Knockout Models Research Articles

Naa80 is required for actin N-terminal acetylation and normal hearing in zebrafish.

Actin is a critical component of the eukaryotic cytoskeleton. In animals, actins undergo unique N-terminal processing by dedicated enzymes resulting in mature acidic and acetylated forms. The final step, N-terminal acetylation, is catalyzed by NAA80 in humans. N-terminal acetylation of actin is crucial for maintaining normal cytoskeletal dynamics and cell motility in human cell lines. However, the physiological impact of actin N-terminal acetylation remains to be fully understood. We developed a zebrafish naa80 knockout model and demonstrated that Naa80 acetylates both muscle and non-muscle actins in vivo. Assays with purified Naa80 revealed a preference for acetylating actin N-termini. Zebrafish lacking actin N-terminal acetylation exhibited normal development, morphology, and behavior. In contrast, humans with pathogenic actin variants can present with hypotonia and hearing impairment. Whereas zebrafish lacking naa80 showed no obvious muscle defects or abnormalities, we observed abnormal inner ear development, small otoliths, and impaired response to sound. In conclusion, we have established that zebrafish Naa80 N-terminally acetylates actins in vitro and in vivo, and that actin N-terminal acetylation is essential for normal hearing.

Deletion of Transmembrane protein 184b leads to retina degeneration in mice.

Transmembrane protein 184b (Tmem184b) has been implicated in axon degeneration and neuromuscular junction dysfunction. Notably, Tmem184b exhibits high expression levels in the retina; however, its specific function within this tissue remains poorly understood. To elucidate the role of Tmem184b in the mammalian visual system, we developed a Tmem184b knockout (KO) model for further investigation. Loss of Tmem184b led to significant decreases in both a and b wave amplitudes of scotopic electroretinogram (ERG) and reduced b wave amplitudes of photopic ERG, respectively, reflecting damage to both the photoreceptors and secondary neuronal cells of the retina. Histologic analyses showed a progressive retinal thinning accompanied by the significantly loss of retinal cells including cone, rod, bipolar, horizontal and retinal ganglion cells. The expression levels of photo-transduction-related proteins were down-regulated in KO retina. TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated Uridine-5'-triphosphate [UTP]nick end labelling) and glial fibrillary acidic protein (GFAP)-labelling results suggested the increased cell death and inflammation in the KO mice. RNA-sequencing analysis and GO enrichment analysis revealed that Tmem184b deletion resulted in down-regulated genes involved in various biological processes such as visual perception, response to hypoxia, regulation of transmembrane transporter activity. Taken together, our study revealed essential roles of Tmem184b in the mammalian retina and confirmed the underlying mechanisms including cell death, inflammation and hypoxia pathway in the absence of Tmem184b, providing a potential target for therapeutic and diagnostic development.

