In this study, we generated a murine model of FNDI that faithfully replicates several features of the human disorder, including autosomal dominant transmission, delayed onset and progressive worsening of the defect in water retention, and loss of AVP-producing neurons in the hypothalamus.
The delayed onset of FNDI has led several groups to speculate that the mutant AVP precursor causes cellular toxicity, leading to eventual loss of AVP-producing neurons (reviewed in ref.35
). This hypothesis is supported by in vitro studies showing accumulation of mutant vasopressin precursors in transfected cell lines (36
) and cytotoxicity of certain mutants (36
). Of note, there is little evidence of apoptosis, suggesting that cell death may occur via other pathways (36
The processing of mutant vasopressin has also been examined using metabolic labeling studies (37
). These experiments document persistent endoglycosidase H sensitivity, consistent with prohormone retention in the ER. The ER becomes distended (43
), and immunoreactive protein forms perinuclear punctate complexes rather than diffuse cytoplasmic staining (36
). Coexpression of WT and mutant AVP precursors results in the formation of WT/mutant dimers, and the mutant prohormone delays WT protein processing (40
). Thus, it appears that the mutant vasopressin precursor may exert at least two distinct cellular effects: (a) formation of complexes with the WT precursor protein that impair its processing, and (b) accumulation of misfolded proteins in the ER, causing cellular toxicity by mechanisms that remain to be fully characterized. Because these in vitro experiments involve high-level expression of the mutant protein and occur in dividing cell lines, it is desirable to develop models that more closely mimic conditions in vivo.
Two animal models have been used previously to study central DI. In the Brattleboro rat, a naturally occurring single base deletion in the NPII coding sequence produces AVP precursors with continued translation into the mRNA poly-A tail, leading to a polylysine stretch of amino acids. In these animals, DI is transmitted in an autosomal recessive manner (44
), suggesting that it is caused by an absolute deficiency of AVP. Although mutant precursors are retained within the ER, there is no apparent loss of AVP-producing neurons (45
). The absence of cell death in the Brattleboro rat might be explained by differences in the effects of various mutant proteins or by species differences in the cellular mechanisms for coping with mutant AVP precursors.
Of interest, the same mutant described here (C67X) has been expressed in transgenic rats (46
). In this case, features of DI were manifest only as a small increase in water intake after repeated dehydration (46
). However, there was evidence of retention of mutant precursors in distended ER. In addition, vesicles contained markers of the lysosomal degradation pathway, suggesting autophagy (47
). There was no evidence of cell death in the transgenic rats. Because the same mutant was expressed in this transgenic model and in our KI mice, it is interesting that the mice exhibit features more typical of the human disorder. Although this may be due to species differences between rats and mice, differences in gene dosage also provide a plausible explanation. The transgenic rats retain two normal vasopressin alleles in addition to the transgenic mutant gene, whereas the KI mice possess one normal vasopressin allele and one mutant allele, analogous to the human condition. Our RT-PCR studies document similar expression levels of the WT and mutant alleles, consistent with the fact that AVP expression is driven by the endogenous gene, which remains in its normal chromosomal locus. In view of evidence for interaction of WT and mutant precursor proteins during processing (40
), gene dosage could affect the ratio of mutant-WT complexes. There may also be differences in the physiologic stimuli for vasopressin expression. For example, chronic mild dehydration in the mice would lead to upregulation of vasopressin gene expression, 50% of which is comprised of mutant precursors. Based on in vitro metabolic studies, processing of the mutant precursor is delayed relative to processing of the WT protein (36
). Thus, a relative excess of mutant protein is predicted to accumulate over time.
The phenotypes of the mice with two different FNDI mutations were distinct. In the case of the signal sequence A(–1)T KI mutant, there was no apparent DI phenotype and we did not detect loss of AVP-producing neurons, even in homozygous mice. This mutation gives rise to aberrant preproAVP that is glycosylated but retains the signal peptide as a result of inefficient cleavage. Consistent with the in vivo results, cell lines expressing the A(–1)T mutant were more viable than cells expressing the C67X mutant (36
). In contrast with the A(–1)T mutant, a severe and consistent phenotype was seen in mice carrying the C67X mutation. The more severe phenotype in the C67X mutant is consistent with in vitro studies that show it is the most cytotoxic among several AVP mutants studied (36
). The C67X mutation was lethal in the homozygous state, presumably due to a complete lack of functional AVP. Heterozygotes were more informative, however. Water intake and urine volumes were increased concomitant with decreased urine osmolality and serum AVP. By 6 months of age, the 24-hour urine volume of the C67X mice was about one-third of their body weight. This is not dissimilar from affected children, who may produce several liters of urine each day and must be provided with easy access to fluids to avoid severe dehydration. Like humans, the mice are able to compensate for profound diuresis by drinking copious amounts of water, indicating that their thirst mechanism remains intact.
One of the advantages of developing an animal model of FNDI is the ability to correlate the phenotype with pathological changes in the brain regions that produce AVP. In C67X heterozygotes, the number of AVP-expressing neurons in the PVN of the hypothalamus decreased beginning at 2 months of age. Comparison with OT-producing cells demonstrated that the neuronal cell loss was specific for AVP-producing neurons. The progressive loss of AVP-producing neurons correlated with the worsening features of DI, suggesting gradual cell loss over time.
Apoptosis was not detected using a TUNEL assay or immunohistochemical analyses of apoptosis markers (caspase-3, cathepsin D; data not shown). However, given the small number of vasopressin-producing neurons and the progressive loss of cells over weeks to months, these assays are unlikely to be sensitive enough to detect apoptosis of a small number of neurons. There was no apparent induction of the ER stress protein Chop (48
), which has been reported to be proapoptotic (49
). In contrast, there was marked induction of BiP, a member of the HSP70 family of molecular chaperones. BiP binds avidly to misfolded proteins whose transport from the ER is blocked, and BiP expression is increased as part of the “unfolded protein response” (reviewed in ref.51
). Thus, elevated levels of BiP are consistent with the retention of mutant AVP precursors in the ER of C67X mice. It is notable that NPII immunoreactivity was confined to the cell bodies of magnocellular neurons. The absence of staining in the neuronal projections suggests that AVP precursors produced from the normal allele may also be retained within the ER through interaction with mutant precursors (40
). Thus, the vasopressinergic neurons of C67X mice exhibit ER accumulation of vasopressin precursors and induction of the BiP chaperone response. These neurons are selectively lost over several months, presumably the consequence of toxic effects of the mutant proteins. However, the ultimate steps that lead to cell loss remain unknown.
We propose that FNDI can be added to a list of neurodegenerative disorders that includes Alzheimer disease, Parkinson disease, and various CAG-repeat diseases (52
). In the case of FNDI, the combination of in vitro and in vivo models suggests cellular dysfunction by several different mechanisms, including dominant negative activity by interactions of mutant and WT precursors, accumulation of mutant precursors in the ER leading to stress protein responses and autophagy, and cellular toxicity by pathways that remain incompletely defined. Ultimately, the loss of AVP-producing neurons depletes AVP production below a level where compensatory responses can prevent DI. Because the C67X KI mouse faithfully replicates many features of FNDI, it provides a useful model for studying the specific molecular and cellular mechanisms responsible for the pathology seen in FNDI, with potential implications for other neurodegenerative diseases.