Endoplasmic reticulum chaperone BiP/GRP78 knockdown leads to autophagy and cell death of arginine vasopressin neurons in mice

The immunoglobulin heavy chain binding protein (BiP), also referred to as 78-kDa glucose-regulated protein (GRP78), is a pivotal endoplasmic reticulum (ER) chaperone which modulates the unfolded protein response under ER stress. Our previous studies showed that BiP is expressed in arginine vasopressin (AVP) neurons under non-stress conditions and that BiP expression is upregulated in proportion to the increased AVP expression under dehydration. To clarify the role of BiP in AVP neurons, we used a viral approach in combination with shRNA interference for BiP knockdown in mouse AVP neurons. Injection of a recombinant adeno-associated virus equipped with a mouse AVP promoter and BiP shRNA cassette provided specific BiP knockdown in AVP neurons of the supraoptic (SON) and paraventricular nuclei (PVN) in mice. AVP neuron-specific BiP knockdown led to ER stress and AVP neuronal loss in the SON and PVN, resulting in increased urine volume due to lack of AVP secretion. Immunoelectron microscopy of AVP neurons revealed that autophagy was activated through the process of AVP neuronal loss, whereas no obvious features characteristic of apoptosis were observed. Pharmacological inhibition of autophagy by chloroquine exacerbated the AVP neuronal loss due to BiP knockdown, indicating a protective role of autophagy in AVP neurons under ER stress. In summary, our results demonstrate that BiP is essential for the AVP neuron system.

leading to UPR activation 13,14 . It was also shown that BiP expression is increased by ER stress 11,15,16 , and has been used as a general indicator of ER stress in addition to the UPR 17 .
Arginine vasopressin (AVP), an antidiuretic hormone that promotes water reabsorption in the kidney, is mainly synthesized in the magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) in the hypothalamus 18 . AVP mRNA expression in the SON and PVN is relatively high, and AVP synthesis and release are upregulated by only a 1-2% increase in plasma osmolality 19 , suggesting that AVP neurons must meet a large demand for AVP production as specialized secretory cells. AVP precursors are subjected to proper folding in the ER 20,21 , and through the folding process, some degree of AVP precursors undergo ER-associated degradation (ERAD) 22 . Furthermore, knockout of the Sel1L-Hrd1 protein complex, a principal ER-resident E3 ligase in mammalian ERAD, is reported to cause marked retention and aggregation of AVP precursors in the ER, resulting in polyuria due to AVP deficiency 23 . These data indicate that ER protein quality control is essential for appropriate AVP synthesis and release. Indeed, ER stress has been implicated in the pathophysiology of some genetic types of central diabetes insipidus such as familial neurohypophysial diabetes insipidus (FNDI) which is caused by the accumulation of mutant AVP precursors in the ER [24][25][26][27][28][29][30] .
Our previous studies showed that BiP is expressed in AVP neurons under non-stress conditions and that its expression is upregulated in proportion to the increase in AVP expression under dehydration 31 . Furthermore, we also demonstrated that AVP release is impaired in dehydrated ATF6α knockout mice in which BiP is not properly upregulated in response to ER stress 29 . While these data suggest that BiP might be involved in the synthesis and release of AVP, the role of BiP in the AVP neuron system has not been fully clarified.
In the present study, we specifically ablated BiP expression in AVP neurons by utilizing virally-mediated shRNA interference and analyzed the morphology of AVP neurons as well as fluid homeostasis in mice.

