Ovol2 promoter mutations in mice and human illuminate species-specific phenotypic divergence.

Pathogenic variants in the highly conserved OVOL2 promoter region cause posterior polymorphous corneal dystrophy (PPCD) 1 by inducing an ectopic expression of the endothelial OVOL2 mRNA. Here we produced an allelic series of Ovol2 promoter mutations in the mouse model including the heterozygous c.-307T>C variant (RefSeq NM_021220.4) causing PPCD1 in humans. Despite the high evolutionary conservation of the Ovol2 promoter, only some alterations of its sequence had phenotypic consequences in mice. Four independent sequence variants in the distal part of the Ovol2 promoter had no significant effect on endothelial Ovol2 mRNA level or caused any ocular phenotype. In contrast, the mutation c.-307T>C resulted in increased Ovol2 expression in the corneal endothelium. However, only a small fraction of adult mice c.-307T>C heterozygotes developed ocular phenotypes such as irido-corneal adhesions, and corneal opacity. Interestingly, phenotypic penetrance was increased at embryonic stages. Notably, c.-307T>C mutation is located next to the Ovol1/Ovol2 transcription factor binding site. Mice carrying an allele with a deletion encompassing the Ovol2 binding site c.-307_-320del showed significant Ovol2 gene upregulation in the cornea endothelium and exhibited phenotypes similar to the c.-307T>C mutation. In conclusion, although the mutations c.-307T>C and -307_-320del lead to a comparably strong increase in endothelial Ovol2 expression as seen in PPCD1 patients, endothelial dystrophy was not observed in the mouse model, implicating species-specific differences in endothelial cell biology. Nonetheless, the emergence of dominant ocular phenotypes associated with Ovol2 promoter variants in mice implies a potential role of this gene in eye development and disease.

Characterization of mice carrying a neurodevelopmental disease-associated GluN2B(L825V) variant.

N-methyl-D-aspartate receptors (NMDARs), encoded by GRIN genes, are ionotropic glutamate receptors playing a critical role in synaptic transmission, plasticity, and synapse development. Genome sequence analyses have identified variants in GRIN genes in patients with neurodevelopmental disorders, but the underlying disease mechanisms are not well understood. Here, we have created and evaluated a transgenic mouse line carrying a missense variant Grin2bL825V , corresponding to a de-novo GRIN2B variant encoding GluN2B(L825V) found in a patient with intellectual disability (ID) and autism spectrum disorder (ASD). We used HEK293T cells expressing recombinant receptors and primary hippocampal neurons prepared from heterozygous Grin2bL825V/+ (L825V/+) and wild-type Grin2b+/+ (+/+) male and female mice to assess the functional impact of the variant. Whole-cell NMDAR currents were reduced in neurons prepared from L825V/+ compared to +/+ mice. Peak amplitude of NMDAR-mediated evoked excitatory postsynaptic currents (NMDAR-eEPSC) was not changed, but NMDAR-eEPSCs in L825V/+ neurons had faster deactivation compared to +/+ neurons and were less sensitive to a GluN2B-selective antagonist ifenprodil. Together, these results suggest a decreased functional contribution of GluN2B subunits to synaptic NMDAR currents in hippocampal neurons from L825V/+ mice. The analysis of the GluN2B(L825V) subunit surface expression and synaptic localization revealed no differences compared to wild-type GluN2B. Behavioral testing of mice of both sexes demonstrated hypoactivity, anxiety, and impaired sensorimotor gating in the L825V/+ strain, particularly affecting males, as well as cognitive symptoms. The heterozygous L825V/+ mouse offers a clinically relevant model of GRIN2B-related ID/ASD and our results suggest synaptic-level functional changes that may contribute to neurodevelopmental pathology.Significance statement Variants in genes for subunits of N-methyl-D-aspartate receptors (NMDARs), a subtype of ionotropic glutamate receptors, are associated with neurodevelopmental disorders. Here we have generated a transgenic mouse model of a de-novo missense GRIN2B gene variant, identified in a patient with intellectual disability and autism, that introduces a single amino acid substitution (L825V) in the NMDAR GluN2B subunit. Di- and triheteromeric NMDARs containing the GluN2B(L825V) subunit have a reduced channel open probability. Synaptic NMDAR currents in neurons from heterozygous L825V/+ mice have accelerated deactivation and reduced ifenprodil sensitivity, suggesting synaptic loss of GluN2B function. L825V/+ mice show increased anxiety, impaired sensorimotor gating, and cognitive deficits, consistent with patient symptoms. Our study describes a clinically relevant mouse model of GRIN2B-related neurodevelopmental pathology.