9378 Knockdown Of The Cdh2 Gene In Zebrafish Determines Its Role In Terminal Hormone Differentiation

Abstract Disclosure: B.V. Fernandes: None. C. MacRae: None. L.R. Carvalho: None. M. Junqueira: None. Introduction: Congenital hypopituitarism (CH) is characterized by the deficiency of one or more pituitary hormones. Whole exome sequencing (WES) has allowed the identification of novel/rare variants in genetic diseases. Among CH patients, 85% lack a molecular diagnosis. WES approach in a patients born to consanguineous parents identified the homozygous variant (c.865G>A, p. Val289Ile) in the CDH2 gene that produce N-Cadherin protein responsible for cell-cell adhesion. This variant was predicted as likely pathogenic allelic variant by ACMG criteria was found in a patient with GH, TSH, ACTH, LF/FSH deficiencies associated with an ectopic neurohypophysis and pituitary hypoplasia. CDH2 analysis in a cohort of 219 patients with CH, followed in a single Brazilian center, identified 3 unrelated families with heterozygous variants and incomplete penetrance where 2 were novel, in addition to the p. Val289Ile. The deleterious effect of the homozygous variant on cell adhesion properties was confirmed in permanent transfection assays in L1 fibroblast cell lines. To determine the role of the cdh2 gene in pituitary development and hormone production and secretion, we selected zebrafish as an animal model due to its 75% genetic homology with humans. Knockout of cdh2 using Crispr/cas9 genomic edition resulted in 85% lethality of animals at 48 hours post-fertilization (hpf), precluding analysis of its role during pituitary development. Objective: To produce a cdh2 knockdown in zebrafish for phenotypic and molecular analysis. Materials and Methods: The pET28B-RfxCas13d-His plasmid was transformed, linearized, and followed by in vitro transcription to produce Cas13 mRNA. Three guide RNAs targeting the cdh2 gene were designed to guide Cas13 to the target. The Cas13/RNA guide complex was injected into single-cell embryos, and phenotypic analyses were performed under a stereomicroscope until 96 hpf. To evaluate cdh2, prop1, pit1 and gh gene expression by RT-PCR, embryos were collected at 24, 48, 72, and 96 hpf. Results: Knockdown animals exhibited microcephaly, cardiac edema, and microphthalmia during embryonic development. CDH2 expression was decreased by 75% in the first 24 hours of life and began to recover by 48 hours, gradually increasing until 96 hpf when it equaled to WT. gh expression was statistically decreased at 48h compared to WT, with an increased gh expression similar to cdh2 increasing reaching similar WT levels by 96 hpf. There was no statistically significant difference in prop1 and pit1 expression compared to WT in the analyzed periods. Conclusion: The cdh2 knockdown in zebrafish using Cas13 enzyme was effective with cdh2 decreased expression by 75% presenting a phenotype similar to the knockout model. Decreased gh expression at 48h suggests an important role of cdh2 in hormonal terminal differentiation, which should be corroborated by analysis of other hormone expressions. Presentation: 6/2/2024

12466 Dual Bone Defect In Patients With Charge Syndrome

Abstract Disclosure: N. Reyes: None. R.J. Santen: None. S.M. Jan De Beur: None. R. Balasubramanian: None. Chromodomain Helicase DNA-binding protein 7 (Chd7) is an ATP-dependent chromatin modifier that mediates nucleosome remodeling. Chd7 mutations result in CHARGE syndrome characterized by coloboma, heart defects, choanal atresia, growth/developmental delay, ear abnormalities, and hypogonadotropic hypogonadism/Kallmann syndrome (IHH/KS). Testosterone has direct and indirect effects (through estrogen), to increase bone formation and decrease bone resorption. Untreated hypogonadism is associated with decreased bone density and fractures. Reports of fractures and low bone density in CHARGE syndrome patients have been attributed to sex hormone deficiency associated with hypogonadism. However, recent data from a Chd7 mouse knockout model suggests an intrinsic Chd7-related bone defect. In wild-type mice, Chd7 promotes osteogenic differentiation of mesenchymal stem cells. Chd7 deficient mice show markedly reduced osteoblasts. Thus, we propose, a dual bone defect in CHARGE syndrome resulting from impaired osteogenesis from CHD7 and sex steroid deficiency; and increased bone resorption due to lack of sex steroids. Here we present two individuals with CHARGE syndrome with markedly low bone density and literature review supporting the dual bone defect concept. Case 1 is a 30 yo male with a pathogenic CHD7 mutation, multiple congenital anomalies of CHARGE syndrome and hypogonadotropic hypogonadism. He had multiple fractures in his teen age years and a bone density Z score of -5.2 at the lumbar spine and -3.3 at the left femoral neck. Because he did not tolerate androgen therapy, a single infusion of zoledronic acid was administered with no further fractures for eight years following infusion. Case 2 is a male evaluated for IHH/KS and ultimately diagnosed with CHARGE Syndrome based on a pathogenic CHD7 mutation. Despite 7 years of testosterone therapy with full virilization, his bone density remained low at Z-score of -1.9 at the spine and -2.6 at the total hip. Results: Both patients had low bone density, one while untreated and the other after full virilization with exogenous androgens. This raises the question whether the bone defects were related to the CHD7 mutation, to androgen deficiency or both. To answer this question, a literature search examined bone density data in untreated patients with CHARGE syndrome versus IHH/KS. We found substantially lower bone density in untreated CHARGE patients Z score -3.46±0.36 (SEM) (n=13) versus IHH/KS -2.16 ±1.0 (n=4). Furthermore, the bone density was also lower in treated CHARGE patients -2.46±0.28 (n=6) versus IHH/KS -0.94±0.26 (n=140). Conclusion: These data, taken together, provide direct human evidence to support the concept that the CHD7-related CHARGE syndrome patients have a defect in both osteogenesis as well as an effect of sex steroid deficiency on bone. Presentation: 6/3/2024