Validation of recombinant adeno-associated virus (rAAV) vectors and BiP shRNA in mouse AVP neurons.
For the validation of our rAAV vectors in mouse AVP neurons, we injected rAAV carrying an AVP promoter and Venus cDNA (rAAV-AVPp-Venus) into the SON and analyzed the proportion of Venusexpressing cells by immunostaining for Venus, AVP and oxytocin (OT) 2 weeks after virus injection (Fig. 1A). Results showed that 95.7% of AVP neurons (314/328 cells) expressed Venus, and 94.3% of Venus-positive cells (314/333 cells) were AVP neurons (Fig. 1B). In contrast, only 1.5% of Venus-positive cells (5/333 cells) expressed OT, which included Venus-positive cells simultaneously expressing AVP and OT (4/333 cells). These data demonstrate that our rAAVs carrying the AVP promoter were almost completely restricted to AVP neurons in mice, as shown in rats 32 .
To specifically ablate BiP in AVP neurons, rAAVs harboring the AVP promoter sequence followed by BiP shRNA were employed. We injected rAAV-AVPp-BiP shRNA into the bilateral SON and PVN for BiP knockdown in AVP neurons and rAAV-AVPp-scrambled shRNA as a control. The knockdown efficiency 2 weeks after rAAV-AVPp-BiP shRNA injection was 51.9% in the SON and 50.2% in the PVN, respectively (Fig. 1C,D).
AVP neuron-specific BiP knockdown increases urine volume. Next, we compared urine volumes, water intake, body weight, plasma osmolality and urine AVP concentrations between mice injected with rAAV-AVPp-scrambled shRNA and rAAV-AVPp-BiP shRNA into the bilateral SON and PVN up to 12 weeks after virus injection. 4 weeks after virus injection, urine volume ( Fig. 2A) as well as water intake (Fig. 2B) increased significantly in mice injected with rAAV-AVPp-BiP shRNA compared with control mice, while no significant differences were observed in body weight (data not shown). Furthermore, urine AVP concentrations were significantly decreased (Fig. 2C) and plasma osmolality was significantly elevated (Fig. 2D) 4 weeks after injection with rAAV-AVPp-BiP shRNA compared to control mice. AVP neuron-specific BiP knockdown leads to loss of AVP neurons. To examine the effects of BiP knockdown on AVP neuronal viability, we counted AVP neurons in the SON and PVN of mice injected with rAAV-AVPp-BiP shRNA or rAAV-AVPp-scrambled shRNA into the bilateral SON and PVN as well as un-injected mice (Fig. 3A). There were no significant differences in the number of AVP neurons between the un-injected and scrambled shRNA groups (Fig. 3B,C). In contrast, while mice injected with rAAV-AVPp-BiP shRNA showed no significant changes 2 weeks after injection, AVP neurons were markedly lost 4 and 12 weeks after virus injection; the degree of neuronal loss was similar between these two time points (Fig. 3B,C). These data indicate that AVP neuronal loss occurred between 2 and 4 weeks after rAAV injection and that BiP is essential for the AVP neuron system. AVP neuron-specific BiP knockdown leads to ER stress in AVP neurons. Immunoelectron microscopy revealed that BiP knockdown in AVP neurons led to dilatation of the ER lumen 2 weeks post-injection at a time point when AVP neurons were not yet lost, whereas a normal ER lumen was observed in AVP neurons of control mice injected with rAAV-AVPp-scrambled shRNA (Fig. 4A-D). Quantitative analyses found that the ratio of ER area to cytoplasm was increased in AVP neurons of mice injected with rAAV-AVPp-BiP shRNA compared to control mice (Fig. 4E). The expression of mRNA for the ER stress markers C/EBP homologous protein (CHOP) and spliced X-box binding protein 1 (XBP1) was upregulated in the PVN of BiP shRNA-injected mice 2 weeks after injection (Fig. 4F,G). These data demonstrate that AVP neuron-specific BiP knockdown leads to ER stress in AVP neurons prior to cell death.
AVP neuron-specific BiP knockdown activates autophagy in AVP neurons through the process of AVP neuronal loss. ER stress and apoptosis are known to be closely related 33 Fig. 1). Given that AVP neuronal loss occurred between 2 and 4 weeks after injection with rAAV-AVPp-BiP shRNA, we performed electron microscopy of AVP neurons 3 and 4 weeks after injection to detect morphological changes in the dying AVP neurons. While nuclear structure was relatively well preserved, autophagic vacuoles containing degraded organelles were observed 3 weeks after BiP shRNA injection (Fig. 5A,B). Furthermore, whereas no obvious morphological changes were found in AVP neurons of control mice 4 weeks after injection with rAAV-AVPp-scrambled shRNA (Fig. 5C), large vacuoles containing various organelles undergoing degradation were detected in some cells by 4 weeks after injection with BiP shRNA (Fig. 5D). For quantification of autophagic activity in AVP neurons under BiP knockdown, we compared the number of autophagic vacuoles in AVP neurons of mice 2 weeks after injection of scrambled or BiP shRNA with or without chloroquine treatment, a lysosomal inhibitor which hampers the autophagic degradation process (Fig, 5E-H). In scrambled shRNA-injected mice, autophagic vacuoles in AVP neurons of chloroquine-treated mice were significantly increased compared to mice without chloroquine administration (Fig. 5I), confirming a constant autophagic flux in AVP neurons and the validity of chloroquine treatment. Autophagic vacuoles in BiP shRNA-injected mice treated with chloroquine were further increased compared to scrambled shRNA-injected mice (Fig. 5I). These results demonstrate that autophagic flux in AVP neurons is activated by AVP neuron specific-BiP knockdown.