A structural model of the iRhom-ADAM17 sheddase complex reveals functional insights into its trafficking and activity.

Several membrane-anchored signal mediators such as cytokines (e.g. TNFα) and growth factors are proteolytically shed from the cell surface by the metalloproteinase ADAM17, which, thus, has an essential role in inflammatory and developmental processes. The membrane proteins iRhom1 and iRhom2 are instrumental for the transport of ADAM17 to the cell surface and its regulation. However, the structure-function determinants of the iRhom-ADAM17 complex are poorly understood. We used AI-based modelling to gain insights into the structure-function relationship of this complex. We identified different regions in the iRhom homology domain (IRHD) that are differentially responsible for iRhom functions. We have supported the validity of the predicted structure-function determinants with several in vitro, ex vivo and in vivo approaches and demonstrated the regulatory role of the IRHD for iRhom-ADAM17 complex cohesion and forward trafficking. Overall, we provide mechanistic insights into the iRhom-ADAM17-mediated shedding event, which is at the centre of several important cytokine and growth factor pathways.

NPFFR2-deficient mice fed a high-fat diet develop strong intolerance to glucose.

A previous study on neuropeptide FF receptor 2 (NPFFR2)-deficient mice has demonstrated that NPFFR2 is involved in the control of energy balance and thermogenesis. Here, we report on the metabolic impact of NPFFR2 deficiency in male and female mice that were fed either a standard diet (STD) or a high-fat diet (HFD) and each experimental group consisted of ten individuals. Both male and female NPFFR2 knockout (KO) mice exhibited severe glucose intolerance that was exacerbated by a HFD diet. In addition, reduced insulin pathway signaling proteins in NPFFR2 KO mice fed a HFD resulted in the development of hypothalamic insulin resistance. HFD feeding did not cause liver steatosis in NPFFR2 KO mice of either sex, but NPFFR2 KO male mice fed a HFD had lower body weights, white adipose tissues, and liver and lower plasma leptin levels compared with their wild-type (WT) controls. Lower liver weight in NPFFR2 KO male mice compensated for HFD-induced metabolic stress by increased liver PPARα and plasma FGF21 hepatokine, which supported fatty acid ß-oxidation in the liver and white adipose tissue. Conversely, NPFFR2 deletion in female mice attenuated the expression of Adra3ß and Pparγ, which inhibited lipolysis in adipose tissue.

Bardet-Biedl Syndrome ciliopathy is linked to altered hematopoiesis and dysregulated self-tolerance.

Bardet-Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by the dysfunction of primary cilia. The immune system of patients with ciliopathies has not been investigated. However, there are multiple indications that the impairment of the processes typically associated with cilia may have influence on the hematopoietic compartment and immunity. In this study, we analyze clinical data of BBS patients and corresponding mouse models carrying mutations in Bbs4 or Bbs18. We find that BBS patients have a higher prevalence of certain autoimmune diseases. Both BBS patients and animal models have altered red blood cell and platelet compartments, as well as elevated white blood cell levels. Some of the hematopoietic system alterations are associated with BBS-induced obesity. Moreover, we observe that the development and homeostasis of B cells in mice is regulated by the transport complex BBSome, whose dysfunction is a common cause of BBS. The BBSome limits canonical WNT signaling and increases CXCL12 levels in bone marrow stromal cells. Taken together, our study reveals a connection between a ciliopathy and dysregulated immune and hematopoietic systems.

CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine remodeling.

Regulation of axon guidance and pruning of inappropriate synapses by class 3 semaphorins are key to the development of neural circuits. Collapsin response mediator protein 2 (CRMP2) has been shown to regulate axon guidance by mediating semaphorin 3A (Sema3A) signaling; however, nothing is known about its role in synapse pruning. Here, using newly generated crmp2-/- mice we demonstrate that CRMP2 has a moderate effect on Sema3A-dependent axon guidance in vivo, and its deficiency leads to a mild defect in axon guidance in peripheral nerves and the corpus callosum. Surprisingly, crmp2-/- mice display prominent defects in stereotyped axon pruning in hippocampus and visual cortex and altered dendritic spine remodeling, which is consistent with impaired Sema3F signaling and with models of autism spectrum disorder (ASD). We demonstrate that CRMP2 mediates Sema3F signaling in primary neurons and that crmp2-/- mice display ASD-related social behavior changes in the early postnatal period as well as in adults. Together, we demonstrate that CRMP2 mediates Sema3F-dependent synapse pruning and its dysfunction shares histological and behavioral features of ASD.