12439 Investigating The Regulation Of Islet Alpha-cell Function Mediated By The ISL1 And LDB1 Transcriptional Complex

Abstract Disclosure: I. Deeba: None. S. Poole: None. Y. Liu: None. C. Hunter: None. Glucose homeostasis is maintained by the action of insulin-secreting β-cells and glucagon-producing α-cells in the pancreatic islet. Diabetes, traditionally considered a dysglycemia centered on the β-cell, also has clear contributions by dysregulated α-cells. However, little is known of the key transcriptional regulators, including transcription factors (TFs) and interacting co-regulators, driving α-cell development and function. A published pancreas-wide knockout (KO) of the LIM-Homeodomain Islet-1 (ISL1) TF in mice led to a dominant β-cell phenotype, including glucose intolerance and reduced insulin secretion. However, significant reductions of glucagon (Gcg) and Arx mRNA and pancreatic GCG content were also noted, suggesting α-cell impacts. Furthermore, we demonstrated that pancreatic ISL1 activity involves interactions with the LDB1 scaffolding co-regulator. However, nothing is known of the comparative α-cell-specific target genes and functions of these protein partners. To first assess ISL1:LDB1 complexes in α-cells, we performed co-IP and proximity ligation assay (PLA) in mouse α-cell lines and found low-glucose stimulated ISL1 and LDB1 interactions. These observations led us to hypothesize that ISL1 and LDB1 complexes regulate transcription of target genes to drive α-cell function. To assess comparative DNA occupancy, we performed ChIP and observed significant ISL1 and LDB1 binding in conserved Gcg promoter and intronic domains. siRNA-mediated Isl1 or Ldb1 knockdown followed by RNA analysis revealed a similar differential expression of α-cell mRNAs (e.g., Gcg, IL6ra, Sox8, Cox5a, Atp5k, Atp5e), suggesting roles in α-cell function. To compare ISL1 and LDB1 importance in vivo, we developed two novel mouse α-cell ISL1 or LDB1 KO models using Gcg-Cre, termed Isl1Δα and Ldb1Δα, respectively. ISL1 and LDB1 loss was confirmed in α-cells by immunofluorescence. Significant fasting hypoglycemia and hypoglucagonemia were found in postnatal Isl1Δα and Ldb1Δα mice, along with decreased GCG+ cell numbers in Ldb1Δα mice, compared to controls. These findings underscore the crucial roles of these factors in regulating GCG production and secretion. Immunostaining for the MAFB α-cell TF in Ldb1Δα islets revealed potential heterogeneous MAFB expression, marked by MAFB+ cells lacking GCG, or co-expressing INS, each indicating a potential loss of α-cell identity. Our lineage tracing analysis revealed α-cell lineage labeled tomato+ cells in Isl1Δα mice appearing to co-express INS, suggesting transdifferentiation. Currently, inducible α-cell-specific KO mice are in development to investigate the role of ISL1 and LDB1 in adult α-cells. In the future, we will also explore the effects of α-cell-specific ISL1 or LDB1 deficiency during embryogenesis. Overall, our study will benefit efforts in identifying new molecular targets and cell-based therapies to combat diabetes. Presentation: 6/1/2024

Transcriptome and proteome profiling reveals TREM2-dependent and -independent glial response and metabolic perturbation in an Alzheimer’s mouse model

Elucidating the intricate molecular mechanisms of Alzheimer's disease (AD) requires a multidimensional analysis incorporating various omics data. In this study, we employed transcriptome and proteome profiling of AppNL-G-F, a human APP knock-in model of amyloidosis, at the early and mid-stages of amyloid-beta (Aβ) pathology to delineate the impacts of Aβ deposition on brain cells. By contrasting AppNL-G-F mice with TREM2 (Triggering receptor expressed on myeloid cells 2) knockout models, our study further investigates the role of TREM2, a well-known AD risk gene, in influencing microglial responses to Aβ pathology. Our results highlight altered microglial states as a central feature of Aβ pathology, characterized by the significant upregulation of microglia-specific genes related to immune responses such as complement system and antigen presentation, and catabolic pathways such as phagosome formation and lysosome biogenesis. The absence of TREM2 markedly diminishes the induction of these genes, impairs Aβ clearance, and exacerbates dystrophic neurite formation. Importantly, TREM2 is required for the microglial engagement with Aβ plaques and the formation of compact Aβ plaque cores. Furthermore, this study reveals substantial disruptions in energy metabolism and protein synthesis, signaling a shift from anabolism to catabolism in response to Aβ deposition. This metabolic alteration, coupled with a decrease in synaptic protein abundance, occurs independently of TREM2, suggesting the direct effects of Aβ deposition on synaptic integrity and plasticity. In summary, our findings demonstrate altered microglial states and metabolic disruption following Aβ deposition, offering mechanistic insights into Aβ pathology and highlighting the potential of targeting these pathways in AD therapy.

