Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins\' physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.

Environmental changes can trigger endoplasmic reticulum (ER) stress and misfolded protein accumulation, potentially leading to pre-eclampsia (PE). Amyloid-β (Aβ) is a crucial misfolded protein that can overactivate autophagy. Our study assessed the expression of Aβ1-42 and autophagic activity in PE placental tissues and trophoblasts under ER stress. Placental tissues were surgically collected from normal pregnant women (NP) and pregnant women with late-onset PE (LOPE) delivering through cesarean section. The expression levels of Aβ1-42 were detected in both PE and NP placental tissues, as well as in tunicamycin (TM)-induced HTR-8/SVneo cells. Autophagy-related proteins, such as Beclin-1, the ratio of LC3-II to LC3-I, ATG5, and SQSTM1/p62 in the placental tissues and HTR-8/SVneo cells were measured by Western blot. The number and morphology of autophagosomes were observed using transmission electron microscopy (TEM). Potential targets associated with the unfolded protein response (UPR) in the placental tissues of NP and PE cases were screened using PCR Arrays. The misfolded protein was significantly upregulated in the PE group. In both PE placental tissues and TM-induced HTR-8/SVneo cells, not only was Aβ1-42 upregulated, but also Beclin-1, ATG5, and LC3BII/I were significantly increased, accompanied by an increase in autophagosome count, while SQSTM1/P62 was downregulated. A total of 17 differentially expressed genes (DEGs) associated with the UPR were identified, among which elevated calnexin (CANX) was validated in the placenta from both PE and TM-induced HTR-8/SVneo cells. Autophagy is significantly upregulated in PE cases due to ER stress-induced Aβ1-42 accumulation, likely mediated by autophagy-related proteins involved in the UPR.

Tau protein misfolding and aggregation are pathological hallmarks of Alzheimer\'s disease and over twenty neurodegenerative disorders. However, the molecular mechanisms of tau aggregation in vivo remain incompletely understood. There are two types of tau aggregates in the brain: soluble aggregates (oligomers and protofibrils) and insoluble filaments (fibrils). Compared to filamentous aggregates, soluble aggregates are more toxic and exhibit prion-like transmission, providing seeds for templated misfolding. Curiously, in its native state, tau is a highly soluble, heat-stable protein that does not form fibrils by itself, not even when hyperphosphorylated. In vitro studies have found that negatively charged molecules such as heparin, RNA, or arachidonic acid are generally required to induce tau aggregation. Two recent breakthroughs have provided new insights into tau aggregation mechanisms. First, as an intrinsically disordered protein, tau is found to undergo liquid-liquid phase separation (LLPS) both in vitro and inside cells. Second, cryo-electron microscopy has revealed diverse fibrillar tau conformations associated with different neurodegenerative disorders. Nonetheless, only the fibrillar core is structurally resolved, and the remainder of the protein appears as a \"fuzzy coat\". From this review, it appears that further studies are required (1) to clarify the role of LLPS in tau aggregation; (2) to unveil the structural features of soluble tau aggregates; (3) to understand the involvement of fuzzy coat regions in oligomer and fibril formation.

Recent studies using cell type-specific knockout mouse models have improved our understanding of the pathophysiological relevance of suppressor of lin-12-like-HMG-CoA reductase degradation 1 (SEL1L-HRD1) endoplasmic reticulum-associated (ER-associated) degradation (ERAD); however, its importance in humans remains unclear, as no disease variant has been identified. Here, we report the identification of 3 biallelic missense variants of SEL1L and HRD1 (or SYVN1) in 6 children from 3 independent families presenting with developmental delay, intellectual disability, microcephaly, facial dysmorphisms, hypotonia, and/or ataxia. These SEL1L (p.Gly585Asp, p.Met528Arg) and HRD1 (p.Pro398Leu) variants were hypomorphic and impaired ERAD function at distinct steps of ERAD, including substrate recruitment (SEL1L p.Gly585Asp), SEL1L-HRD1 complex formation (SEL1L p.Met528Arg), and HRD1 activity (HRD1 p.Pro398Leu). Our study not only provides insights into the structure-function relationship of SEL1L-HRD1 ERAD, but also establishes the importance of SEL1L-HRD1 ERAD in humans.

