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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 24, 2000 as doi:10.1096/fj.99-0892fje. |
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1
* Molecular Neuroendocrinology Research Group, Department of Medicine, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, U.K.;
Neuropeptide Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Republic of Singapore;
Rudolf Magnus Institute for Neuroscience, Department of Medical Pharmacology, 3584 CG Utrecht, The Netherlands; and
§ Netherlands Institute for Brain Research, 1105 AZ, Amsterdam, The Netherlands
1Correspondence: Molecular Neuroendocrinology Research Group, Department of Medicine, University of Bristol, Bristol Royal Infirmary, Marlborough Street, Bristol BS2 8HW, U.K. E-mail d.murphy{at}bristol.ac.uk
SPECIFIC AIM
To test hypotheses regarding the etiology of dominant autosomal familial neurohypophyseal diabetes insipidus (FNDI) in new transgenic rat models.
PRINCIPAL FINDINGS
1. Human familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant endocrine disorder that presents in early childhood as excessive drinking and urination as a consequence of a progressive loss of secretion of vasopressin (VP) from posterior pituitary nerve terminals.
2. Mutations in the VP gene have been implicated as the cause of FNDI, but the mechanisms by which these mutants manifest their pathology and prevent the secretion of the coexpressed wild-type protein are unknown.
3. One hypothesis suggests that mutant precursors are toxic and stop the synthesis of wild-type VP by killing expressing cells. Another hypothesis suggests that aberrant interactions between mutant and wild-type precursors might inhibit the elaboration or secretion of the products of the normal allele.
4. We have tested these hypotheses using new transgenic rat models that
express an FNDI mutant VP gene (3-VCAT-3-Cys67stop; Fig. 1c
) encoding a truncated VP precursor (Cys67stop).
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5. In situ hybridization (Fig. 1d
) revealed
appropriate cell-specific expression of the transgene in hypothalamic
magnocellular neurons of the supraoptic nucleus (SON) and
paraventricular nucleus (PVN). Transgene transcript levels were
dramatically induced by the physiological stimulation of dehydration.
6. The transgene RNA is translated into a peptide that can be detected
in SON and PVN cell bodies, but not processes, of dehydrated rats using
a specific antibody that recognizes the unique exposed
carboxyl-terminal epitope found in the truncated Cys67stop
precursor (Fig. 1e
).
7. Rats were assessed for effects of transgene expression on
water balance using a protocol that ensured sustained transgene
induction. After 6 cycles of chronic intermittent dehydration (CID; a
CID cycle consists of 72 h of complete fluid deprivation followed
by 96 h of rehydration), the 3-VCAT-3-Cys67stop rats drank
significantly more and produced significantly more urine than control
animals (Fig 1f
).
8. Cell-specific and -inducible expression of the Cys67stop mutation in rat VP hypothalamic neurons does not result in cell death or atrophy.
9. Expression of the FNDI mutant causes a neuronal pathology
characterized by distorted structures in the cell body (Fig. 2a
) that are labeled by antisera that recognize endoplasmic
reticulum (ER) markers (Fig. 2b
), and that accumulate mutant
gene products (Fig. 2a, b
).
|
10. Two ER phenotypes were evident (Fig. 2b
). The
first was characterized by a diffuse ER staining that colocalized with
Cys67stop-like immunoreactivity. The second phenotype was distinguished
by a swollen and distorted ER packed with Cys67stop protein and
endogenous VP. Transmission electron microscopy studies revealed that
these bodies were delimited by a thick membrane and contained
structurally intact ER and aggregates of mutant protein, and may
represent preautophagic vesicles.
11. Intracellular pathology is accompanied by an increase in the
abundance of the mannose-6-phosphate receptor (MPR), a marker of
endosome-lysosome activity (Fig. 2c, d
).
