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. 2014 Mar 27;5(3):e1148.
doi: 10.1038/cddis.2014.124.

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

D Hagiwara  1 , H Arima  1 , Y Morishita  1 , L Wenjun  1 , Y Azuma  1 , Y Ito  1 , H Suga  1 , M Goto  1 , R Banno  1 , Y Sugimura  1 , A Shiota  2 , N Asai  3 , M Takahashi  3 , Y Oiso  1
Affiliations

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

D Hagiwara et al. Cell Death Dis. .

Abstract

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.

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Figures

Figure 1
Figure 1
Changes in urine volume and AVP. (a) Urine volumes in WT (dashed line) and FNDI mice (solid line) with water access ad libitum (ad lib, open circles) or subjected to WD (closed circles). (b and c) Urine AVP in ad lib and WD groups in WT (b) and FNDI mice (c). Results are expressed as means±S.E. *P<0.05, **P<0.01 compared with ad lib group at each corresponding time point. n=5–6 animals per group. % BW, percent body weight
Figure 2
Figure 2
Effects of WD on inclusion body formation in AVP neurons of FNDI mice. (a) Toluidine blue staining of the SON in 3-month-old FNDI mice with water access ad libitum (ad lib) or WD for 4 weeks (WD4). (b) The number of inclusion bodies with a diameter >4.5 μm in the SON. The data of 3-month-old FNDI mice with water access ad libitum (ad lib) and of those subjected to WD for 1, 2, and 4 weeks (WD1, 2, 4) are shown. Results are expressed as means±S.E. **P<0.01 compared with ad lib group. n=5–7 animals per group. Scale bars, 10 μm
Figure 3
Figure 3
Electron microscopic analysis in AVP neurons of 3-month-old FNDI mice. AVP neurons of the SON in 3-month-old FNDI mice with water access ad libitum (a–d) and of those subjected to WD for 4 weeks (e–h). Higher magnification images of boxed areas in (a, c, e and g) are shown in (b, d, f and h), respectively. The arrowheads in (d) indicate irregular-shaped aggregates confined to some compartments of the ER, which seem to communicate with intact ER lumen, and those in (h) indicate phagophores engulfing the ER, which contains scattered aggregates. Scale bars, 2 μm (a, e and g), 1 μm (c)
Figure 4
Figure 4
Autophagosome formation and p62 expression in AVP neurons of FNDI/GFP-LC3 mice. (a) Immunofluorescence staining for GFP (green) and mutant NPII (magenta) in the SON of 3-month-old FNDI/GFP-LC3 mice with water access ad libitum (ad lib) and of those subjected to WD for 4 weeks (WD4). (b) Immunofluorescence staining for p62 (magenta) and NPII (green) in the SON of 3-month-old FNDI mice in ad lib and WD4 groups. (c) Immunofluorescence staining for p62 (magenta) and GFP-LC3 (green) in the SON of 3-month-old FNDI/GFP-LC3 mice in WD4 group. Scale bars, 10 μm (a and b), 5 μm (c)
Figure 5
Figure 5
Autophagosome formation and p62 expression under ER stress in mouse hypothalamic slice explants. (a) Immunofluorescence staining for GFP in hypothalamic slice explants of GFP-LC3 transgenic mice cultured with vehicle (control) and 1 μM thapsigargin for 24 h. (b) The number of GFP-LC3 puncta per cell. GFP-LC3 puncta in 60–70 cells were counted in each group in a series of equally acquired images. (c) Quantitative real-time RT-PCR analysis for p62 mRNA in hypothalamic slice explants of WT mice cultured with vehicle (veh) and 1 μM thapsigargin for 6, 12 and 24 h. (d) Immunoblot analysis of protein lysates from hypothalamic slice explants of WT mice immunolabeled for p62. Explants were incubated with vehicle (veh) and 1 μM thapsigargin for 6, 12 and 24 h. Results are expressed as means±S.E. *P<0.05, ***P<0.001 compared with vehicle-treated explants. Scale bars, 10 μm
Figure 6
Figure 6
AVP neuronal loss in FNDI mice subjected to WD. (a and b) Immunohistochemistry for NPII in the SON of 5-month-old FNDI mice with water access ad libitum (ad lib) and of those subjected to WD for 12 weeks (WD12). (c) The number of NPII-expressing cells in the SON counted with immunohistochemistry. (d and e) In situ hybridization for AVP hnRNA in the SON of 5-month-old FNDI mice in ad lib and WD12 groups. (f) The number of AVP hnRNA-expressing cells in the SON counted with in situ hybridization. (g and h) Higher magnification images of boxed areas in (d and e), respectively. (i) The numbers of AVP hnRNA grains per cell are shown. Results are expressed as means±S.E. *P<0.05, ***P<0.001 compared with ad lib group. n=5–8 animals per group. Scale bars, 100 μm (a and b), 50 μm (d and e), 20 μm (g and h)
Figure 7
Figure 7
Electron microscopic analysis in AVP neurons of 5-month-old FNDI mice. AVP neurons of the SON in 5-month-old FNDI mice with water access ad libitum (a–d) and of those subjected to WD for 12 weeks (e–h). Higher magnification images of boxed areas in (a, c and e) are shown in (b, d and f), respectively. Scale bars, 2 μm (a, e, g and h), 1 μm (c)
Figure 8
Figure 8
Possible mechanisms of cell death of AVP neurons in FNDI mice. Aggregates are confined to a sub-compartment of the ER, so-called ERAC, in AVP neurons of the SON in FNDI mice. Failure of ERAC formation leads to autophagy. When autophagy continues, the organelles including ER are lost over time, leading to autophagy-associated cell death
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