Familial Neurohypophysial Diabetes Insipidus Research Articles

Autosomal Dominant Familial Neurohypophyseal Diabetes Insipidus in Three Generations

Familial neurohypophysial diabetes insipidus (FNDI), an autosomal dominant disorder, comes in many forms that are differentiated by the inheritance pattern and the underlying genetic lesion. The disease is caused by mutations in the vasopressin-neurophysin 2- copeptin protein (AVP-NPII), in wolframin (WFS1) or in proprotein convertase subtilisin/kexin type 1 (PCSK1) genes. Materials and methods: In this study, we report a case of familial neurohypophyseal DI in three generations; followed in unit of the endocrinology, diabetology, metabolic diseases and nutrition department of the Mohammed VI University Hospital of Marrakesh. Results: This was a 45-year-old patient who had been suffering from polyuro-polydipsia syndrome since the age of 12, with daily urine volumes ranging from 8.0 to 15.0 litres, but had not seen a doctor. The patient was born without complications and had normal puberty. In the family history, three other members of the patient’s family have also had polypolypolydispic syndrome since adolescence, covering three generations, including the patient’s mother, her younger sister and daughter. A water restriction test was performed but the patient did not tolerate it. We completed with the minirin test with a good clinical response: urine concentrated with a volume of 500ml, and urinary osmolarity at H4 of 276.13 (Fig 2). As presented in (Fig 3), cranial MRI revealed a hypersignal region in the posterior pituitary lobe. Serum cortisol, thyroid function, and estrogen were all in the normal range The growth hormone (GH), insulin like growth factor (IGF)-1 levels and the genetic testing were not performed due to limited financial resources. After treatment with oral desmopressin 60 ug two times daily was started, the symptoms of polydipsia and polyuria were satisfactorily controlled.

Response to endoplasmic reticulum stress in arginine vasopressin neurons.

Arginine vasopressin (AVP) is an antidiuretic hormone synthesized principally in the hypothalamic supraoptic and paraventricular nuclei. The immunoglobulin heavy chain binding protein (BiP), one of the most abundant endoplasmic reticulum (ER) chaperones, is highly expressed in AVP neurons, even under basal conditions. Moreover, its expression is upregulated in proportion to the increase in AVP expression under dehydration. These data suggest that AVP neurons are constantly exposed to ER stress. BiP knockdown in AVP neurons induces ER stress and autophagy, resulting in AVP neuronal loss, indicating that BiP is pivotal in maintaining the AVP neuron system. Furthermore, inhibition of autophagy after BiP knockdown exacerbates AVP neuronal loss, suggesting that autophagy induced under ER stress is a protective cellular mechanism by which AVP neurons cope with ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the AVP gene. It is characterized by delayed-onset progressive polyuria and eventual AVP neuronal loss. In AVP neurons of FNDI model mice, mutant protein aggregates are confined to a specific compartment of the ER, called the ER-associated compartment (ERAC). The formation of ERACs contributes to maintaining the function of the remaining intact ER, and mutant protein aggregates in ERACs undergo autophagic-lysosomal degradation without isolation or translocation from the ER, representing a novel protein degradation system in the ER.

Degradation of Mutant Protein Aggregates within the Endoplasmic Reticulum of Vasopressin Neurons.

SummaryMisfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER.

A novel mechanism of autophagy-associated cell death of vasopressin neurons in familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI), characterized by delayed-onset progressive polyuria and loss of arginine vasopressin (AVP) neuron, is an autosomal dominant disorder caused by AVP gene mutations. We previously generated a knock-in mouse model for FNDI, which recapitulated the phenotype of human FNDI. To address the mechanisms underlying AVP neuron loss, we subjected FNDI mice to intermittent water deprivation, which accelerated the phenotype and induced AVP neuron loss within a relative short period. Electron microscopic analyses revealed that aggregates were confined to a sub-compartment of the endoplasmic reticulum (ER), ER-associated compartment (ERAC), in AVP neurons of FNDI mice under normal conditions. In contrast, aggregates scattered throughout the dilated ER lumen, and phagophores, autophagosome precursors, emerged and surrounded the ER containing scattered aggregates in FNDI mice subjected to water deprivation for 4weeks, suggesting that failure of ERAC formation leads to autophagy induction for degradation of aggregates. Furthermore, the cytoplasm was entirely occupied with large vacuoles in AVP neurons of FNDI mice subjected to water deprivation for 12weeks, at which stage 30-40% of AVP neurons were lost. Our data demonstrated that although autophagy should primarily be a protective mechanism, continuous autophagy leads to gradual loss of organelles including ER, resulting in autophagy-associated cell death of AVP neurons in FNDI mice.