Comparison of the Effects of Inappropriate Meal Timing-Induced and Genetic Models of Circadian Clock Disruption on Uterine mRNA Expression Profiles

BackgroundAccumulating evidence reveals that inappropriate meal timing contributes to the development of lifestyle-related diseases. An underlying mechanism is thought to be the disruption of the intracellular circadian clock in various tissues based on observations in both systemic and tissue-specific clock gene-deficient mice. However, whether the effects of conditional clock gene knockout are comparable to those of inappropriate meal timing remains unclear. ObjectivesThis study aimed to compare the effects of a recently developed 28-h feeding cycle model with those of a core clock gene Bmal1 uterine conditional knockout (Bmal1 cKO) model on uterine mRNA expression profiles. MethodsThe models were generated by subjecting C57BL/6J mice to an 8-h/20-h feeding/fasting cycle for 2 wk and crossing Bmal1-floxed mice with PR-Cre mice. Microarray analyses were conducted using uterine samples obtained at the beginning of the dark and light periods. ResultsThe analyses identified 516 and 346, significantly 4-fold and 2-fold, up- or downregulated genes in the 28-h feeding cycle and Bmal1 cKO groups, respectively, compared with each control group. Among these genes, only 7 (1.4%) and 63 (18.2%) were significantly up- or downregulated in the other model. Moreover, most (n = 44, 62.9%) of these genes were oppositely regulated. These findings were confirmed by gene set enrichment analyses. ConclusionsThis study reveals that a 28-h feeding cycle and Bmal1 cKO differently affect gene expression profiles and highlights the need for considering this difference to assess the pathophysiology of diseases associated with inappropriate meal timing.

Sleepless nights and social plights: medial septum GABAergic hyperactivity in a neuroligin 3-deficient autism model.

Social deficits represent a core symptom domain of autism spectrum disorder (ASD), which is often comorbid with sleep disturbances. In this issue of the JCI, Sun et al. explored a medial septum (MS) circuit linking these behaviors in a neuroligin 3 conditional knockout model of autism. They identified GABAergic neuron hyperactivity following neuroligin 3 deletion in the MS. This hyperactivity resulted in the inhibition of the downstream preoptic area (POA) and hippocampal CA2 region, resulting in sleep loss and social memory deficits, respectively. Inactivating the hyperactive MS GABA neurons or activating the POA or CA2 rescued the behavioral deficits. Together, these findings deepen our understanding of neural circuits underlying social and sleep deficits in ASD.

Deubiquitinases in skeletal muscle - The Underappreciated Side of the Ubiquitination Coin.

Ubiquitination is a post-translational modification that plays important roles in regulating protein stability, function, localization, and protein-protein interactions. Proteins are ubiquitinated via a process involving specific E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. Simultaneously, protein ubiquitination is opposed by deubiquitinating enzymes (DUBs). DUB-mediated deubiquitination can change protein function or fate and recycle ubiquitin to maintain the free ubiquitin pool. Approximately 100 DUBs have been identified in the mammalian genome, and characterized into seven classes (USP, OTU, UCH, MJD, JAMM, MINDY and ZUP classes). Of these 100 DUBs, there has only been relatively limited investigation of 19 specifically in skeletal muscle cells, in vitro or in vivo, using overexpression, knockdown, and knockout models. To date, evidence indicates roles for individual DUBs in regulating aspects of myogenesis, protein turnover, muscle mass, and muscle metabolism. However, the exact mechanism by which these DUBs act (i.e. the specific targets of these DUBs and the type of ubiquitin chains they target) is still largely unknown, underscoring how little we know about DUBs in skeletal muscle. This review endeavors to comprehensively summarize the current state of knowledge of the function of DUBs in skeletal muscle and highlight the opportunities for gaining a greater understanding through further research into this important area of skeletal muscle and ubiquitin biology.