CONCLUSIONS AND SIGNIFICANCE
Any hypothesis that seeks to explain the etiology of FNDI
must address its dominant and progressive nature. The two current
hypotheses are not entirely consistent with clinical and experimental
observations. The first hypothesis suggests that aberrantly folded
mutant protein may be toxic to the cell. We could find no evidence of
atrophy or apoptosis in transgenic rats. Whereas neuronal atrophy might
be a long-term consequence in some cases of FNDI, it is probably not
the primary cause of the disease. The second hypothesis suggests that
heterodimerization of mutant with wild-type precursors might disrupt
the elaboration of VP gene products. However, this is difficult to
reconcile with the progressive nature of the disease, and it has been
shown that there is in fact little specific interaction between
wild-type and Cys67stop precursors. Based on our new evidence, we
suggest that FNDI may be initiated by the trapping of wild-type VP gene
products within an ER that is targeted for lysosomal degradation by
autophagy. A schematic diagram illustrating this process is shown in
Fig. 3
.
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Magnocellular neurons expressing the Cys67stop transgene develop a subcellular pathology characterized by a grossly distorted ER that accumulates both mutant and wild-type NP-II. The Cys67stop protein is probably retained in the ER by thiol retention, which involves disulfide interchange interactions with matrix proteins. Mutant Cys67stop possesses an unpaired Cys at residue 61 that is likely to be exposed and is eminently vulnerable to participation in disulfide interchanges. Mutations that result in uneven numbers of Cys residues can also subject other proteins to thiol retention. The Cys67stop protein may be involved in intermolecular disulfide bonding with other mutant VP prohormones presenting an exposed Cys 61 or with wild-type prohormones at an intermediate folding stage when disulfide interchanges are reversible. Thus, whereas there is no evidence for specific interactions between wild-type and mutant NPs, nonspecific disulfide interactions, leading to the formation of inappropriate nonselective aggregates, cannot be ruled out. Thus might the Cys67stop protein be responsible for the trapping of the wild-type VP precursor in the ER.
Proteins that have been retained in the ER are targeted for degradation. Two pathways are available: the autophagic lysosomal-endosomal pathway, and the proteasome system. Most substrates, including a majority of the ER retained proteins, are marked for proteasomal degradation by covalent linkage to multiple molecules of ubiquitin. There is prominent ubiquitin staining in the SON of dehydrated wild-type and transgenic rats, but transgenesis does not alter the staining pattern (not shown). The mutant protein is thus probably selectively targeted to the normally functioning cytosolic proteasome system, leaving most of the wild-type protein intact and capable of being delivered through the secretory pathway.
In contrast, autophagy is a nonselective bulk process whereby whole regions of cytoplasm become enveloped to form closed vacuoles, from which the sequestered material is subsequently delivered to lysosomes for degradation. Sequestration is energy dependent and starts with the formation of double-membrane sheets, possibly derived from the ribosome-free regions of the ER or the Golgi cisternae. Acidification of the autophagic vacuole is accomplished by acquisition or activation of an H+-ATPase, after which acid hydrolases are delivered, either by MPR-mediated transfer from endosomes or fusion with preexisting MPR deficient lysosomes. The increased number of MPR-positive endosomal compartments in chronically stimulated transgenic rats demonstrates a marked activation of the endosomal-lysosomal system, and we suggest that this correlates with increased autophagy.
We propose a new hypothesis to explain how the expression of a
mutant FNDI allele can cause the loss of hormone production in VP
neurons that continue to express the wild-type gene (Fig. 3)
. The
mutant Cys67stop possesses an unpaired Cys residue that will result in
capture in the ER, probably by the thiol retention quality control
system. The captive mutant protein is likely to be targeted to the
cytosolic ubiquitin-proteasome system, leaving the wild-type
protein intact and capable of being delivered through the secretory
pathway. However, as mutant proteins accumulate in the ER, aggregates
will form that will also contain wild-type molecules, trapped by
aberrant disulfide-bond interactions with Cys67stop. This results in
the development of a subcellular pathology characterized by a grossly
distorted ER that accumulates both mutant and wild-type NP-II. We
propose that under these circumstances, the selective degradation
system cannot cope, and a more general degradation
system—autophagy—is activated to remove the deranged
ER. Wild-type prohormone will be eliminated when the deranged organelle
is destroyed, resulting in VP deficiency.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.99-0892fje
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0.05) is shown by an
asterisk.