Chemical chaperone 4-phenylbutylate reduces mutant protein accumulation in the endoplasmic reticulum of arginine vasopressin neurons in a mouse model for familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI), characterized by progressive polyuria and loss of arginine vasopressin (AVP) neurons, is an autosomal dominant disorder caused by AVP gene mutations. Our previous studies with FNDI model mice demonstrated that mutant proteins accumulated in the endoplasmic reticulum (ER) of AVP neurons. Here, we examined therapeutic effects of the chemical chaperone 4-phenylbutylate (4-PBA) in FNDI mice. Treatment with 4-PBA reduced mutant protein accumulation in the ER of FNDI mice and increased AVP release, leading to reduced urine volumes. Furthermore, AVP neuron loss under salt loading was attenuated by 4-PBA treatment. These data suggest that 4-PBA ameliorated mutant protein accumulation in the ER of AVP neurons and thereby prevented FNDI phenotype progression.

Activating transcription factor 6α is required for the vasopressin neuron system to maintain water balance under dehydration in male mice.

Activating transcription factor 6α (ATF6α) is a sensor of endoplasmic reticulum (ER) stress and increases the expression of ER chaperones and molecules related to the ER-associated degradation of unfolded/misfolded proteins. In this study, we used ATF6α knockout (ATF6α(-/-)) mice to clarify the role of ATF6α in the arginine vasopressin (AVP) neuron system. Although urine volumes were not different between ATF6α(-/-) and wild-type (ATF6α(+/+)) mice with access to water ad libitum, they were increased in ATF6α(-/-) mice compared with those in ATF6α(+/+) mice under intermittent water deprivation (WD) and accompanied by less urine AVP in ATF6α(-/-) mice. The mRNA expression of immunoglobulin heavy chain binding protein, an ER chaperone, was significantly increased in the supraoptic nucleus in ATF6α(+/+) but not ATF6α(-/-) mice after WD. Electron microscopic analyses demonstrated that the ER lumen of AVP neurons was more dilated in ATF6α(-/-) mice than in ATF6α(+/+) mice after WD. ATF6α(-/-) mice that were mated with mice possessing a mutation causing familial neurohypophysial diabetes insipidus (FNDI), which is characterized by progressive polyuria and AVP neuronal loss due to the accumulation of mutant AVP precursor in the ER, manifested increased urine volume under intermittent WD. The aggregate formation in the ER of AVP neurons was further impaired in FNDI/ATF6α(-/-) mice compared with that in FNDI mice, and AVP neuronal loss was accelerated in FNDI/ATF6α(-/-) mice under WD. These data suggest that ATF6α is required for the AVP neuron system to maintain water balance under dehydration.

Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30–40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.

The hypothalamic–neurohypophysial system: genomes, genes and genetics

The hypothalamic–neurohypophysial system: genomes, genes and genetics

Endoplasmic reticulum stress in vasopressin neurons of familial diabetes insipidus model mice: aggregate formation and mRNA poly(A) tail shortening

The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone, which binds to newly synthesized secretory and transmembrane proteins to facilitate protein folding. BiP mRNA is expressed in the arginine vasopressin (AVP) neurons in the supraoptic nucleus of wild-type mice even in basal conditions, and the expression levels increase in response to dehydration. These data suggest that AVP neurons are subjected to ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is caused by mutations in the gene locus of AVP. The mutant proteins could accumulate in the ER and possibly increase ER stress in the AVP neurons. We bred mice possessing a mutation causing FNDI, which manifested progressive polyuria, as do the patients with FNDI. Electron microscopic analyses demonstrated that aggregates accumulated in the ER of AVP neurons in FNDI mice. Despite polyuria, which could potentially induce dehydration, AVP mRNA expression was decreased in the supraoptic nucleus, and the AVP mRNA poly(A) tail length was shortened in FNDI mice compared with wild-type mice. Incubation of hypothalamic explants of wild-type mice with ER stressors caused shortening of the poly(A) tail length of AVP mRNA, accompanied by decreases in the expression. These data revealed a mechanism by which ER stress decreases poly(A) tail length of AVP mRNA, and this reduces the load of unfolded proteins that form the aggregates in ER of the AVP neurons in FNDI mice.