SOST/Sclerostin impairs the osteogenesis and angiogesis in glucocorticoid-associated osteonecrosis of femoral head.

Glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH) is a progressive bone disorder which frequently results in femoral head collapse and hip joint dysfunction. Sclerostin (SOST) is principally secreted by osteocytes in bone and plays an important role in bone homeostasis and homeostasis of skeletal integrity. Our previous study reported that short-term use of glucocorticoid increased serum sclerostin levels. Here this study is aimed to identify whether sclerostin played an essential role in the occurrence and development of GA-ONFH. Glucocorticoid-induced osteonecrosis of femoral head (ARCO stage II) samples were collected and sclerostin staining was conducted. Osteocyte cell line Ocy454, MC3T3-E1 and endothelial cells was used. MC3T3-E1 or endothelial cells were co-cultured with Ocy454 or SOST-silencing Ocy454 in presence of dexamethasone to mimic the crosstalk of various cells in the bone niche. GA-ONFH rat model and SOST knockout model was built to better understand the phenomenon in vivo. Sclerostin was highly concentrated in osteonecrosis patient sample in the necrotic area. Co-culture with osteocytes aggravated the inhibition of dexamethasone on MC3T3-E1 and endothelial cells. Sclerostin derived from osteocytes impaired osteogenesis and angiogenesis via inhibiting the Wnt pathway. In GA-ONFH rat model, SOST knockout ameliorated the incidence of osteonecrosis and improved bone metabolism compared with the wild type group through histological, immunohistochemical and bone metabolic analyses. Sclerostin contribute to pathologic process of GA-ONFH by impairing osteogenesis and angiogenesis.

Functionally redundant roles of ID family proteins in spermatogonial stem cells

Spermatogonial stem cells (SSCs) are essential for sustained sperm production, but SSC regulatory mechanisms and markers remain poorly defined. Studies have suggested that the Id family transcriptional regulator Id4 is expressed in SSCs and involved in SSC maintenance. Here, we used reporter and knockout models to define the expression and function of Id4 in the adult male germline. Within the spermatogonial pool, Id4 reporter expression and inhibitor of DNA-binding 4 (ID4) protein are found throughout the GFRα1+ fraction, comprising the self-renewing population. However, Id4 deletion is tolerated by adult SSCs while revealing roles in meiotic spermatocytes. Cultures of undifferentiated spermatogonia could be established following Id4 deletion. Importantly, ID4 loss in undifferentiated spermatogonia triggers ID3 upregulation, and both ID proteins associate with transcription factor partner TCF3 in wild-type cells. Combined inhibition of IDs in cultured spermatogonia disrupts the stem cell state and blocks proliferation. Our data therefore demonstrate critical but functionally redundant roles of IDs in SSC function.

Fmo5 plays a sex-specific role in goblet cell maturation and mucus barrier formation.

The intestine plays a key role in metabolism, nutrient and water absorption, and provides both physical and immunological defense against dietary and luminal antigens. The protective mucosal lining in the intestine is a critical component of intestinal barrier that when compromised, can lead to increased permeability, a defining characteristic of inflammatory bowel disease (IBD), among other intestinal diseases. Here, we define a new role for the flavin-containing monooxygenase (FMO) family of enzymes in maintaining a healthy intestinal epithelium. Using Caenorhabditis elegans we measure intestinal barrier function, actin expression, and intestinal damage response. In mice, we utilize an intestine-specific, tamoxifen- inducible knockout model of the mammalian homolog of Cefmo-2 , Fmo5, and assess histology, mucus barrier thickness, and goblet cell physiology. We also treat mice with the ER chaperone Tauroursodeoxycholic acid (TUDCA). In nematodes, we find Cefmo-2 is necessary and sufficient for intestinal barrier function, intestinal actin expression, and is induced by intestinal damage. In mice, we find striking changes to the intestine within two weeks following Fmo5 disruption. Alterations include sex-dependent changes in colon epithelial histology, goblet cell localization, and mucus barrier formation. These changes are significantly more severe in female mice, mirroring differences observed in IBD patients. Furthermore, we find increased protein folding stress in Fmo5 knockout animals and successfully rescue the severe female phenotype with addition of a chemical ER chaperone. Together, our results identify a highly conserved and novel role for Fmo5 in the mammalian intestine and support a key role for Fmo5 in maintenance of ER/protein homeostasis and proper mucus barrier formation.

