| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 21, 2005 as doi:10.1096/fj.04-3163fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Molecular Neuroendocrinology Research Group, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, England, UK; and
"Amedeo Avogadro" University, Laboratory of Molecular Pathology, Department of Medical Sciences, Novara, Italy
1Correspondence: Molecular Neuroendocrinology Research Group, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, England, UK. E-mail: d.murphy{at}bristol.ac.uk
SPECIFIC AIMS
Mutations in the gene encoding the antidiuretic hormone vasopressin (VP) gene cause human autosomal familial neurohypophyseal diabetes insipidus (adFNDI), a rare inherited disorder that presents as polydipsia and polyuria as a consequence of a loss of secretion of VP from posterior pituitary nerve terminals. Work from our laboratories has shown that adFNDI, like other neurodegenerative diseases, is associated with autophagy. We have recently shown that the activation of autophagy in mouse neuroblastoma Neuro2a cells after adenoviral vector-mediated delivery of an adFNDI mutant VP transgene (Cys67stop) is a cell survival mechanism and that its inhibition induces apoptosis. In the present report, we have further explored the relationship between mutant protein accumulation, autophagy, cell survival, and cell death. These studies were based on the premise that neuronal cells in vivo are continuously exposed to environmental and metabolic insults, such as excitotoxic and/or oxidative stress. We have tested the hypothesis that frail neurons, already undergoing autophagy in order to clear mutant proteins, might be more prone to cell suicide as a consequence of exposure to such stressors.
PRINCIPAL FINDINGS
1. The expression of Cys67stop sensitizes Neuro2a cells to a second insult in an autophagy-dependent manner
Mouse neuroblastoma Neuro2a cells were infected for 10 h with an adenoviral vector, Ad-VCAT-Cys67stop, which encodes the Cys67stop adFNDI-truncated VP precursor, or with the control virus Ad-VCAT, which encodes a C-terminally tagged wild-type VP precursor. Cells were incubated with DA for a further 8, 14, and 40 h, and cell viability was assessed. We previously showed that the expression of Cys67stop on its own has no effect on cell viability. We now show that Ad-VCAT-Cys67stop-infected cells are sensitized to the toxic effects of DA; cell death was faster and more pronounced at all time points compared with uninfected cells or cells infected with the Ad-VCAT control virus. We then asked whether the cell death induced by DA in Neuro2A cells expressing the Cys67stop protein is related to autophagy. As previously demonstrated, blockade of autophagy using either Asn, which prevents fusion of autophagosomes with lysosomes, or 3MA, which blocks autophagosome formation, has no effect on the viability of Ad-VCAT-infected cells at any time point. However, both 3MA and Asn induce cell death in cells infected with Ad-Cys67stop, suggesting that autophagy is a prosurvival mechanism in cells accumulating a mutant protein. DA toxicity in Ad-VCAT-infected cells becomes apparent after 40 h of exposure, but simultaneous treatment with 3MA or Asn has no effect on viability, suggesting that DA-induced death in these cells is autophagy independent. In contrast, blockade of autophagy dramatically reduced cell death in DA-treated cultures infected with the Ad-VCATCys67stop virus.
2. Cell death induced by DA in Cys67stop-expressing cells has apoptotic characteristics
We asked whether the cell death induced by DA in Cys67stop-infected cells was related to classic apoptosis. Flow cytometry was used to quantify cell populations labeled with FITC-annexin V that identify early apoptotic cells. Treatment of Cys67stop-expressing cells with DA quadrupled the association of FITC-annexin V with the surface phosphatidylserine, indicative of early apoptosis (Fig. 1
A). In addition, flow cytometry of PI-labeled cells was used to demonstrate an increase in the subG1 population in Ad-VCAT-Cys67stop-infected Neuro2a but not in control-infected cells (Fig. 1B
). Apoptosis is associated with activation of caspases by a cytosolic multiprotein complex formed upon cytochrome c release from permeabilized mitochondria; immunocytochemistry revealed massive cytochrome c release from mitochondria in DA-treated Ad-VCAT-Cys67stop-infected cells (Fig. 1C, D
). Flow cytometry revealed a strong activation of caspases in DA-treated cells expressing Cys67stop, which was abolished by treatment with 3MA or Asn (Fig. 2
A). In contrast, in control Ad-VCAT-infected cells, DA treatment was associated with a small increase of caspase activity, which was insensitive to 3M and Asn (Fig. 2A
). We then used the specific inhibitor ZVAD-fmk to show that the death of DA-treated Ad-VCAT-Cys67stop-infected cells was caspase dependent. As expected, ZVAD-fmk blocks caspase activity in DA-treated cells expressing Cys67stop (Fig. 2B
). This results in the rescue of cell viability, as assessed by the Trypan blue exclusion test (Fig. 2C
). The increase in Trypan blue-positive cells in DA-treated VCAT cultures is unchanged by caspase inhibition.
|
|
CONCLUSIONS AND SIGNIFICANCE
We have previously shown that the specific expression of an adFNDI mutant transgene (Cys67stop) in rat VP hypothalamic neurons activates autophagy, a process that is mimicked after viral delivery of Cys67stop to Neuro2a cells. In the latter model inhibition of autophagy triggers apoptosis, suggesting a role for this bulk degradation process in cell survival after the accumulation of a toxic mutant protein. We now show that autophagy, induced as a consequence of mutant Cys67stop protein expression, sensitizes the cell to the lethal effects of DA. This mode of cell death exhibits features typically associated with classical apoptosis, including caspase-dependence. Yet, inhibition of autophagy reversed these effects and rescued cell viability. We propose that autophagy-mediated cell death is a "two-hit" process: after the cellular stress of the accumulation of a misfolded mutant protein, autophagy is prosurvival. However, a second insult triggers an autophagy-dependent apoptosis.
We have previously speculated as to the role of autophagy in the etiology of adFNDI (Fig. 3
). We suggested that the accumulation of mutant Cys67stop protein in the ER causes insoluble aggregates to form. This results in the development of a pathology characterized by a grossly deranged endoplasmic reticulum in which accumulates mutant and trapped wild-type protein. Under this circumstance, autophagy, acting as a cell survival mechanism, removes the deranged structures. Wild-type prohormone will be eliminated when the deranged organelle is destroyed, resulting in progressive VP-deficiency that leads to a chronic increase in plasma osmolality. Overstimulation of VP neurons by endogenous, cell autonomous, mechanisms and by excitatory afferents might then trigger a pathologic sequence of neurotoxic events that ultimately leads to autophagy-dependent cell death. Consistent with this hypothesis, a limited number of autopsy examinations have described a paucity of VP neurons in the hypothalami of adFNDI patients. Similarly, a recent report has shown that "knock-in" mice expressing an adFNDI mutant VP gene exhibit a progressive loss of VP-expression.
|
Even though adFNDI is a rare disease, we suggest that our studies of this intriguing model have broader implications for our understanding of more common, and more devastating, neurodegenerative disorders. We have shown that autophagy has a bimodal role in the etiology and pathogenesis of protein aggregation disease. Initially, autophagy is triggered to destroy potentially toxic aggregates. For example, as in our adFNDI model, the induction of autophagy by polyglutamine aggregates in Huntington’s disease models decreases their accumulation and toxicity. However, this ongoing autophagic activity sensitizes the neuron to the toxic effects of a second insult. Under these circumstances, autophagy switches from a prosurvival to a pro-death mode. The signaling pathways that flick this switch are currently under investigation.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3163fje; doi: 10.1096/fj.04-3163fje
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