Poly(A) Tail Length of Neurohypophysial Hormones Is Shortened Under Endoplasmic Reticulum Stress

Familial neurohypophysial diabetes insipidus (FNDI) is caused by mutations in the gene locus of arginine vasopressin (AVP), an antidiuretic hormone. Although the carriers are normal at birth, polyuria and polydipsia appear several months or years later. Previously, we made mice possessing a mutation causing FNDI and reported that the mice manifested progressive polyuria as do the patients with FNDI. Here, we report that decreases in AVP mRNA expression in the supraoptic nucleus were accompanied by shortening of the AVP mRNA poly(A) tail length in the FNDI mice, a case in which aggregates accumulated in the endoplasmic reticulum (ER) of the hypothalamic AVP neurons. Expression levels of AVP heteronuclear RNA in the supraoptic nucleus, a sensitive indicator for gene transcription, were not significantly different between FNDI and wild-type mice. Incubation of hypothalamic explants of wild-type mice with ER stressors (thapsigargin and tunicamycin) caused shortening of the poly(A) tail length of AVP and oxytocin mRNA, accompanied by decreases in their expression. On the other hand, an ER stress-reducing molecule (tauroursodeoxycholate) increased the poly(A) tail length as well as the expression levels of AVP and oxytocin mRNA. These data reveal a novel mechanism by which ER stress decreases poly(A) tail length of neurohypophysial hormones, probably to reduce the load of unfolded proteins.

Mechanisms Underlying Progressive Polyuria in Familial Neurohypophysial Diabetes Insipidus

Familial neurohypophysial diabetes insipidus (FNDI), an autosomal dominant disorder, is mostly caused by mutations in the gene of neurophysin II (NPII), the carrier protein of arginine vasopressin (AVP). The analyses of knock-in mice expressing a mutant NPII that causes FNDI in humans demonstrated that polyuria progressed substantially in the absence of loss of AVP neurones. Morphological analyses revealed that inclusion bodies were present in the AVP neurones in the supraoptic nucleus and that the size and numbers of inclusion bodies gradually increased in parallel with the increases in urine volume. Electron microscopic analyses showed that aggregates existed in the endoplasmic reticulum (ER) of AVP neurones. These data suggest that cell death is not the primary cause of polyuria in FNDI, and that the aggregate formation in the ER is likely to be related to the pathogenesis of the progressive polyuria.

Activation of vasopressin neurons leads to phenotype progression in a mouse model for familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI) is a rare disease that is inherited in an autosomal dominant manner. In a previous study, we made a mouse model for FNDI, which showed progressive polyuria accompanied by inclusion bodies in the arginine vasopressin (AVP) neurons formed by aggregates in the endoplasmic reticulum. The present study was conducted to determine whether the activities of AVP neurons are related to the phenotype progression in the FNDI model. In the first experiment, female heterozygous mice were administered either desmopressin (dDAVP) or a vehicle (control) subcutaneously with osmotic minipumps for 30 days. The dDAVP treatment significantly decreased the urine volume, AVP mRNA expression, and inclusion bodies in the AVP neurons. Urine volume in the dDAVP group remained significantly less than the control for 14 days even after the minipumps were removed. In the second experiment, the males were fed either a 0.2% Na or 2.0% Na diet for 6 mo. Urine AVP excretion was significantly increased in the 2.0% Na group compared with the 0.2% Na group for the first 2 mo but gradually decreased thereafter. Throughout the experiments, urine volume increased progressively in the 2.0% Na group but not in the 0.2% Na group. Immunohistochemical analyses revealed that inclusion bodies in the AVP cells had significantly increased in the 2.0% Na compared with the 0.2% Na group. These data demonstrated that activation of AVP neurons could accelerate the aggregate formation as well as the progression of the polyuria in the FNDI model mice.

Progressive polyuria without vasopressin neuron loss in a mouse model for familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI), an autosomal dominant disorder, is mostly caused by mutations in the gene of neurophysin II (NPII), the carrier protein of arginine vasopressin (AVP). Previous studies suggest that loss of AVP neurons might be the cause of polyuria in FNDI. Here we analyzed knockin mice expressing mutant NPII that causes FNDI in humans. The heterozygous mice manifested progressive polyuria as do patients with FNDI. Immunohistochemical analyses revealed that inclusion bodies that were not immunostained with antibodies for mutant NPII, normal NPII, or AVP were present in the AVP cells in the supraoptic nucleus (SON), and that the size of inclusion bodies gradually increased in parallel with the increases in urine volume. Electron microscopic analyses showed that aggregates existed in the endoplasmic reticulum (ER) as well as in the nucleus of AVP neurons in 1-mo-old heterozygous mice. At 12 mo, dilated ER filled with aggregates occupied the cytoplasm of AVP cells, while few aggregates were found in the nucleus. Analyses with in situ hybridization revealed that expression of AVP mRNA was significantly decreased in the SON in the heterozygous mice compared with that in wild-type mice. Counting cells expressing AVP mRNA in the SON indicated that polyuria had progressed substantially in the absence of neuronal loss. These data suggest that cell death is not the primary cause of polyuria in FNDI, and that the aggregates accumulated in the ER might be involved in the dysfunction of AVP neurons that lead to the progressive polyuria.

