. The SDR was very small in GLT-1 knockout mice but was maintained in gerbil hippocampi in which postsynaptic neurons were absent because of ischemia. Because GLT-1 transporters are exclusively expressed in astrocytes, our results provide direct evidence that astrocytes play the dominant role in sequestering synaptically released glutamate.

Functional properties of two bombesin-like peptide receptors revealed by the analysis of mice lacking neuromedin B receptor.

The neuromedin B-preferring receptor (NMB-R) is one of the members of the bombesin (BN)-like peptide receptor subfamily in mammals. Previously, we have generated and characterized mice with targeted disruption of the two other BN-like peptide receptors, bombesin receptor subtype-3 (BRS-3) and gastrin-releasing peptide-preferring receptor (GRP-R). Here we describe the generation and analysis of NMB-R-deficient mice to investigate how NMB-R differs from BRS-3 and GRP-R. Compensation for NMB-R deficiency by overexpression of GRP-R and/or BRS-3 was not detected. Although the hypothermic effect of NMB was reduced by 50% in NMB-R-deficient mice, the effect of GRP infusion was comparable to the wild-type mice. In contrast, fundic smooth muscle contraction on stimulation with NMB or GRP was normal in NMB-R-deficient mice. Administration of GRP but not NMB suppressed glucose intake in both normal and NMB-R-deficient mice. These results suggest that the NMB-R has an essential role in thermoregulation, but not for smooth muscle contraction of the fundus or for the suppression of feeding behavior. In addition, the behavioral phenotypes of GRP-R-deficient mice were not observed in NMB-R-deficient mice. These data show that the functions of NMB-R and GRP-R are distinct, with only partial overlap.

Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice.

To study the function of GLAST, a glutamate transporter highly expressed in the cerebellar Bergmann astrocytes, the mouse GLAST gene was inactivated. GLAST-deficient mice developed normally and could manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging task such as staying on a quickly rotating rod. Electrophysiological examination revealed that Purkinje cells in the mutant mice remained to be multiply innervated by climbing fibres even at the adult stage. We also found that oedema volumes in the mutant mice increased significantly after cerebellar injury. These results indicate that GLAST plays active roles both in the cerebellar climbing fibre synapse formation and in preventing excitotoxic cerebellar damage after acute brain injury.

Functions of the two glutamate transporters GLAST and GLT-1 in the retina.

In the retina, the glutamate transporter GLAST is expressed in Müller cells, whereas the glutamate transporter GLT-1 is found only in cones and various types of bipolar cells. To investigate the functional role of this differential distribution of glutamate transporters, we have analyzed GLAST and GLT-1 mutant mice. In GLAST-deficient mice, the electroretinogram b-wave and oscillatory potentials are reduced and retinal damage after ischemia is exacerbated, whereas GLT-1-deficient mice show almost normal electroretinograms and mild increased retinal damage after ischemia. These results demonstrate that GLAST is required for normal signal transmission between photoreceptors and bipolar cells and that both GLAST and GLT-1 play a neuroprotective role during ischemia in the retina.

Generation and characterization of mice lacking gastrin-releasing peptide receptor.

Gastrin-releasing peptide (GRP) is a mammalian bombesin-like peptide which is widely distributed in the central nervous system as well as in the gastrointestinal tract. GRP binds to its high affinity receptor (GRPR) to elicit a wide spectrum of biological effects on behavior, digestion, and metabolism. To define the in vivo function of GRPR, we generated GRPR null mutant mice by gene targeting. The intracerebroventricular administration of GRP caused hypothermia in wild-type mice, but not in mutant mice. The GRPR deficient mice showed significantly increased locomotor activity during the dark period, and social responses scored by sniffing, mounting, and approaching behaviors against an intruder. Aggressive scores such as fighting and biting were not altered in the mutant mice. These phenotypes were observed in mice generated from two independent ES cell clones and backcrossed to a C57BL/6J background. The GRPR deficient mice should be useful for studying the bombesin system in vivo.

Mice lacking bombesin receptor subtype-3 develop metabolic defects and obesity.

Mammalian bombesin-like peptides are widely distributed in the central nervous system as well as in the gastrointestinal tract, where they modulate smooth-muscle contraction, exocrine and endocrine processes, metabolism and behaviour. They bind to G-protein-coupled receptors on the cell surface to elicit their effects. Bombesin-like peptide receptors cloned so far include, gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). However, despite the molecular characterization of BRS-3, determination of its function has been difficult as a result of its low affinity for bombesin and its lack of an identified natural ligand. We have generated BRS-3-deficient mice in an attempt to determine the in vivo function of the receptor. Mice lacking functional BRS-3 developed a mild obesity, associated with hypertension and impairment of glucose metabolism. They also exhibited reduced metabolic rate, increased feeding efficiency and subsequent hyperphagia. Our data suggest that BRS-3 is required for the regulation of endocrine processes and metabolism responsible for energy balance and adiposity. BRS-3-deficient mice provide a useful new model for the investigation of human obesity and associated diseases.

Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1.

Extracellular levels of the excitatory neurotransmitter glutamate in the nervous system are maintained by transporters that actively remove glutamate from the extracellular space. Homozygous mice deficient in GLT-1, a widely distributed astrocytic glutamate transporter, show lethal spontaneous seizures and increased susceptibility to acute cortical injury. These effects can be attributed to elevated levels of residual glutamate in the brains of these mice.

Dominant negative mutant of ionotropic glutamate receptor subunit GluR3: implications for the role of a cysteine residue for its channel activity and pharmacological properties.

We reported that a 33-amino-acid deletion (from tyrosine-715 to glycine-747) in a putative extracellular loop of GluR3 produced a mutant that exhibited dominant negative effects upon the functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [Sekiguchi et al. (1994) J. Biol. Chem. 269, 14559-14565]. In this study, we searched for a key residue in the dominant negative effects to explore the mechanism and examined the role of the residue in the function of the AMPA receptor. We prepared 20 GluR3 mutants with amino acid substitutions within the 33-amino-acid-region, and dominant negative effects were tested electrophysiologically in Xenopus oocytes co-expressing the mutant and normal subunits. Among the mutants, only a GluR3 mutant in which an original cysteine (Cys)-722 was replaced by alanine exhibited a dominant negative effect comparable with that of the original mutant in which the entire 33-amino-acid segment is deleted. The co-expression of the Cys-722 mutant did not inhibit the translation of normal subunits in oocytes. The Cys-722 mutant formed a functional homomeric receptor with significantly higher affinity for glutamate or kainate than a homomeric GluR3 receptor. The Cys-722 mutation greatly enhanced the sensitivity of GluR3 for aniracetam, which alters kinetic properties of AMPA receptors. The kainate-induced currents in oocytes expressing the Cys-722 mutant alone showed strong inward rectification. These results suggest that the Cys-722 in GluR3 is important for dominant negative effects and plays a crucial role in the determination of pharmacological properties in AMPA receptor function.

A deletion in the second cytoplasmic loop of GluR3 produces a dominant negative mutant of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor.

We have characterized a GluR3 mutant (sGluR3) which has a 33-amino acid deletion in its second cytoplasmic loop (deficit from Tyr-715 to Gly-747 of GluR3-flop). Xenopus oocytes injected with cRNA transcribed from cDNA for this mutant did not respond to kainate and glutamate (each 100 microM) at a holding potential of -70 mV. In oocytes coinjected with cRNAs for this mutant and for normal alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits, the current response to both kainate and glutamate was much weaker than that observed in oocytes injected with cRNA for the normal subunits alone. This inhibitory action was not accompanied by a significant change of ED50 value and shape of the I-V curve and was AMPA receptor-specific. A comparable deletion in GluR1 produced a mutant with properties similar to those of sGluR3, while a 33-amino acid deletion elsewhere in the second cytoplasmic loop of GluR3 (Met-674 to Phe-706) gave a mutant with a weaker effect. These results suggest that sGluR3 acts as a dominant negative mutant on the functional expression of normal AMPA receptors in oocytes by assembling with the normal subunits to produce an essentially nonfunctional receptor complex.

A kindred of hereditary adult-onset leukodystrophy with sparing of the optic radiations.

We describe a family with hereditary adult-onset leukodystrophy with sparing of the optic radiations, shown clinically, neuroradiologically and electrophysiologically. All five affected members developed their leukodystrophy in their fourth decade and clinical features steadily progressed. The sparing of the optic radiations is not typical of other leukodystrophies. We suggest that this is a new clinical entity.

[A surgical case of thoracic aortic aneurysm due to Takayasu's aortitis associated with ulcerative colitis].

We experienced a case of thoracic aortic aneurysm due to Takayasu's aortitis associated with ulcerative colitis. Steroid was medicated to control inflammation and operation was performed. The ascending aorta, aortic arch, brachiocephalic artery, and left carotid artery were replaced by artificial graft. We made elephant trunk type anastomosis at the distal side of the graft to provide for growth of the aneurysm after operation. This was a rare case considered autoimmune overlapping syndrome, and its background was complicated by HLA-Bw52 and parasitic Metagonimus Yokogawai. Relationship between steroid medication and progression of the disease is not certain yet. Postoperative course is uneventful, no recurrence of inflammation is seen and the aneurysm is not enlarged until now.