Autophagic inhibition exacerbates AVP neuronal loss in AVP neuron-specific BiP knockdown mice.
To examine the role of autophagy through the process of AVP neuronal loss under AVP neuron-specific BiP knockdown, we counted AVP neurons in the SON and PVN of chloroquine-treated mice injected with rAAV-AVPp-BiP shRNA or rAAV-AVPp-scrambled shRNA into the bilateral SON and PVN (Fig. 6A). There were no significant differences in the number of AVP neurons between chloroquine-treated mice 2 and 4 weeks after scrambled shRNA injection (Fig. 6B,C), and the values were similar to those in un-injected mice (Fig. 3B, C). In contrast, AVP neurons were markedly lost in chloroquine-treated mice 2 weeks after BiP shRNA injection (

Discussion
In the present study, we performed BiP knockdown in mouse AVP neurons by injecting rAAV vectors expressing BiP shRNA under the control of an AVP promoter into the SON and PVN. Our data demonstrated that AVP neuron-specific BiP knockdown leads to ER stress and activates autophagy in AVP neurons followed by AVP neuronal loss. Since BiP whole-body knockout mice were reported to be embryonically lethal due to apoptosis of the inner cell mass of the embryo 34 , BiP conditional knockout has been utilized to investigate the role of BiP in a wide range of specific cell types. Previous studies demonstrated that BiP knockout leads to ER stress and the death of various cell types including hepatocytes 35 , adipocytes 36 , myocytes 37 , respiratory epithelial cells 38,39 , hematopoietic cells 40 , Purkinje cells 41 , oligodendrocytes and Schwann cells 42 . In the current study, we also showed that BiP conditional knockdown in AVP neurons led to dilatation of the ER lumen and upregulation of ER stress markers followed by AVP neuronal loss. Furthermore, it is of note that approximately 90% of AVP neurons in the SON and 70% in the PVN were lost by partial knockdown (about 50%) of BiP expression. This finding contrasts with previous studies in which cell death resulted from complete deletion of BiP expression using a conditional knockout strategy. We previously reported that the basal expression of BiP is relatively high in AVP neurons 31 , suggesting that AVP neurons are exposed to ER stress even under basal conditions. This is likely due to the need for AVP neurons to continuously synthesize large amounts of peptides in the ER for the maintenance of water homeostasis. Taken together, this suggests that AVP neurons are vulnerable to ER stress, and that BiP is pivotal in maintaining the AVP neuron system. www.nature.com/scientificreports/ ER stress, UPR and apoptosis are known to be closely related 33 . In almost all previous in vivo BiP whole-body and conditional knockout studies, apoptosis was reported to be involved in the cell death 34,35,[37][38][39][40][41][42] , mainly based on TUNEL assay results. In contrast, we observed almost no TUNEL-positive cells in the SON and PVN of AVP neuron-specific BiP knockdown mice. Cell death has historically been classified into three different forms based on its morphological features: (1) type I cell death or apoptosis, manifesting as cytoplasmic shrinkage, chromatin condensation, nuclear fragmentation and plasma membrane blebbing; (2) type II cell death or autophagy, displaying extensive cytoplasmic vacuolization; and (3) type III cell death or necrosis, exhibiting no specific features of type I or II cell death 43 . Our observations of the ultrastructural morphology of dying AVP neurons revealed the emergence of autophagosome membranes and autolysosomes. In some cells, large vacuoles containing various www.nature.com/scientificreports/ organelles undergoing degradation were present throughout the cytoplasm. Furthermore, we did not find any morphological features characteristic of apoptosis or necrosis. These data suggest that autophagy was activated in AVP neurons during the cell death process induced by BiP knockdown. Investigation into the presence and absence of autophagic flux inhibitors can reveal the dynamic changes in autophagic processes 44 . In the current study, we showed that autophagic vacuoles were significantly increased in AVP neurons of AVP neuron-specific BiP knockdown mice under the pharmacological inhibition of autophagy by chloroquine treatment. These results demonstrate that autophagic flux was activated in AVP neurons after BiP knockdown. The finding that autophagy was activated in AVP neurons following BiP knockdown-induced ER stress is consistent with our previous study showing that ER stress induces autophagy in organotypic cultures of the mouse hypothalamus 28 . ER stress-induced autophagy should primarily be a protective and adaptive mechanism by which misfolded/unfolded proteins and damaged organelles are cleared 45 . However, if excessive autophagy occurs over an extended period due to sustained ER stress, a greater proportion of organelles will be degraded, leading to the perturbation of cellular homeostasis and eventual cell death. This is exemplified in FNDI model mice, for which we previously reported that sustained ER stress caused by the accumulation of mutant AVP precursors in the ER results in the autophagy-associated cell death of AVP neurons 28 . In the present study, BiP knockdown in AVP neurons induced autophagy and AVP neuronal loss; however, pharmacological inhibition of autophagy by chloroquine exacerbated AVP neuronal loss. This indicates a protective role of autophagy in AVP neurons under conditions of ER stress.
Glial cells are the first elements that react to insults of the central nervous system 46 . Activated microglia migrate to and surround damaged or dead cells, clearing cellular debris and activating astrocytes through the production and release of pro-inflammatory cytokines 47 . In the current study, microglia and astrocytes were activated as indicated by the reactive gliosis that occurred in the SON and PVN of AVP neuron-specific BiP knockdown mice. Upregulation of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β was also observed in the PVN after BiP knockdown.  SON (B) and PVN (C) in the cont sh 2wk + CQ, cont sh 4wk + CQ, BiP sh 2wk + CQ and BiP sh 4wk + CQ groups. Results were analyzed by two-way ANOVA followed by a Bonferroni test and are expressed as the means ± SE (n = 3 per group). n.s. not significant.