Osteocyte Sptbn1 Deficiency Alters Cell Survival and Mechanotransduction Following Formation of Plasma Membrane Disruptions (PMD) from Mechanical Loading.

We and others have shown that application of high-level mechanical loading promotes the formation of transient plasma membrane disruptions (PMD) which initiate mechanotransduction. We hypothesized that increasing osteocyte cell membrane fragility, by disrupting the cytoskeleton-associated protein β2-spectrin (Sptbn1), could alter osteocytic responses and bone adaptation to loading in a PMD-related fashion. In MLO-Y4 cells, treatment with the spectrin-disrupting agent diamide or knockdown of Sptbn1 via siRNA increased the number of PMD formed by fluid shear stress. Primary osteocytes from an osteocyte-targeted DMP1-Cre Sptbn1 conditional knockout (CKO) model mimicked trends seen with diamide and siRNA treatment and suggested the creation of larger PMD, which repaired more slowly, for a given level of stimulus. Post-wounding cell survival was impaired in all three models, and calcium signaling responses from the wounded osteocyte were mildly altered in Sptbn1 CKO cultures. Although Sptbn1 CKO mice did not demonstrate an altered skeletal phenotype as compared to WT littermates under baseline conditions, they showed a blunted increase in cortical thickness when subjected to an osteogenic tibial loading protocol as well as evidence of increased osteocyte death (increased lacunar vacancy) in the loaded limb after 2weeks of loading. The impaired post-wounding cell viability and impaired bone adaptation seen with Sptbn1 disruption support the existence of an important role for Sptbn1, and PMD formation, in osteocyte mechanotransduction and bone adaptation to mechanical loading.

Influenza induces lung lymphangiogenesis independent of YAP/TAZ activity in lymphatic endothelial cells

The lymphatic system consists of a vessel network lined by specialized lymphatic endothelial cells (LECs) that are responsible for tissue fluid homeostasis and immune cell trafficking. The mechanisms for organ-specific LEC responses to environmental cues are not well understood. We found robust lymphangiogenesis during influenza A virus infection in the adult mouse lung. We show that the number of LECs increases twofold at 7 days post-influenza infection (dpi) and threefold at 21 dpi, and that lymphangiogenesis is preceded by lymphatic dilation. We also show that the expanded lymphatic network enhances fluid drainage to mediastinal lymph nodes. Using EdU labeling, we found that a significantly higher number of pulmonary LECs are proliferating at 7 dpi compared to LECs in homeostatic conditions. Lineage tracing during influenza indicates that new pulmonary LECs are derived from preexisting LECs rather than non-LEC progenitors. Lastly, using a conditional LEC-specific YAP/TAZ knockout model, we established that lymphangiogenesis, fluid transport and the immune response to influenza are independent of YAP/TAZ activity in LECs. These findings were unexpected, as they indicate that YAP/TAZ signaling is not crucial for these processes.

A single-domain antibody targeting factor XII inhibits both thrombosis and inflammation

Factor XII (FXII) is the zymogen of the plasma protease FXIIa that activates the intrinsic coagulation pathway and the kallikrein kinin-system. The role of FXII in inflammation has been obscure. Here, we report a single-domain antibody (nanobody, Nb) fused to the Fc region of a human immunoglobulin (Nb-Fc) that recognizes FXII in a conformation-dependent manner and interferes with FXIIa formation. Nb-Fc treatment inhibited arterial thrombosis in male mice without affecting hemostasis. In a mouse model of extracorporeal membrane oxygenation (ECMO), FXII inhibition or knockout reduced thrombus deposition on oxygenator membranes and systemic microvascular thrombi. ECMO increased circulating levels of D-dimer, alkaline phosphatase, creatinine and TNF-α and triggered microvascular neutrophil adherence, platelet aggregation and their interaction, which were substantially attenuated by FXII blockade. Both Nb-Fc treatment and FXII knockout markedly ameliorated immune complex-induced local vasculitis and anti-neutrophil cytoplasmic antibody-induced systemic vasculitis, consistent with selectively suppressed neutrophil migration. In human blood microfluidic analysis, Nb-Fc treatment prevented collagen-induced fibrin deposition and neutrophil adhesion/activation. Thus, FXII is an important mediator of inflammatory responses in vasculitis and ECMO, and Nb-Fc provides a promising approach to alleviate thrombo-inflammatory disorders.

