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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 21, 2005 as doi:10.1096/fj.04-3162fje. |
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* Molecular Neuroendocrinology Research Group, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, England, UK; and
"Amedeo Avogadro" University, Laboratory of Molecular Pathology, Department of Medical Sciences, Novara, Italy
2Correspondence: Molecular Neuroendocrinology Research Group, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Whitson St., Bristol BS1 3NY, England, UK. E-mail: d.murphy{at}bristol.ac.uk
SPECIFIC AIMS
Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is caused by mutations in the gene encoding the neuropeptide hormone vasopressin (VP) and presents as excessive drinking and urination as a consequence of a progressive loss of secretion of VP from posterior pituitary nerve terminals. We have previously shown that expression of an adFNDI mutant transgene encoding a truncated precursor (Cys67stop) in rat VP neurons induces autophagy, a conserved bulk process that delivers regions of cytosol to lysosomes for degradation. Thus, adFNDI, like other neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s, is associated with autophagy, although its role in these disorders is unclear. To address the relationships between mutant protein accumulation, autophagy, cell survival, and cell death, we have developed a tractable model system consisting of the adenoviral (Ad) vector-mediated delivery of an adFNDI mutant protein (Cys67stop) or an epitope-tagged wild-type VP precursor into mouse neuroblastoma Neuro2a cells.
PRINCIPAL FINDINGS
1. Cys67stop mutant protein accumulates in cathepsin D positive compartments
Transgene VCAT encodes the wild-type VP precursor tagged with a C-terminal epitope (DR-12-EK). In the VCAT-Cys67stop adFNDI mutant transgene, a C to A transition replaces a Cys at position 67 of the neurophysin portion of the VP precursor with a translation termination codon. VCAT-Cys67stop thus encodes a truncated VP precursor (Cys67stop) implicated as the cause of adFNDI in a Japanese kindred. Wild-type and mutant transgenes were expressed in the VP neurons of germline transgenic rats under the control of VP gene regulatory sequences (3-VCAT-3 and 3-VCAT-3-Cys67stop, respectively) or delivered to Neuro2a cell using Ad vectors (Ad-VCAT and Ad-VCAT-Cys67stop, respectively). We first used antisera that recognize the Cys67stop mutant protein and the DR-12-EK epitope to ask about the fate of transgene proteins in Ad-infected Neuro2a cells, and this was compared with rats expressing the same transgenes in VP-expressing neurons. In 3-VCAT-3 rats, transgene proteins follow a parallel path with the endogenous wild-type VP through the regulated secretory pathway. Similarly, in Neuro2a cells, Ad-VCAT expression elicits DR-12-EK staining in the cell body, with material entering neurite processes and accumulating in terminals. In contrast, and similar to 3-VCAT-3-Cys67stop transgenic rats, the truncated protein encoded by Ad-VCAT-Cys67stop is confined to the cell body, often within enlarged vacuolar compartments of endoplasmic reticulum (ER) origin, as revealed by colocalization with calnexin and protein disulphide isomerase (PDI). Staining with acridine orange (AO), a dye that identifies acid vacuolar compartments, showed a diffuse punctate staining in noninfected and in Ad-VCAT-infected cells, as expected for lysosomes. However, in Ad-VCAT-Cys67STOP transfected cells, AO revealed enlarged acidic compartments. The Cys67stop protein expressed in Neuro2a cell shows some overlap with a marker of the trans-Golgi network (TGN), and is strongly colocalized with the lysosomal marker cathepsin D. These data suggest that in Neuro2a cells, as in the VP neurons of transgenic rats, the mutant Cys67stop protein accumulates in the ER and in acidic degradative compartments.
2. Cys67stop expression triggers autophagy
Monodansylcadaverine (MDC), a spontaneously fluorescent dye, selectively labels autophagosomes and autolysosomes. In noninfected and Ad-VCAT-infected Neuro2a cells MDC staining revealed a diffuse punctuate pattern, which is likely to be indicative of basal, constitutive autophagy. In contrast, Ad-VCAT-Cys67stop infection results in the accumulation of large MDC-stained vesicles, suggesting up-regulation of autophagy. Western blot analysis of the expression of Beclin-1, the mammalian analog of yeast autophagy gene Atg6, a regulator of the autophagic pathway that associates with apoptosis inhibitor bcl-2, revealed significant up-regulation in Ad-VCAT-Cys67stop infected cells, consistent with an up-regulation of autophagic processes.
