Arginine vasopressin (AVP) is an antidiuretic hormone synthesized principally in the hypothalamic supraoptic and paraventricular nuclei. The immunoglobulin heavy chain binding protein (BiP), one of the most abundant endoplasmic reticulum (ER) chaperones, is highly expressed in AVP neurons, even under basal conditions. Moreover, its expression is upregulated in proportion to the increase in AVP expression under dehydration. These data suggest that AVP neurons are constantly exposed to ER stress. BiP knockdown in AVP neurons induces ER stress and autophagy, resulting in AVP neuronal loss, indicating that BiP is pivotal in maintaining the AVP neuron system. Furthermore, inhibition of autophagy after BiP knockdown exacerbates AVP neuronal loss, suggesting that autophagy induced under ER stress is a protective cellular mechanism by which AVP neurons cope with ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the AVP gene. It is characterized by delayed-onset progressive polyuria and eventual AVP neuronal loss. In AVP neurons of FNDI model mice, mutant protein aggregates are confined to a specific compartment of the ER, called the ER-associated compartment (ERAC). The formation of ERACs contributes to maintaining the function of the remaining intact ER, and mutant protein aggregates in ERACs undergo autophagic-lysosomal degradation without isolation or translocation from the ER, representing a novel protein degradation system in the ER.

Arginine vasopressin (AVP) is synthesized principally in the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) as a precursor protein called prepro-AVP. This precursor contains a signal peptide, AVP, neurophysin II (NPII), and copeptin. Prepro-AVP is then processed into pro-AVP, which is folded into its native conformation in the endoplasmic reticulum (ER) and packed into secretory granules. AVP, NPII, and copeptin are then cleaved from pro-AVP in the vesicles during axonal transport.

Magnocellular AVP neurons in the SON and PVN project their axons to the posterior pituitary, where AVP is released into the bloodstream [1]. This systemic AVP plays a critical role in water balance homeostasis by promoting the reabsorption of free water in the kidney. Parvocellular AVP neurons, located in the PVN, release AVP into the hypophyseal portal vein. This vein carries AVP to corticotrophs in the anterior pituitary, where AVP stimulates ACTH release in a coordinated manner with CRH [2, 3]. AVP synthesis in and release from the magnocellular AVP neurons are regulated in response to changes in plasma osmolality and blood pressure/volume [4], while the activity of parvocellular AVP neurons is elevated under glucocorticoid deficiency [5, 6].

The ER is an essential organelle that is responsible for protein synthesis, folding, assembly, and transport. Properly folded proteins are transported to the cellular membrane or packed into secretory granules for secretion [7, 8]. However, when misfolded or unfolded proteins accumulate in the ER lumen, it leads to a condition called ER stress [9, 10]. The unfolded protein response (UPR) is a cellular defense mechanism for coping with ER stress whereby the folding capacity and ER-associated degradation (ERAD) are upregulated [11] and the protein load is decreased in the ER [12]. UPR is thus primarily a protective mechanism, but could induce cell death if severe ER stress is prolonged [13].

The mRNA expression of AVP in the SON and PVN is relatively high, and only a 1–2% increase in plasma osmolality can upregulate AVP synthesis and release [14], indicating that AVP neurons have to meet a high demand for AVP production as specialized secretory cells. Indeed, the immunoglobulin heavy chain binding protein (BiP), one of the most abundant ER chaperones and also referred to as the 78-kDa glucose-regulated protein (GRP78) [15-17], was highly expressed in AVP neurons even under basal conditions, and its expression was further upregulated in proportion to the increase in AVP expression under dehydration [18]. These data indicate that AVP neurons are constantly exposed to ER stress, and that elevated AVP synthesis itself causes ER stress, as some proportions of AVP precursors (prepro- and pro-AVP) fail to mature and undergo ERAD through the folding process [19]. ER stress reduced the stability and expression of AVP mRNA by shortening the mRNA poly(A) tail length, resulting in decreases in AVP synthesis [20]. Furthermore, AVP deficiency occurs in patients with Wolfram syndrome [21] caused by mutations of the ER transmembrane protein wolframin (WFS1) [22], which has been shown to play a key role in maintaining ER homeostasis [23], and increased ER stress and AVP deficiency were observed under dehydration in WFS1 knockout mice [24]. These data demonstrate the importance of ER protein quality control for appropriate AVP synthesis and release.