Minor disturbances in central nervous system function in familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI) is caused by a defect in vasopressin synthesis and release as a result of a heterozygous mutation in the gene for the vasopressin prohormone. The predominant characteristic of FNDI is excessive thirst and urine production. However, vasopressin not only has peripheral endocrine effects, but also regulates numerous brain functions. We investigated whether central functions are affected in FNDI, by studying neuropsychological functioning of 23 affected members (15 males, 8 females) of a large family carrying a T/G transition mutation at nucleotide 2110 (codon 116) of the vasopressin prohormone gene (Cys116Gly). The relatively large number of family members with FNDI made it possible to compare cognitive and other CNS effects in these subjects with those of family members without FNDI. Thirty-seven adult volunteers (20 males, 17 females) from the same family and 11 non-family members (2 males, 9 females) from northern part of The Netherlands were tested. The mean age of the subjects was 35+/-12 years. Of the 63 quantified neuropsychological parameters few were statistically different between the subjects with FDNI and control subjects. Memory retrieval processes and sustained attention were worse in the subjects with FDNI. Moreover, these individuals reported significantly fewer symptoms of agoraphobia and miscellaneous symptoms, and had significantly lower scores on a scale measuring anger. The performance of FNDI subjects on an auditory verbal learning test (the 15-word test learning trial) was worse, but not significantly so, than that of the subjects without FDNI. There were subjective complaints of forgetfulness and slow recalls and those were observed in daily life by non-affected family members. These moderate differences in neuropsychological performance indicate that in human FNDI parvocellular vasopressin systems that supply the brain may be less affected or give no such serious disabilities, than the magnocellular hypothalamo-neurohypophysial system that provides vasopressin for endocrine regulation of water homeostasis.

Autophagy in Hypothalamic Neurones of Rats Expressing a Familial Neurohypophysial Diabetes Insipidus Transgene

We have tested the hypothesis that familial neurohypophysial diabetes insipidus (FNDI) is initiated by a process of autophagy. FNDI is a dominant, progressive inherited disorder characterized by pronounced drinking and urination caused by loss of secretion of antidiuretic hormone (vasopressin). In rats expressing an FNDI mutant transgene (Cys67stop) in vasopressin magnocellular neurones, the mutant protein fails to enter the regulated secretory pathway, and accumulates in a swollen and distended endoplasmic reticulum (ER) that also contains wild-type, endogenous vasopressin. Transmission electron microscopy suggested that these are autophagic vesicles. We have now examined the expression of vesicular markers in our transgenic rats, and demonstrate that activation of autolysosomal processes is a consequence of the expression of Cys67stop. Swollen vesicles containing Cys67stop are immunoreactive for cathepsin D (a lysosomal protease), endolyn (a marker of late endosomes) and lysosomal associated membrane protein 1, suggesting that they may be degradative autolysosomes. In addition, there is an up-regulation of lysosomal markers specifically in cells expressing Cys67stop. The expression of Cys67stop affects neither the trans-Golgi network nor early endosomes. These data support the proposal that Cys67stop mutant protein aggregates within the ER, which is targeted for lysosomal degradation by autophagy.

Familial Neurohypophysial Diabetes Insipidus in a Large Dutch Kindred: Effect of the Onset of Diabetes on Growth in Children and Cell Biological Defects of the Mutant Vasopressin Prohormone1

Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant trait in which expression of a mutant vasopressin prohormone reduces vasopressin production. We investigated the NP85 Cys-->Gly mutant vasopressin prohormone in a large kindred in The Netherlands. We demonstrate that growth retardation is an important early sign in two children from this kindred, which recuperates by substitution therapy with 1-desamino-8-D-arginine vasopressin. To obtain clues about the basis for the dominant inheritance of FNDI, we analyzed the trafficking and processing of the mutant vasopressin prohormone in cell lines by metabolic labeling and immunoprecipitation. The mutant vasopressin prohormone was retained in the endoplasmic reticulum and thus was not processed to vasopressin. This defect was not caused by dimerization of the vasopressin prohormone via its unpaired cysteine residue. High level expression of the mutant vasopressin prohormone in cell lines resulted in strong accumulation in the endoplasmic reticulum and an altered morphology of this organelle. We hypothesize that disturbance of the endoplasmic reticulum results in dysfunction and ultimately cell death of the cells expressing the mutant prohormone. Our data support the hypothesis that FNDI is a progressive neurodegenerative disease with delayed onset of symptoms. Its treatment requires early detection of symptoms for which growth parameters are useful.