Primary bud necrosis of grape buds is a physiological disorder that leads to decreased berry yield and has a catastrophic impact on the double cropping system in sub-tropical areas. The pathogenic mechanisms and potential solutions remain unknown. In this study, the progression and irreversibility patterns of primary bud necrosis in \'Summer Black\' were examined via staining and transmission electron microscopy observation. Primary bud necrosis was initiated at 60 days after bud break and was characterized by plasmolysis, mitochondrial swelling, and severe damage to other organelles. To reveal the underlying regulatory networks, winter buds were collected during primary bud necrosis progression for integrated transcriptome and metabolome analysis. The accumulation of reactive oxygen species and subsequent signaling cascades disrupted the regulation systems for cellular protein quality. ROS cascade reactions were related to mitochondrial stress that can lead to mitochondrial dysfunction, lipid peroxidation causing damage to membrane structure, and endoplasmic reticulum stress leading to misfolded protein aggregates. All these factors ultimately resulted in primary bud necrosis. Visible tissue browning was associated with the oxidation and decreased levels of flavonoids during primary bud necrosis, while the products of polyunsaturated fatty acids and stilbenes exhibited an increasing trend, leading to a shift in carbon flow from flavonoids to stilbene. Increased ethylene may be closely related to primary bud necrosis, while auxin accelerated cell growth and alleviated necrosis by co-chaperone VvP23-regulated redistribution of auxin in meristem cells. Altogether, this study provides important clues for further study on primary bud necrosis.

Mutations in γ-aminobutyric acid (GABA) transporter 1 (GAT-1)-encoding SLC6A1 have been associated with myoclonic atonic epilepsy and other phenotypes. We determined the patho-mechanisms of the mutant GAT-1, in order to identify treatment targets.
We conducted whole-exome sequencing of patients with myoclonic atonic epilepsy (MAE) and characterized the seizure phenotypes and EEG patterns. We studied the protein stability and structural changes with homology modeling and machine learning tools. We characterized the function and trafficking of the mutant GAT-1 with 3H radioactive GABA uptake assay and confocal microscopy. We utilized different models including a knockin mouse and human astrocytes derived from induced pluripotent stem cells (iPSCs). We focused on astrocytes because of their direct impact of astrocytic GAT-1 in seizures.
We identified four novel SLC6A1 variants associated with MAE and 2 to 4 Hz spike-wave discharges as a common EEG feature. Machine learning tools predicted that the variant proteins are destabilized. The variant protein had reduced expression and reduced GABA uptake due to endoplasmic reticular retention. The consistent observation was made in cortical and thalamic astrocytes from variant-knockin mice and human iPSC-derived astrocytes. The Slc6a+/A288V mouse, representative of MAE, had increased 5-7 Hz spike-wave discharges and absence seizures.
SLC6A1 variants in various locations of the protein peptides can cause MAE with similar seizure phenotypes and EEG features. Reduced GABA uptake is due to decreased functional GAT-1, which, in thalamic astrocytes, could result in increased extracellular GABA accumulation and enhanced tonic inhibition, leading to seizures and abnormal EEGs.

Congenital hypothyroidism with biallelic thyroglobulin (Tg protein, encoded by the TG gene) mutation is an endoplasmic reticulum (ER) storage disease. Many patients (and animal models) grow an enlarged thyroid (goiter), yet some do not. In adulthood, hypothyroid TGcog/cog mice (bearing a Tg-L2263P mutation) exhibit a large goiter, whereas adult WIC rats bearing the TGrdw/rdw mutation (Tg-G2298R) exhibit a hypoplastic thyroid. Homozygous TG mutation has been linked to thyroid cell death, and cytotoxicity of the Tg-G2298R protein was previously thought to explain the lack of goiter in WIC-TGrdw/rdw rats. However, recent studies revealed that TGcog/cog mice also exhibit widespread ER stress-mediated thyrocyte death, yet under continuous feedback stimulation, thyroid cells proliferate in excess of their demise. Here, to examine the relative proteotoxicity of the Tg-G2298R protein, we have used CRISPR-CRISPR-associated protein 9 technology to generate homozygous TGrdw/rdw knock-in mice in a strain background identical to that of TGcog/cog mice. TGrdw/rdw mice exhibit similar phenotypes of defective Tg protein folding, thyroid histological abnormalities, hypothyroidism, and growth retardation. TGrdw/rdw mice do not show evidence of greater ER stress response or stress-mediated cell death than TGcog/cog mice, and both mouse models exhibit sustained thyrocyte proliferation, with comparable goiter growth. In contrast, in WIC-TGrdw/rdw rats, as a function of aging, the thyrocyte proliferation rate declines precipitously. We conclude that the mutant Tg-G2298R protein is not intrinsically more proteotoxic than Tg-L2263P; rather, aging-dependent difference in maintenance of cell proliferation is the limiting factor, which accounts for the absence of goiter in adult WIC-TGrdw/rdw rats.

