The antidiuretic hormone, arginine vasopressin (VP), is synthesized in hypothalamic neurons as a prepro-hormone consisting of the 19 amino-acid signal sequence, the nonapeptide hormone, the carrier protein neurophysin II (NPII), and a 39 amino-acid glycopeptide with a single N-glycosylation site. The signal sequence is cleaved cotranslationally. Prior to secretion, the prohormone is cleaved into its three moieties. Via action on V₂ receptors on renal distal tubule and collecting duct cells, VP increases free water reabsorption through apical aquaporin-2 water channels. In individuals carrying heterozygous mutations in the VP gene, autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) develops gradually. There is histological and experimental evidence that mutant VP precursors exert a chronically toxic effect on vasopressinergic neurons, which would explain the clinical phenotype and the dominant inheritance. We have identified several novel pathogenic mutations and studied their intracellular transport and degradation. When expressed in transfected cells, the mutant precursor is retained in the endoplasmic reticulum (ER) and forms aggregates. It is retrotranslocated from the ER to the cytosol and degraded by the proteasome, along with wild-type precursor mistargeted due to the relatively inefficient native signal peptide.

In the first part of our project, we aim to define minimal requirements for cytotoxicity of VP-NPII mutants and to study the cell’s response to pathogenic mutants. To test cytotoxicity, we will use conditionally immortalized rat hypothalamic cells. Deletion mutants will provide a means to define the structural requirements necessary for cytotoxicity. Using mutants targeted exclusively to the cytosol or the ER, we will determine whether it is the cytosolic or ER-associated population of mutant precursor which triggers cell death. The unfolded protein response, triggered by ER retention of mutant proteins, will be studied in a model using inducible expression of mutants in human embryonic kidney (Hek) cells. Our experiments will help elucidate the pathomechanisms of ADNDI in the context of intracellular accumulation of mutant protein, which is a hallmark of neurodegeneration.

The second part of the project aims to define the role of the vasopressin and glycopeptide domains in folding, secretion efficiency and sorting of the precursor to the regulated secretory pathway. To address this question, we will express in a neuronal cell line (PC-12) various artificial VP-NPII deletion mutants. These experiments will allow the characterization of the vasopressin prohormone with respect to its structural properties needed for regulated secretion, a fundamental process common to all endocrine cells.