RING1 missense variants reveal sensitivity of DNA damage repair to H2A monoubiquitination dosage during neurogenesis

Polycomb repressive complex 1 (PRC1) modifies chromatin through catalysis of histone H2A lysine 119 monoubiquitination (H2AK119ub1). RING1 and RNF2 interchangeably serve as the catalytic subunit within PRC1. Pathogenic missense variants in PRC1 core components reveal functions of these proteins that are obscured in knockout models. While Ring1a knockout models remain healthy, the microcephaly and neuropsychiatric phenotypes associated with a pathogenic RING1 missense variant implicate unappreciated functions. Using an in vitro model of neurodevelopment, we observe that RING1 contributes to the broad placement of H2AK119ub1, and that its targets overlap with those of RNF2. PRC1 complexes harboring hypomorphic RING1 bind target loci but do not catalyze H2AK119ub1, reducing H2AK119ub1 by preventing catalytically active complexes from accessing the locus. This results in delayed DNA damage repair and cell cycle progression in neural progenitor cells (NPCs). Conversely, reduced H2AK119ub1 due to hypomorphic RING1 does not generate differential expression that impacts NPC differentiation. In contrast, hypomorphic RNF2 generates a greater reduction in H2AK119ub1 that results in both delayed DNA repair and widespread transcriptional changes. These findings suggest that the DNA damage response is more sensitive to H2AK119ub1 dosage change than is regulation of gene expression.

Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer's disease mouse model.

Characteristic cerebral pathological changes of Alzheimer's disease (AD) such as glucose hypometabolism or the accumulation of cleavage products of the amyloid precursor protein (APP), known as Aβ peptides, lead to sustained endoplasmic reticulum (ER) stress and neurodegeneration. To preserve ER homeostasis, cells activate their unfolded protein response (UPR). The rhomboid-like-protease 4 (RHBDL4) is an enzyme that participates in the UPR by targeting proteins for proteasomal degradation. We demonstrated previously that RHBLD4 cleaves APP in HEK293T cells, leading to decreased total APP and Aβ. More recently, we showed that RHBDL4 processes APP in mouse primary mixed cortical cultures as well. Here, we aim to examine the physiological relevance of RHBDL4 in the brain. We first found that brain samples from AD patients and an AD mouse model (APPtg) showed increased RHBDL4 mRNA and protein expression. To determine the effects of RHBDL4's absence on APP physiology in vivo, we crossed APPtg mice to a RHBDL4 knockout (R4-/-) model. RHBDL4 deficiency in APPtg mice led to increased total cerebral APP and amyloidogenic processing when compared to APPtg controls. Contrary to expectations, as assessed by cognitive tests, RHBDL4 absence rescued cognition in 5-month-old female APPtg mice. Informed by unbiased RNAseq data, we demonstrated in vitro and in vivo that RHBDL4 absence leads to greater levels of active β-catenin due to decreased proteasomal clearance. Decreased β-catenin activity is known to underlie cognitive defects in APPtg mice and AD. Our work suggests that RHBDL4's increased expression in AD, in addition to regulating APP levels, leads to aberrant degradation of β-catenin, contributing to cognitive impairment.

Cerebellar activity in PINK1 knockout rats during volitional gait.

