Caenorhabditis elegans UNC-40, Drosophila Frazzled, and vertebrate Neogenin and DCC constitute a subgroup of the immunoglobulin superfamily (IgSF). They possess four immunoglobulin-like domains and six fibronectin-type III repeats at the extracellular region, a single transmembrane region, and a approximately 300 amino-acid intracellular region. UNC-40, Frazzled and DCC can function in axon guidance as the receptor of Netrin (Cell Mol. Life Sci. 56 (1999) 62; Curr. Opin. Cell Biol. 10 (1998) 609). Neogenin binds to Netrin-1 with the same affinity as DCC in vitro (Cell 87 (1996) 175), and is expressed by neurons as they project axons (J. Cell Biol. 127 (1994) 2009), suggesting that it is also a DCC-like Netrin receptor. A zebrafish homologue of DCC (zDCC) is reported recently (Mech. Dev. 109 (2001) 105), but so far there is no report of zebrafish Neogenin. To elucidate a possible neural function of vertebrate Neogenin, we cloned and characterized a zebrafish homologue of neogenin, zneo1, and identified four alternative splice sites within it. In the adult, despite broad tissue distribution, our reverse transcription polymerase chain reaction and Northern analyses demonstrated the dominant expression of zneo1 mRNA in brain. We detected zneo1 mRNA in the embryos from 10 hpf onward and revealed its spatiotemporally regulated expression pattern in both neuronal and non-neuronal tissues by in situ hybridization. Our data showed that during early brain development, zneo1 mRNA was not only present in the proliferative ventricular zones but also in the domains of several first postmitotic neuron clusters when they extended axons. Alternative splicing generates several isoforms of zneo1. Most of them are developmentally regulated, showing distinct distribution in brain and other tissues.
The germ line insertion of a defective retrovirus into the Mpv17 gene of mice is associated with a recessive phenotype. Mice homozygous for the integration develop glomerulosclerosis at a young age. The phenotype resembles human glomerulosclerosis in its physiological parameters as well as in histology. A human homologue of the Mpv17 gene has been identified, isolated and analyzed. We here show that this gene, which has a role in the production of reactive oxygen species, can rescue the phenotype of Mpv17 deficient mice when introduced by transgenesis. This provides formal proof for the hypothesis that the phenotype is caused by the loss of function of the Mpv17 gene. It also provides evidence for the functional conservation of the Mpv17 gene in mammals and points to a potential role of this gene in human kidney disease.
The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.
The transgenic mouse strain Mpv17 develops severe morphological degeneration of the inner ear and nephrotic syndrome at a young age (Meyer zum Gottesberge et al., 1996; Weiher et al., 1990). The audiograms (1-32 kHz) of Mpv17-negative mice were determined from auditory brain stem responses in young (2 months) and old (7 months) animals. Audiograms of age-matched wild-type mice with the same genetic background, but wild-type at the Mpv17 locus, were also determined. Furthermore, young Mpv17-negative mice that carried a human Mpv17 homologue gene were studied. NMRI mice served as a reference for normal hearing. Mpv17-negative mice suffer from severe sensorineural hearing loss as early as 2 months after birth. In the old Mpv17-negative mice no responses could be elicited at all. The 2 month old wild-type mice had normal audiograms, at 7 months only high threshold responses were seen. The poor audiograms of the Mpv17-negative mice are assumed to be the functional correlate of the morphological degeneration of the cochlea described earlier (Meyer zum Gottesberge et al., 1996). The finding that 2 out of 4 Mpv17-negative mice with the human Mpv17 gene had normal audiograms, shows that the gene inactivation can be functionally compensated by the human Mpv17 gene product.
Ultrastructural investigations were performed in young (approximately 2 months) and old (7 months) Mpv17-negative and wild-type mice. The onset, the severity and the pattern of the degeneration significantly differed between both mice strains. In the wild-type mouse strain the degenerative changes of the cochlear structures were similar to the aging pattern described for other species. In contrast, the Mpv17 mutants showed degenerative changes of the cochlear structures already at the age of 2 months. The degenerative changes were patchy arranged throughout the entire length of the cochlea and involved the organ of Corti as well as the stria vascularis epithelia with alterations of the basement membrane of the capillaries. The severe sensorineural hearing loss and degenerative changes of the cochlear structures indicate that cochlear structures, especially the outer hair cells and the intermediate cells of the stria vascularis, are vulnerable to the missing Mpv17 gene product.
During regeneration, retinal ganglion cell axons in fish upregulate a cell surface protein that is recognized by the monoclonal antibody (mAB) M802. M802 antigen appeared to be linked to the intracellular, membrane-associated lipid raft/microdomain proteins reggie-1 and reggie-2 that were previously shown to be reexpressed in axon-regenerating neurons [Development 124 (1997), 577]. Here, we report the isolation of the M802 antigen and its identification as the teleost homolog of mammalian Thy-1. Fish Thy-1 is detected in the same detergent-insoluble lipid raft fractions from a fibroblast cell line and from axon regenerating retinae as reggie-1 and 2. Importantly, mAB M802 coimmunoprecipitates reggie-1 and 2 from this lipid raft fraction, implying that fish Thy-1 and reggies interact. This correlates with their colocalization in growing cell processes after M802 antigen/Thy-1 activation with mAB M802. These findings suggest a role of clustered M802 antigen/Thy-1 in reggie raft microdomains for cell growth and axon regeneration.
The mutant mouse strain Mpv17-/-, carries a retroviral germline integration that inactivates the Mpv17 gene. Mpv17-deficient mice develop progressive glomerulosclerosis and sensineural deafness at early age. Characteristic basement membrane alterations are found in both sites of pathology. Mpv17 is a peroxisomal protein involved in the metabolism of reactive oxygen species, yet its molecular function is unknown. Dysregulation of antioxidant enzymes and basal membrane components has been established in this model and successful therapeutic intervention with antioxidants prove the causal role of reactive oxygen species in the development of the disease phenotype. We here investigated if the Mpv17-/- mice might be hypertensive. Indeed, our study revealed that Mpv17-/- mice developed significant systemic hypertension and tachycardia between 4 weeks and 5 months of age, accompanied by polyuria and elevated natriuresis. Judging from serum and urine parameters, the hypertensive condition develops concomitantly with the renal disease. Biochemical and pharmacological studies that used the endothelin receptor antagonist bosentan and the angiotensin converting enzyme inhibitor cilazapril indicated no involvement of the endothelin and renin-angiotensin systems in this hypertension, suggesting a potential novel mechanism of blood pressure regulation in this new murine hypertension model. Thus, Mpv17-/- mice unravel an intriguing new association between a defect in reactive oxygen metabolism and the age-dependent development of hypertension.