Scientific Reports
| (2020) 10:19730 | https://doi.org/10.1038/s41598-020-76839-z www.nature.com/scientificreports/ Astrocytes are known to be important components in the regulation of AVP release 48 . Hyperosmotic challenges retract the astrocytic processes surrounding AVP neurons, which facilitates neuronal interactions through synapses and dendritic gap junctions, increasing AVP neuronal excitability and promoting synchronous AVP secretion 49 . In contrast, hypoosmotic challenges inhibit AVP neuronal activity by evoking the expansion of astrocytic processes, wedging them in between neuronal dendrites and lessening dendritic gap junctional connectivity 49 . In the present study, AVP neuron-specific BiP knockdown led not only to the death of AVP neurons but also to the activation of astrocytes. While the increase in urine volume could be mainly attributed to the substantial loss of AVP neurons, activated astrocytes might also contribute to decreased AVP secretion from the residual AVP neurons, as reactive astrogliosis has been reported to induce subsequent perturbation of synaptic homeostasis and the neuronal-glial network 50,51 .
In conclusion, our data demonstrate that BiP knockdown in AVP neurons leads to ER stress and activates autophagy in AVP neurons followed by AVP neuronal loss, suggesting that BiP is essential for the AVP neuron system. In addition, autophagic inhibition exacerbates AVP neuronal loss due to BiP knockdown in AVP neurons, indicating a protective role of autophagy in AVP neurons under conditions of ER stress.

Methods
Animals. C57BL/6J mice were purchased from Chubu Science Materials (Nagoya, Japan). Mice were maintained under controlled conditions (23.0 ± 0.5 °C, lights on 09:00 to 21:00); male mice were used in all experiments. All procedures were approved by the Animal Experimentation Committee of the Nagoya University Graduate School of Medicine and performed in accordance with institutional guidelines for animal care and use.
Stereotaxic targeting of rAAVs into the mouse SON and PVN. Two-month-old mice were anesthetized with 1-2% isoflurane (Wako, Osaka, Japan) using an animal anesthetization device (MA-AT210D, Muromachi Kikai, Tokyo, Japan) and placed on a stereotaxic apparatus (Model 900LS; Kopf Instruments, Tujunga, CA, USA). rAAV injections were performed using glass pipettes prepared from 1-5 μl micropipettes (708707; Brand, Wertheim, Germany) using a glass pipette puller (PC-100; Narishige, Tokyo, Japan). The injection volume of the rAAVs was 200 nl per nucleus. The injection coordinates were as follows, in accordance with the mouse brain atlas 54  Immunohistochemistry. As described previously [28][29][30] , mice were deeply anesthetized and transcardially perfused with a cold fixative containing 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.4. After fixation, brains were removed and immersed in the same fixative for 24 h at 4 °C, then dissected and cut into 50-μm sections on a vibratome (VT1200 S; Leica Biosystems, Wetzlar, Germany). The sections were washed with PBS and 0.3% Triton X-100 in PBS, followed by blocking with a mixture of 5% normal goat serum and 3% bovine serum albumin in PBS for 1 h at room temperature (RT). For immunofluorescence staining, the sections were incubated with primary antibodies overnight at 4 ℃. After being rinsed in PBS with 0.05% Tween 20, slices were treated with corresponding secondary antibodies for 2 h at RT. Fluorescence images were acquired with a laser-scanning confocal microscope (LSM 5 Pascal; Carl Zeiss, Oberkochen, Germany) or a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan) and processed using Adobe Photoshop CS5 (Adobe Systems, San In situ hybridization and quantification. The BiP exonic probe was produced from a 922-bp fragment containing bases 852 to 1773 of the mouse BiP cDNA as described previously 31  The radiolabeled RNA probe was purified using Quick Spin Columns for radiolabeled RNA purification (Roche Diagnostics, Basel, Switzerland), precipitated with ethanol, and resuspended in 100 μl of 10 mM Tris-HCl, pH 7.5 containing 20 mM dithiothreitol. Mice were deeply anesthetized and transcardially perfused with cold 4% PFA in PBS, then brains were removed and immersed in the same fixative for 3 h at 4 °C, followed by cryoprotection in PBS containing 10-20% sucrose at 4 °C. Cryoprotected brains were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical, Tokyo, Japan) and stored at − 80 °C until sectioning. For sectioning, brains were cut into 16-μm sections on a cryostat (CM3050 S; Leica Biosystems) at − 20 °C, thaw-mounted on Superfrost Plus microscope slides (Matsunami Glass Ind., Osaka, Japan), and slices were stored at − 80 °C until in situ hybridization. Prehybridization, hybridization, and post-hybridization procedures were performed as described previously 26 . The sections were exposed to BioMax MR film (Carestream Health, Rochester, NY, USA) for 4 days. Expression levels in the SON and PVN were quantified by measuring the integrated optimal densities (optical densities × area) from the film images using ImageJ software (NIH).

