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.

The gracile axonal dystrophy (gad) mouse is an autosomal recessive mutant that shows sensory ataxia at an early stage, followed by motor ataxia at a later stage. Pathologically, the mutant is characterized by 'dying-back' type axonal degeneration and formation of spheroid bodies in nerve terminals. Recent pathological observations have associated brain ageing and neurodegenerative diseases with progressive accumulation of ubiquitinated protein conjugates. In gad mice, accumulation of amyloid beta-protein and ubiquitin-positive deposits occur retrogradely along the sensory and motor nervous systems. We previously reported that the gad mutation was transmitted by a gene on chromosome 5 (refs 10,11). Here we find that the gad mutation is caused by an in-frame deletion including exons 7 and 8 of Uchl1, encoding the ubiquitin carboxy-terminal hydrolase (UCH) isozyme (Uch-l1) selectively expressed in the nervous system and testis. The gad allele encodes a truncated Uch-l1 lacking a segment of 42 amino acids containing a catalytic residue. As Uch-l1 is thought to stimulate protein degradation by generating free monomeric ubiquitin, the gad mutation appears to affect protein turnover. Our data suggest that altered function of the ubiquitin system directly causes neurodegeneration. The gad mouse provides a useful model for investigating human neurodegenerative disorders.

Caveolin-3 is a muscle-specific protein integrated in the caveolae, which are small invaginations of the plasma membrane. Mutations of the caveolin-3 gene, localized at 3p25, have been reported to be involved in the pathogenesis of limb-girdle muscular dystrophy (LGMD1C or caveolinopathy) with mild clinical symptoms, inherited through an autosomal dominant form of genetic transmission. To elucidate the pathogenetic mechanism, we developed caveolin-3-deficient mice for use as animal models of caveolinopathy. Caveolin-3 mRNA and its protein were absent in homozygous mutant mice. In heterozygous mutant mice, both the mRNA and its protein were normal in size, but their amounts were reduced by about half. The density of caveolae in skeletal muscle plasma membrane was roughly proportional to the amount of caveolin-3. In homozygous mutant mice, muscle degeneration was recognized in soleus muscle at 8 weeks of age and in the diaphragm from 8 to 30 weeks, although there was no difference in growth and movement between wild-type and mutant mice. No apparent muscle degeneration was observed in heterozygous mutant mice, indicating that pathological changes caused by caveolin-3 gene disruption were inherited through the recessive form of genetic transmission.

In birds, differentiation of embryonic gonads is not as strictly determined by the genetic sex as it is in mammals, and can be influenced by early manipulation with a sex steroid hormone. Thus administration of an aromatase inhibitor induces testis development in the genetic female, and administration of estrogen induces a left ovotestis in the genetic male embryo. Another feature of avian gonadogenesis is that only the left ovary develops in most species. Molecular mechanisms underlying these features at the level of gene expression have not been elucidated. In this paper, we present evidence that a gene for aromatase cytochrome P-450, an enzyme required for the last step in the synthesis of estradiol-17 , is expressed in medullae of the left and right gonads of a female chicken embryo, but not in those of a male chicken embryo, and that an estrogen receptor gene is expressed only in epithelium (and cortex later, in the female) of the left, not the right, gonad of both sexes, but the expression in the male left gonad is temporary and restricted to an early stage of development. Differential expression of these two genes serves well to explain the above features of gonadal development in birds. Furthermore, in ovo administration of estradiol-17 from the 5th to the 14th day of incubation does not cause expression of the estrogen receptor gene in the right gonad of chicken embryos of either sex, suggesting that the absence of expression of the estrogen receptor gene in the right gonad is not the result of down-regulation, but may be regarded as an important cause of the unilateral ovarian development.

Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase (GAA), a glycogen degrading lysosomal enzyme. GAA-deficient (AMD) Japanese quails exhibit progressive myopathy and cannot lift their wings, fly, or right themselves from the supine position (flip test). Six 4-wk-old acid maltase-deficient quails, with the clinical symptoms listed, were intravenously injected with 14 or 4.2 mg/kg of precursor form of recombinant human GAA or buffer alone every 2-3 d for 18 d (seven injections). On day 18, both high dose-treated birds (14 mg/kg) scored positive flip tests and flapped their wings, and one bird flew up more than 100 cm. GAA activity increased in most of the tissues examined. In heart and liver, glycogen levels dropped to normal and histopathology was normal. In pectoralis muscle, morphology was essentially normal, except for increased glycogen granules. In sharp contrast, sham-treated quail muscle had markedly increased glycogen granules, multi-vesicular autophagosomes, and inter- and intrafascicular fatty infiltrations. Low dose-treated birds (4.2 mg/kg) improved less biochemically and histopathologically than high dose birds, indicating a dose-dependent response. Additional experiment with intermediate doses and extended treatment (four birds, 5.7-9 mg/kg for 45 d) halted the progression of the disease. Our data is the first to show that an exogenous protein can target to muscle and produce muscle improvement. These data also suggest enzyme replacement with recombinant human GAA is a promising therapy for human Pompe disease.

No abstract

Protein palmitoylation has emerged as an important mechanism for regulating protein trafficking, stability, and protein–protein interactions; however, its relevance to disease processes is not clear. Using a genome-wide, phenotype driven N-ethyl-N-nitrosourea–mediated mutagenesis screen, we identified mice with failure to thrive, shortened life span, skin and hair abnormalities including alopecia, severe osteoporosis, and systemic amyloidosis (both AA and AL amyloids depositions). Whole-genome homozygosity mapping with 295 SNP markers and fine mapping with an additional 50 SNPs localized the disease gene to chromosome 7 between 53.9 and 56.3 Mb. A nonsense mutation (c.1273A>T) was located in exon 12 of the Zdhhc13 gene (Zinc finger, DHHC domain containing 13), a gene coding for palmitoyl transferase. The mutation predicted a truncated protein (R425X), and real-time PCR showed markedly reduced Zdhhc13 mRNA. A second gene trap allele of Zdhhc13 has the same phenotypes, suggesting that this is a loss of function allele. This is the first report that palmitoyl transferase deficiency causes a severe phenotype, and it establishes a direct link between protein palmitoylation and regulation of diverse physiologic functions where its absence can result in profound disease pathology. This mouse model can be used to investigate mechanisms where improper palmitoylation leads to disease processes and to understand molecular mechanisms underlying human alopecia, osteoporosis, and amyloidosis and many other neurodegenerative diseases caused by protein misfolding and amyloidosis.

Altered function of the ubiquitin pathway has been implicated in the etiology of neurodegeneration. For example, gracile axonal dystrophy (gad) mutant mice, which harbor a deletion within the gene encoding ubiquitin C-terminal hydrolase L1 (Uch-L1), display sensory ataxia followed by posterior paralysis and lethality. We previously showed that mice homozygous for a targeted deletion of the related Uch-L3 gene are indistinguishable from wild-type. To assess whether the two hydrolases have redundant function, we generated mice homozygous for both Uch-L1gad and Uch-L3Delta3-7. The double homozygotes weigh 30% less than single homozygotes and display an earlier onset of lethality, possibly due to dysphagia, a progressive loss in the ability to swallow food. This is consistent with histological analysis that revealed axonal degeneration of the nucleus tractus solitarius (NTS) and area postrema (AP) of the medulla. The NTS is essential for central nervous system control of swallowing. The double homozygotes also display a more severe axonal degeneration of the gracile tract of the medulla and spinal cord than had been observed in Uch-L1gad single homozygotes. In addition, degeneration of dorsal root ganglia cell bodies was detected in both the double homozygotes and Uch-L3Delta3-7 single homozygotes. Given that both Uch-L1gad and Uch-L3Delta3-7 single homozygotes display distinct degenerative defects that are exacerbated in the double homozygotes, we conclude that Uch-L1 and Uch-L3 have both separate and overlapping functions in the maintenance of neurons of the gracile tract, NTS and AP. This study is the first to successfully document dysphagia in the mouse and is a potentially valuable resource for understanding human neurodegenerative disorders that cause swallowing defects.