Familial Neurohypophysial Diabetes Insipidus in a Large Dutch Kindred: Effect of the Onset of Diabetes on Growth in Children and Cell Biological Defects of the Mutant Vasopressin Prohormone

Familial Neurohypophysial Diabetes Insipidus in a Large Dutch Kindred: Effect of the Onset of Diabetes on Growth in Children and Cell Biological Defects of the Mutant Vasopressin Prohormone

A diabetes insipidus vasopressin prohormone altered outside the central core of neurophysin accumulates in the endoplasmic reticulum

Over 20 mutations affecting the neurophysin moiety of the vasopressin prohormone, have been identified in families suffering from familial neurohypophysial diabetes insipidus (FNDI). Only one of these, NP87E→stop, is located outside the central conserved domain implicated in sorting of the vasopressin prohormone. To obtain clues about the mechanism of induction of FNDI by this atypical mutant we stably expressed wild type and NP87E→stop vasopressin prohormones in (neuro)endocrine cell lines. Metabolic labeling and immunoprecipitation demonstrated reduced processing of the mutant prohormone to neurophysin. In addition, evoked secretion of neurophysin and vasopressin was diminished, suggesting that part of the mutant is retained in another intracellular compartment than the secretory granules. Indeed, immunofluorescence demonstrated accumulation of the truncated vasopressin prohormone in the endoplasmic reticulum. We conclude that the presence of the vasopressin moiety and the central conserved core of the neurophysin domain suffices for sorting and processing, but not for efficient endoplasmic reticulum exit of the vasopressin-neurophysin molecule.

Genetic models of vasopressin deficiency.

Animal models of genetic hormone deficiency are useful as models for physiological studies of hormone deficiency and hormone action. Structure-function studies of the specific underlying gene defect may help in understanding mechanisms regulating gene expression and secretion of the peptide product. Spontaneous genetic models of vasopressin deficiency, such as the Brattleboro rat and human familial diabetes insipidus, have facilitated many studies of vasopressin. However, the Brattleboro rat may not be an ideal model of genetic vasopressin deficiency and therefore could be less useful for studies of the central nervous system or as a background strain for the introduction of new vasopressin gene constructs. The human model is appropriately limited by the constraints of human studies, so that engineered animal models of specific diseases, such as familial neurohypophysial diabetes insipidus, are required. The recent development of a vasopressin-null mouse may provide insights into the various roles of vasopressin in the stress response, cardiovascular regulation and behaviour. Additionally, animals with a complete genetic deficiency of vasopressin can serve as a background strain for introduction of novel vasopressin gene constructs to enable sophisticated studies of the regulation of vasopressin expression and the intracellular processes required for appropriate secretion of vasopressin peptide. As advanced techniques of genetic manipulation become more reliable, conditional expression of vasopressin, regulated by time or body site will permit even more detailed studies in this field.

Mutations in the Vasopressin Prohormone Involved in Diabetes Insipidus Impair Endoplasmic Reticulum Export but Not Sorting

Familial neurohypophysial diabetes insipidus is characterized by vasopressin deficiency caused by heterozygous expression of a mutated vasopressin prohormone gene. To elucidate the mechanism of this disease, we stably expressed five vasopressin prohormones with a mutation in the neurophysin moiety (NP14G-->R, NP47E-->G, NP47DeltaE, NP57G-->S, and NP65G-->V) in the neuroendocrine cell lines Neuro-2A and PC12/PC2. Metabolic labeling demonstrated that processing and secretion of all five mutants was impaired, albeit to different extents (NP65G-->V >/= NP14G-->R > NP47DeltaE >/= NP47E-->G > NP57G-->S). Persisting endoglycosidase H sensitivity revealed these defects to be due to retention of mutant prohormone in the endoplasmic reticulum. Mutant prohormones that partially passed the endoplasmic reticulum were normally targeted to the regulated secretory pathway. Surprisingly, this also included mutants with mutations in residues involved in binding of vasopressin to neurophysin, a process implicated in targeting of the prohormone. To mimick the high expression in vasopressin-producing neurons, mutant vasopressin prohormones were transiently expressed in Neuro-2A cells. Immunofluorescence displayed formation of large accumulations of mutant prohormone in the endoplasmic reticulum, accompanied by redistribution of an endoplasmic reticulum marker. Our data suggest that prolonged perturbation of the endoplasmic reticulum eventually leads to degeneration of neurons expressing mutant vasopressin prohormones, explaining the dominant nature of the disease.