The activating transcription factor 6α (ATF6α) is one of the ER stress sensors [25] that upregulates the transcription of ER chaperones such as BiP and ERAD components to enhance protein folding and degradation when the UPR is under ER stress [25-27]. In ATF6α-knockout mice under dehydration, BiP expression in AVP neurons was not upregulated, leading to an increase in ER stress compared to wild-type mice [28]. ATF6α-knockout mice also showed increased urine volumes after intermittent water deprivation due to the lack of increase in AVP secretion in response to dehydration [28], suggesting that BiP is involved in the synthesis and release of AVP. However, the role of BiP in AVP neurons has yet to be fully clarified because BiP expression is preserved under basal conditions in ATF6α-knockout mice.

To further investigate the role of BiP in AVP neurons, we employed AVP neuron-specific BiP knockdown mice by injecting recombinant adeno-associated virus vectors harboring the AVP promoter sequence followed by BiP shRNA into the bilateral SON and PVN. In AVP neuron-specific BiP knockdown mice, BiP expression levels in the SON and PVN were decreased by approximately 50%, and the ER lumen in AVP neurons was dilated under basal conditions [29].

AVP neuron-specific BiP knockdown induced a loss of approximately 90% of the magnocellular AVP neurons, resulting in increased urine volumes due to AVP deficiency [29]. Notably, a 50% knockdown of BiP expression led to the loss of 90% of the magnocellular AVP neurons, suggesting that magnocellular AVP neurons are vulnerable to ER stress, and that BiP is pivotal in maintaining the magnocellular AVP neuron system. In contrast, only 30% of parvocellular AVP/CRH neurons were lost in AVP neuron-specific BiP knockdown mice, in which the hypothalamic-pituitary-adrenal response was maintained [30]. These differences in the ratios of cell death might be explained by the differences in the amount of protein synthesized within the cells.

ER stress, UPR, and apoptosis are known to be closely related [31]. Furthermore, apoptosis is reported to be involved in the death of various cell types in almost all BiP whole-body [32] and conditional knockout studies [33-39], mainly based on the results of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. In contrast, almost no TUNEL-positive cells were observed in the SON and PVN of AVP neuron-specific BiP knockdown mice [29]. Electron microscopy of AVP neurons revealed no morphological features characteristic of apoptosis or necrosis, but the emergence of autophagosome membranes and autolysosomes was observed [29]. Furthermore, autophagic vacuoles were increased in AVP neurons of AVP neuron-specific BiP knockdown mice under the pharmacological inhibition of autophagy by chloroquine treatment [29]. These results demonstrate that autophagic flux is 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 [40]. Indeed, while BiP knockdown in AVP neurons induced autophagy and AVP neuronal loss in AVP neuron-specific BiP knockdown mice, pharmacological inhibition of autophagy exacerbated AVP neuronal loss [29], indicating a protective role of autophagy in AVP neurons under ER stress conditions (Fig. 1).

Fig. 1

Effects of BiP knockdown in AVP neurons

In AVP neuron-specific BiP knockdown mice, ER stress was induced and autophagy was activated in AVP neurons followed by AVP neuronal loss. Subsequent pharmacological inhibition of autophagy exacerbated AVP neuronal loss, indicating a protective role of autophagy in AVP neurons under ER stress conditions.

Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the AVP gene [41]. Over 80 causal mutations have been identified so far, and most of them are located in the NPII-coding region [42]. FNDI is characterized by gradual appearance of polyuria and eventual loss of AVP neurons. Even though carriers of the disorder show no symptoms at birth, polyuria and polydipsia can appear within several months or years after birth, despite the presence of one normal allele [43]. Autopsies have shown AVP neuronal loss in the hypothalamus in patients with FNDI [44-47]. To better understand the pathophysiology of FNDI, we previously developed an FNDI mouse model by introducing an NPII mutation that causes FNDI in humans [48]. The FNDI mice with heterozygous NPII mutation exhibit increased urine volumes and water intake due to AVP deficiency, which recapitulates the phenotypes of patients with FNDI [48].