A hybrid allele that originated from homologous recombination between CEL and its pseudogene (CELP), CEL-HYB1 increases the risk of chronic pancreatitis (CP). Although suggested to cause digestive enzyme misfolding, definitive in vivo evidence for this postulate has been lacking.
CRISPR-Cas9 was used to generate humanized mice harboring the CEL-HYB1 allele on a C57BL/6J background. Humanized CEL mice and C57BL/6J mice were used as controls. Pancreata were collected and analyzed by histology, immunohistochemistry, immunoblotting, and transcriptomics. Isolated pancreatic acini were cultured in vitro to measure the secretion and aggregation of CEL-HYB1 protein. Mice were given caerulein injections to induce acute pancreatitis (AP) and CP.
Pancreata from mice expressing CEL-HYB1 developed pathological features characteristic of focal pancreatitis that included acinar atrophy and vacuolization, inflammatory infiltrates, and fibrosis in a time-dependent manner. CEL-HYB1 expression in pancreatic acini led to decreased secretion and increased intracellular aggregation and triggered endoplasmic reticulum stress compared with CEL. The autophagy levels of pancreata from mice expressing CEL-HYB1 changed at different developmental stages; some aged CEL-HYB1 mice exhibited an accumulation of large autophagic vesicles and impaired autophagy in acinar cells. Administration of caerulein increased the severity of AP/CP in mice expressing CEL-HYB1 compared with control mice, accompanied by higher levels of endoplasmic reticulum stress.
Expression of a humanized form of CEL-HYB1 in mice promotes endoplasmic reticulum stress and pancreatitis through a misfolding-dependent pathway. Impaired autophagy appears to be involved in the pancreatic injury in aged CEL-HYB1 mice. These mice have the potential to be used as a model to identify therapeutic targets for CP.

Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer\'s disease and Parkinson\'s disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD+ synthase-nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including α-synuclein, Tau (K19), amyloid β, and islet amyloid polypeptide. By combining NMR spectroscopy, cross-linking mass spectrometry, and computational modeling, we further reveal that mN3 uses different region of its amphiphilic surface near the active site to directly bind different amyloid client proteins. Our work demonstrates a client recognition mechanism of NMNAT via which it chaperones different amyloid client proteins against pathological aggregation and implies a potential protective role for NMNAT in different amyloid-associated diseases.

Amyloidosis involves stepwise growth of fibrils assembled from soluble precursors. Transthyretin (TTR) naturally folds into a stable tetramer, whereas conditions and mutations that foster aberrant monomer formations facilitate TTR oligomeric aggregation and subsequent fibril extension. We investigated the early assembly of oligomers by WT TTR compared with its V30M and V122I variants. We monitored time-dependent redistribution among monomer, dimer, tetramer, and oligomer contents in the presence and absence of multimeric TTR seeds. The seeds were artificially constructed recombinant multimers that contained 20-40 TTR subunits via engineered biotin-streptavidin (SA) interactions. As expected, these multimer seeds rapidly nucleated TTR monomers into larger complexes, while having less effect on dimers and tetramers. In vivo, SA-induced multimers formed TTR-like deposits in the heart and the kidney following i.v. injection in mice. While all 3 variants prominently deposited glomerulus in the kidney, only V30M resulted in extensive deposition in the heart. The cardiac TTR deposits varied in size and shape and were localized in the intermyofibrillar space along the capillaries. These results are consistent with the notion of monomeric TTR engaging in high-avidity interactions with tissue amyloids. Our multimeric induction approach provides a model for studying the initiation of TTR deposition in the heart.