Measurements of urine volume, AVP concentration and plasma osmolality.
Mice were housed in metabolic cages, and 24-h pooled urine was collected and measured throughout the experimental period. Urine AVP concentrations were measured with a radioimmunoassay kit (AVP kit YAMASA; Yamasa, Chiba, Japan). Blood was collected from the submandibular vein and immediately centrifuged for plasma separation. Plasma osmolality was determined using the cryoscopic method (Oriental Yeast Co., Ltd, Tokyo, Japan).
Quantification of AVP neurons. The best-matched slices for the SON and PVN at 0.70 and 0.82 mm caudal from the bregma, respectively, in accordance with the mouse brain atlas 54 , were selected from each mouse for the analyses. The number of cells labeled with an anti-AVP-NP antibody by immunohistochemistry were counted per SON and PVN, and the mean values for each mouse were subjected to statistical analyses. Electron microscopy. As described previously [28][29][30] , mice were deeply anesthetized and transcardially perfused with 4% PFA and 2% glutaraldehyde (GA) in PBS. Brains were then immersed in the same fixative for 3 h at 4 °C. After fixation, brains were cut into 100-μm sections on a vibratome (VT1200 S). Free-floating sections were washed with 0.1 M phosphate buffer (PB) and 0.1% Triton X-100 in PB, followed by incubation with a mouse anti-AVP-NP antibody (PS41) overnight at 4 °C. Sections were then washed with 0.1 M PB and incubated with biotinylated horse anti-mouse IgG (H + L) (1:200) for 2 h at RT. Sections were washed then treated with avidin-biotin complex solution (1:100; Vectastain ABC-HRP kit; PK-4000; Vector Laboratories) for 90 min at RT. Signals were developed with 0.1 M PB containing 0.1% 3,3'-diaminobenzidine dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and 0.004% hydrogen peroxide. The stained sections were further fixed in 2.5% GA in 0.1 M PB overnight at 4 °C, followed by post-fixation with 2% osmium tetroxide for 20 min at 4 °C. Each section was dehydrated in a graded ethanol series, treated with propylene oxide and embedded in epoxy resin (TAAB 812 resin; TAAB Laboratories Equipment, Aldermaston, UK). The resin was polymerized for 48 h at 60 °C. Ultrathin sections (70-nm thickness) including the SON were prepared using an ultramicrotome with a diamond knife (Reichert Ultracut S; Leica Biosystems) and counterstained with lead citrate before analysis with an electron microscope (JEM-1400EX; JEOL, Tokyo, Japan). For quantification of the ER, the total ER area was analyzed using immunoelectron microscopic images from 5 AVP neurons in each group. The area of the ER relative to that of the cytoplasm was determined from digitized images using ImageJ software. Autophagic vacuoles were counted using immunoelectron microscopic images 44 from 7 to 8 AVP neurons in each group, and results were expressed per 10 µm 2 of the cytoplasm.

Scientific Reports
| (2020) 10:19730 | https://doi.org/10.1038/s41598-020-76839-z www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.