Efficient allele conversion in mouse zygotes and primary cells based on electroporation of Cre protein.

Cre-loxP recombination system is a powerful tool for genome engineering. One of its applications is found in genetic mouse models that often require to induce Cre recombination in preimplantation embryos. Here, we describe a technically simple, affordable and highly efficient protocol for Cre protein delivery into mouse zygotes by electroporation. We show that electroporation based delivery of Cre has no negative impact on embryo survival and the method can be easily combined with in vitro fertilization resulting in a significantly faster generation of desired models. Lastly, we demonstrate that Cre protein electroporation is suitable for allelic conversion in primary cells derived from conditional mouse models.

The iRhom homology domain is indispensable for ADAM17-mediated TNFα and EGF receptor ligand release.

Membrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.

CRL4-DCAF12 Ubiquitin Ligase Controls MOV10 RNA Helicase during Spermatogenesis and T Cell Activation.

Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an "ancient" RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.

KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype.

Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy.

Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif.

The formation of mineralized tissues is governed by extracellular matrix proteins that assemble into a 3D organic matrix directing the deposition of hydroxyapatite. Although the formation of bones and dentin depends on the self-assembly of type I collagen via the Gly-X-Y motif, the molecular mechanism by which enamel matrix proteins (EMPs) assemble into the organic matrix remains poorly understood. Here we identified a Y/F-x-x-Y/L/F-x-Y/F motif, evolutionarily conserved from the first tetrapods to man, that is crucial for higher order structure self-assembly of the key intrinsically disordered EMPs, ameloblastin and amelogenin. Using targeted mutations in mice and high-resolution imaging, we show that impairment of ameloblastin self-assembly causes disorganization of the enamel organic matrix and yields enamel with disordered hydroxyapatite crystallites. These findings define a paradigm for the molecular mechanism by which the EMPs self-assemble into supramolecular structures and demonstrate that this process is crucial for organization of the organic matrix and formation of properly structured enamel.

The iRhom2/ADAM17 Axis Attenuates Bacterial Uptake by Phagocytes in a Cell Autonomous Manner.

Uptake of bacteria by phagocytes is a crucial step in innate immune defence. Members of the disintegrin and metalloproteinase (ADAM) family critically control the immune response by limited proteolysis of surface expressed mediator molecules. Here, we investigated the significance of ADAM17 and its regulatory adapter molecule iRhom2 for bacterial uptake by phagocytes. Inhibition of metalloproteinase activity led to increased phagocytosis of pHrodo labelled Gram-negative and -positive bacteria (E. coli and S. aureus, respectively) by human and murine monocytic cell lines or primary phagocytes. Bone marrow-derived macrophages showed enhanced uptake of heat-inactivated and living E. coli when they lacked either ADAM17 or iRhom2 but not upon ADAM10-deficiency. In monocytic THP-1 cells, corresponding short hairpin RNA (shRNA)-mediated knockdown confirmed that ADAM17, but not ADAM10, promoted phagocytosis of E. coli. The augmented bacterial uptake occurred in a cell autonomous manner and was accompanied by increased release of the chemokine CXCL8, less TNFα release and only minimal changes in the surface expression of the receptors TNFR1, TLR6 and CD36. Inhibition experiments indicated that the enhanced bacterial phagocytosis after ADAM17 knockdown was partially dependent on TNFα-activity but not on CXCL8. This novel role of ADAM17 in bacterial uptake needs to be considered in the development of ADAM17 inhibitors as therapeutics.

A novel PSMA/GCPII-deficient mouse model shows enlarged seminal vesicles upon aging.