We then characterized the intracellular distribution of key regulators of autophagy, comparing 3-VCAT-3 and 3-VCAT-3-Cys67stop transgenic rats with Neuro2a cells infected with Ad-VCAT or Ad-VCAT-Cys67stop (Fig. 1
). We examined Beclin 1, the class III phosphatidylinositol 3-kinase (PI3K) Vps34, and Rab24. Beclin-1 interacts with Vps34, and, together, these molecules control autophagy as a complex at the TGN. The GTPase Rab24 is thought to be involved in the regulation of vesicular transport associated with autophagy. In 3-VCAT-3 transgenic rats and in Ad-VCAT-infected cells, Beclin 1, Vps34, and Rab24 show a typical perinuclear reticular localization. There is no colocalization with DR-12-EK-like immunoreactivity. In contrast, expression of the Cys67stop protein, either in 3-VCAT-3-Cys67stop transgenic rats or following infection of Neuro2a cells with the Ad-VCAT-Cys67stop viral vector, dramatically alters the distribution of these markers, which now strongly localize with either the Cys67stop protein or the ER marker PDI in large vesicles.
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3. Inhibition of the autophagic-lysosomal pathway in Cys67stop-expressing cells induces apoptosis
We then asked whether expression of the Cys67stop mutant protein and activation of autophagy in Neuro2a cells affected cell viability. On its own, Ad-VCAT-Cys67 had no effect on the viability of Neuro2a cells as assessed by visual microscopic examination, cell growth assessed by counting, or incorporation of Trypan blue over a time course of 2 or 8 days. However, inhibition of autophagosome formation with 3-methyl adenine (3MA), autophagosome-lysosme fusion with asparagine (Asn), or cathepsin D activity with pepstatin A (Pst) all markedly increased cell death.
To ascertain whether the cell death induced by the inhibition of autophagic-lysosomal protein degradation in Ad-VCAT-Cys67stop infected Neuro2a cells was classically apoptotic in nature, we used flow cytometry to quantify cell populations labeled with either FITC-Annexin V (Fig. 2
A), to identify early apoptotic cells, or PI, to identify the subG1 hypodiploid population (Fig. 2B
). Inhibition of the autophagic-lysosomal pathway with 3MA, Asn, or Pst had no effect on annexin-V binding in Neuro2a cells infected with Ad-VCAT. However, blockade of the autophagy-lysosomal pathway in Cys67stop expressing cells doubled the association of FITC-annexin V with the surface phosphatidylserine, indicative of early apoptosis. Similarly, treatment of Ad-VCAT-Cys67stop-infected Neuro2a cells with Asn or Pst significantly increased the subG1 population while having no effect on Ad-VCAT-infected cells. The presence of characteristic chromatin alterations was also evidenced by DAPI staining, which revealed nuclear condensation and fragmentation in Asn- and Pst-treated Ad-VCAT-Cys67stop-infected, but not Ad-VCAT-infected cells (Fig. 2C
). We then assessed cytochrome c release from mitochondria and caspase activation. Asn and Pst treatment resulted in a reduction in mitochondrial integrity, as assessed by cytochrome c immunocytochemistry in Ad-VCAT-Cys67stop-infected, but not Ad-VCAT-infected cells (Fig. 2D
). Finally, we showed that 3MA, Asn, and Pst treatments all activated caspases in Ad-VCAT-Cys67stop-infected Neuro2a cells, but not in Ad-VCAT-infected cells (Fig. 2E
).
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CONCLUSIONS AND SIGNIFICANCE
This study provides evidence that the activation of autophagy by the cellular stress of the expression of an adFNDI mutant protein gene is a pro-survival mechanism. Following delivery into Neuro2a cells, wild-type VP gene products enter neurite processes and accumulate in terminals. In contrast, the Cys67stop adFNDI mutant protein is confined to enlarged vesicles of ER origin in the cell body (Fig. 3
), and colocalizes with markers of autophagy (Fig. 3)
. Cys67stop expression had no effect on cell viability, but inhibition of autophagy or lysosomal protein degradation significantly increased apoptotic cell death.
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We suggest that autophagy might play a role in the etiology of adFNDI (Fig. 3)
. The accumulation of mutant Cys67stop protein in the ER results in the development of a pathology characterized by a grossly deranged ER that accumulates both mutant and trapped wild-type protein. 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. By analogy, we can hypothesize that the activation of autophagy seen in Parkinson’s, Huntington’s and Alzheimer’s neurons may also have a pro-survival role, at least in the early stages of these diseases. Indeed, recent evidence suggests that, as in our adFNDI model, the induction of autophagy in Huntington’s disease decreases the accumulation and toxicity of polyglutamine aggregates.
FOOTNOTES
1 Present address: Cambridge Institute for Medical Research (CIMR), Wellcome Trust/MRC Building, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2XY, England. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3162fje; doi: 10.1096/fj.04-3162fje
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