To assay for secretion, wild-type pro-vasopressin, the recessive mutant P7L, and the dominant mutants ⌬E47, Y2H, C28Y and ⌬G227 were expressed in COS-1 cells, pulse-labeled with [ 35 S]methionine/cysteine, chased for 2 hours, isolated from the cells and from the media by immunoprecipitation, and analyzed by SDSgel electrophoresis and autoradiography ( Fig. 2A). As expected, a considerable fraction of the wild type and of P7L was recovered from the media, reflecting their ability to fold and pass ER quality control. By contrast, the dominant mutants ⌬E47, Y2H and C28YAutosomal dominant neurohypophyseal diabetes insipidus results from mutations in the precursor protein of the antidiuretic hormone arginine vasopressin. Mutant prohormone is retained in the endoplasmic reticulum of vasopressinergic neurons and causes their progressive degeneration by an unknown mechanism. Here, we show that several dominant provasopressin mutants form disulfide-linked homo-oligomers and develop large aggregations visible by immunofluorescence and immunogold electron microscopy, both in a fibroblast and a neuronal cell line. Double-labeling showed the pro-vasopressin aggregates to colocalize with the chaperone calreticulin, indicating that they originated from the endoplasmic reticulum.The aggregates revealed a remarkable fibrillar substructure. Bacterially expressed and purified mutant pro-vasopressin spontaneously formed fibrils under oxidizing conditions. Mutagenesis experiments showed that the presence of cysteines, but no specific single cysteine, is essential for disulfide oligomerization and aggregation in vivo. Our findings assign autosomal dominant diabetes insipidus to the group of neurodegenerative diseases associated with the formation of fibrillar protein aggregates.

Mutations in the gene encoding the antidiuretic hormone arginine vasopressin cause autosomal dominant neurogenic diabetes insipidus. Autoptic data in affected individuals suggest that the neurons expressing mutant vasopressin undergo selective degeneration. Expression studies have shown that the mutants are retained in the endoplasmic reticulum, but how this trafficking defect is linked to neurotoxicity is unknown. One possibility is that unsecreted mutant precursors, or degradation products thereof, are cytotoxic. We therefore investigated the fate of endoplasmic reticulum-retained pathogenic mutants. Our data show that the mutants are retrotranslocated to the cytosol and degraded by the proteasome. In the presence of proteasomal inhibitors, three distinct un-or deglycosylated cytosolic species of vasopressin precursors were stabilized: pre-pro-vasopressin, pro-vasopressin, and an N-terminally truncated form. In addition to the retrotranslocated forms, a fraction of the newly synthesized precursor was not translocated, but was synthesized into the cytosol due to inefficient function of the vasopressin signal peptide. As a result, cytosolic pre-pro-vasopressin and its degradation product were also recovered when wild-type vasopressin was expressed. Cytosolic forms of vasopressin might trigger cytotoxicity in vivo, as has been proposed in the case of prion protein, which also contains an inefficient N-terminal signal peptide.The antidiuretic hormone, arginine vasopressin, is synthesized in vasopressinergic neurons of the hypothalamus as a precursor consisting of three moieties ( Fig. 1): the 19-aminoacid signal sequence, the nonapeptide hormone, the vasopressin-associated carrier protein neurophysin II, and a 39-aminoacid glycopeptide (copeptide) with a single N-glycosylation site (1). The precursor is cotranslationally targeted to the ER, 1 where the signal is cleaved off by signal peptidase and the copeptide is core glycosylated. The prohormone contains a total of eight disulfide bonds. After complex glycosylation in the Golgi apparatus, the matured precursor is cleaved into its three moieties and targeted to secretory granules at the distal end of the axons. From there, the hormone is released into the circulation upon osmotic and non-osmotic stimuli. Vasopressin binds to its receptor on cells of the renal collecting duct, initiating a signaling cascade that leads to the mobilization of aquaporin-2 water channels, allowing regulated water reabsorption. Through this mechanism, vasopressin mediates the conservation of as much as ϳ20 liters of fluid/day, thereby playing an important role in water homeostasis.Lack of circulating vasopressin causes diabetes insipidus. Affected individuals suffer from polyuria and polydipsia due to the inability to concentrate their urine. In rare cases, the condition is caused by mutations in the vasopressin gene and is inherited in an autosomal dominant manner (2, 3). Around 40 mutations have been reported that alter the signal peptide (4, 5), the hormone (6, 7), or the neurophysi...

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