Preclinical models of Parkinson's disease are imperative to gain insight into the neural circuits that contribute to gait dysfunction in advanced stages of the disease. A PTEN-induced putative kinase 1 knockout early-onset model of Parkinson's disease may be a useful rodent model to study the effects of neurotransmitter degeneration caused by a loss of PTEN-induced putative kinase 1 function on brain activity during volitional gait. The goal of this study was to measure changes in neural activity at the cerebellar vermis at 8 months of age. It was found that gait deficits, except run speed, were not significantly different from age-matched wild-type controls, as previously reported. PTEN-induced putative kinase 1 knockout (n = 4) and wild-type (n = 4) rats were implanted with a micro-electrocorticographic array placed over cerebellar vermis Lobules VI (a-c) and VII. Local field potential recordings were obtained during volitional gait across a runway. Power spectral analysis and coherence analysis were used to quantify network oscillatory activity in frequency bands of interest. Cerebellar vermis power was hypoactive in the beta (VIb, VIc and VII) and alpha (VII) bands at cerebellar vermis Lobules VIb, VIc and VII in PTEN-induced putative kinase 1 knockout rats compared with wild-type controls during gait (P < 0.05). These results suggest that gait improvement in PTEN-induced putative kinase 1 knockout rats at 8 months may be a compensatory mechanism attributed to movement corrections caused by a decreased inhibition of the alpha band of cerebellar vermis Lobule VII and beta band of Lobules VIb, VIc and VII. The PTEN-induced putative kinase 1 knockout model may be a valuable tool for understanding the circuit mechanisms underlying gait dysfunction in patients with early-onset Parkinson's disease with a functional loss of PTEN-induced putative kinase 1. Future studies investigating the cerebellar vermis as a potential biomarker and therapeutic target for the treatment of gait dysfunction in Parkinson's disease are warranted.

The Sperm-Associated Antigen 11A (Spag11a) Knockout Mice Display Sub-Fertility and Perturbations in the Sperm Proteome.

Spermatogenesis and sperm maturation that occur in the testis and epididymis respectively are crucial for fertility. Factors secreted by the testicular and epididymal epithelial cells govern the processes of gametogenesis and maturation. Proteins encoded by the sperm-associated antigen 11a (Spag11a) gene are implicated as having a possible role in sperm maturation. However, studies that demonstrate their definite role in fertility and sperm function using knockout models have not yet reported. In this study, Spag11a knockout mice were generated, genotyped and the reproductive parameters (fecundity, sperm count, capacitation, and acrosome reaction) and sperm proteome were determined. Litter size and sperm count were decreased in the Spag11a knockout mice when compared to the wild-type controls. Spermatozoa from the knockout mice were able to undergo capacitation. However, acrosome reaction did not occur in sperm obtained from knockout mice. Structural abnormalities in the head and tail structures were evident in the spermatozoa of knockout mice. Perturbations in the expression of sperm proteins that are involved in gametogenesis were evident. The subfertility observed in Spag11a knockout mice could be a manifestation of lower sperm count, impaired acrosome reactions, and disturbances in the sperm proteome. The results of this study lend further support to the role of Spag11a gene in male gamete function.

Genetic deletion of the apoptosis associated speck like protein containing a card in LysM+ macrophages attenuates spinal cord injury by regulating M1/M2 polarization through ASC-dependent inflammasome signaling axis

Apoptosis associated speck like protein containing a card (ASC), the key adaptor protein of the assembly and activation of canonical inflammasomes, has been found to play a significant role in neuroinflammation after spinal cord injury (SCI). The previous studies indicated that widely block or knockout ASC can ameliorate SCI. However, ASC is ubiquitously expressed in infiltrated macrophages and local microglia, so further exploration is needed on which type of cell playing the key role. In this study, using the LysMcre;Ascflox/flox mice with macrophage-specifc ASC conditional knockout (CKO) and contusive SCI model, we focus on evaluating the specific role of ASC in lysozyme 2 (LysM)+ myeloid cells (mainly infiltrated macrophages) in this pathology. The results revealed that macrophage-specifc Asc CKO exhibited the follow effects: (1) A significant reduction in the numbers of infiltrated macrophages in the all phases of SCI, and activated microglia in the acute and subacute phases. (2) A significant reduction in ASC, caspase-1, interleukin (IL)-1β, and IL-18 compared to control mice. (3) In the acute and subacute phases of SCI, M1 subset differentiation was inhibited, and M2 differentiation was increased. (4) Histology and hindlimb motor recoveries were improved. In conclusion, this study elucidates that macrophage-specific ASC CKO can improve nerve function recovery after SCI by regulating M1/M2 polarization through inhibiting ASC-dependent inflammasome signaling axis. This indicates that ASC in peripheral infiltrated macrophages may play an important role in SCI pathology, at least in mice, could be a potential target for treatment.