Electron microscopic analyses of AVP neurons in FNDI mice revealed that aggregates were confined to a specific compartment of the rough ER, termed the ER-associated compartment (ERAC) [49]. As FNDI mice aged, the size of ERACs in AVP neurons increased in proportion to the rise in urine volumes [48]. In addition, the size of ERACs decreased when AVP synthesis was suppressed in FNDI mice treated with desmopressin, an AVP antagonist [50]. These observations suggest that mutant proteins accumulated in ERACs of AVP neurons in FNDI mice. Interestingly, despite the presence of massive aggregates in the ER of AVP neurons as ERACs, the expression levels of BiP, an ER stress marker, in AVP neurons did not differ between wild-type and FNDI mice at 3 months of age [49]. This suggests that ERACs are formed in order to maintain function in the remainder of the ER by sequestering and confining mutant proteins to the ERACs. Notably, ERAC-like structures have also been reported in other diseases, such as α1-antitrypsin (AT) deficiency [51], familial encephalopathy with neuroserpin inclusion bodies [52], seipinopathy [53], and autosomal dominant retinitis pigmentosa [54, 55]. ERAC formation is considered to mitigate ER stress and improve cellular viability in α1-AT deficiency [51], indicating that it could be a common UPR mechanism shared by several cell types to cope with ER stress.

The degradation of mutant protein aggregates within ERACs was investigated through three-dimensional electron microscopic analyses of AVP neurons in FNDI mice using serial block-face scanning electron microscopy. The results revealed that ERACs were connected to the intact ER lumen through small protrusions and to lysosomes in the cytosol through another protrusion originating from the ER-connected ERAC [56]. This suggests that ERACs have direct connections with both the ER lumen and lysosomes in the cytosolic compartment of AVP neurons, and that mutant protein aggregates in the ER could undergo degradation by lysosomes. Immunofluorescence and immunoelectron microscopy showed that lysosome-associated membrane protein 2 and lysosomal degradation enzyme cathepsin D were localized inside the mutant NPII-positive ERACs [56]. The number of ERACs was decreased by rapamycin treatment, which induced the autophagic-lysosomal degradation system, and increased by the administration of chloroquine, a lysosomal inhibitor [56].

Misfolded or unfolded proteins in the ER are assumed to be targeted for ERAD, which involves translocation of substrates from the ER to the cytosol and degradation by the ubiquitin-proteasome system [57-59]. Knockout of the Sel1L-Hrd1 complex, a principal ER-resident E3 ligase in mammalian ERAD, results in marked retention and aggregation of AVP precursors in the ER, leading to polyuria due to AVP deficiency [60]. While these findings indicate that ERAD is essential to the cellular function of AVP neurons, the evidence from FNDI mice shows that, in addition to ERAD, there exists another mechanism by which protein aggregates could be degraded within ERACs without translocation from the ER to the cytosol in AVP neurons (Fig. 2).

Fig. 2

ERAC formation and protein aggregate degradation in the ERAC

Mutant protein aggregates were confined to the ERAC, a specific compartment of the rough ER, in the AVP neurons of FNDI mice. The pathophysiological significance of the ERACs is to maintain function in the remainder of the ER. ERACs had direct connections with both the ER lumen and lysosomes in the cytosolic compartment, and mutant protein aggregates in the ER underwent degradation by lysosomes.

AVP neurons are constantly exposed to ER stress even under basal conditions since AVP neurons have to meet a large demand for AVP production as specialized secretory cells. BiP knockdown in AVP neurons induced further ER stress and autophagy, resulting in AVP neuronal loss [29, 30]. This indicates that BiP is pivotal in maintaining the AVP neuron system. Furthermore, inhibition of autophagy after BiP knockdown exacerbated AVP neuronal loss [29]. These findings suggest that autophagy induced under ER stress has a protective role as UPR in AVP neurons.

In AVP neurons of FNDI mice, mutant proteins were confined to and accumulated in the specific compartment of the rough ER called the ERAC [49]. ERAC formation is a common UPR shared by several cell types to cope with ER stress, and its pathophysiological significance is to maintain function in the remainder of the ER. Furthermore, mutant protein aggregates in ERACs underwent autophagic-lysosomal degradation without isolation or translocation from the ER [56], which represents a novel protein degradation system in the ER. Further studies on ERAC formation and protein degradation in the ER could lead to the development of novel therapeutic strategies for diseases related to ER stress.

This work was supported in part by JSPS KAKENHI Grant Numbers JP15K19530 (to D.H.); JP16H06280 (to D.H.), Grant-in-Aid for Scientific Research on Innovative Areas—Platforms for Advanced Technologies and Research Resources “Advanced Bioimaging Support”; JP21K08552 (to D.H.); the Alexander von Humboldt Foundation Research Fellowship (to D.H.); the Yamaguchi Endocrine Research Foundation (to D.H.); JSPS KAKENHI Grant Number JP21K20930 (to Y.K.); the Suzuken Memorial Foundation (to H.A.); and the Cooperative Study Programs of the National Institute for Physiological Sciences (to H.A.).

The authors declare no competing interests.

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