BACKGROUND: Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is an important diagnostic and therapeutic target in prostate cancer. PSMA/GCPII is also expressed in many healthy tissues, but its function has only been established in the brain and small intestine. Several research groups have attempted to produce PSMA/GCPII-deficient mice to study the physiological role of PSMA/GCPII in detail. The outcomes of these studies differ dramatically, ranging from embryonic lethality to production of viable PSMA/GCPII-deficient mice without any obvious phenotype. METHODS: We produced PSMA/GCPII-deficient mice (hereafter also referred as Folh1-/- mice) by TALEN-mediated mutagenesis on a C57BL/6NCrl background. Using Western blot and an enzyme activity assay, we confirmed the absence of PSMA/GCPII in our Folh1-/- mice. We performed anatomical and histopathological examination of selected tissues with a focus on urogenital system. We also examined the PSMA/GCPII expression profile within the mouse urogenital system using an enzyme activity assay and confirmed the presence of PSMA/GCPII in selected tissues by immunohistochemistry. RESULTS: Our Folh1-/- mice are viable, breed normally, and do not show any obvious phenotype. Nevertheless, aged Folh1-/- mice of 69-72 weeks exhibit seminal vesicle dilation, which is caused by accumulation of luminal fluid. This phenotype was also observed in Folh1+/- mice; the overall difference between our three cohorts (Folh1-/- , Folh1+/- , and Folh1+/+ ) was highly significant (P < 0.002). Of all studied tissues of the mouse urogenital system, only the epididymis appeared to have a physiologically relevant level of PSMA/GCPII expression. Additional experiments demonstrated that PSMA/GCPII is also present in the human epididymis. CONCLUSIONS: In this study, we provide the first evidence characterizing the reproductive tissue phenotype of PSMA/GCPII-deficient mice. These findings will help lay the groundwork for future studies to reveal PSMA/GCPII function in human reproduction.

In vivo regulation of the A disintegrin and metalloproteinase 10 (ADAM10) by the tetraspanin 15.

A disintegrin and metalloproteinase 10 (ADAM10) plays a major role in the ectodomain shedding of important surface molecules with physiological and pathological relevance including the amyloid precursor protein (APP), the cellular prion protein, and different cadherins. Despite its therapeutic potential, there is still a considerable lack of knowledge how this protease is regulated. We have previously identified tetraspanin15 (Tspan15) as a member of the TspanC8 family to specifically associate with ADAM10. Cell-based overexpression experiments revealed that this binding affected the maturation process and surface expression of the protease. Our current study shows that Tspan15 is abundantly expressed in mouse brain, where it specifically interacts with endogenous ADAM10. Tspan15 knockout mice did not reveal an overt phenotype but showed a pronounced decrease of the active and mature form of ADAM10, an effect which augmented with aging. The decreased expression of active ADAM10 correlated with an age-dependent reduced shedding of neuronal (N)-cadherin and the cellular prion protein. APP α-secretase cleavage and the expression of Notch-dependent genes were not affected by the loss of Tspan15, which is consistent with the hypothesis that different TspanC8s cause ADAM10 to preferentially cleave particular substrates. Analyzing spine morphology revealed no obvious differences between Tspan15 knockout and wild-type mice. However, Tspan15 expression was elevated in brains of an Alzheimer's disease mouse model and of patients, suggesting that upregulation of Tspan15 expression reflects a cellular response in a disease state. In conclusion, our data show that Tspan15 and most likely also other members of the TspanC8 family are central modulators of ADAM10-mediated ectodomain shedding in vivo.

Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.

Kallikrein-related peptidase 5 and seasonal influenza viruses, limitations of the experimental models for activating proteases.

Every year, influenza A virus (IAV) affects and kills many people worldwide. The viral hemagglutinin (HA) is a critical actor in influenza virus infectivity which needs to be cleaved by host serine proteases to exert its activity. KLK5 has been identified as an activating protease in humans with a preference for the H3N2 IAV subtype. We investigated the origin of this preference using influenza A/Puerto Rico/8/34 (PR8, H1N1) and A/Scotland/20/74 (Scotland, H3N2) viruses. Pretreatment of noninfectious virions with human KLK5 increased infectivity of Scotland IAV in MDCK cells and triggered influenza pneumonia in mice. These effects were not observed with the PR8 IAV. Molecular modeling and in vitro enzymatic studies of peptide substrates and recombinant HAs revealed that the sequences around the cleavage site do not represent the sole determinant of the KLK5 preference for the H3N2 subtype. Using mouse Klk5 and Klk5-deficient mice, we demonstrated in vitro and in vivo that the mouse ortholog protease is not an IAV activating enzyme. This may be explained by unfavorable interactions between H3 HA and mKlk5. Our data highlight the limitations of some approaches used to identify IAV-activating proteases.

Profiling system for skin kallikrein proteolysis applied in gene-deficient mouse models.

Kallikrein-related proteases (KLKs) play a critical role in epidermis physiology and have been implicated in skin pathologies such as Netherton syndrome. The contribution of individual KLKs to skin proteolysis is poorly understood. Monitoring of their activities in skin proteome is hampered by overlapping substrate specificities, and there is a need for novel assays. Here, we present a platform of selective and sensitive fluorogenic substrates and inhibitors for profiling KLK5, KLK7 and KLK14. These chemical tools were evaluated using recombinant KLKs and tissue from a unique set of mice deficient in eight combinations of KLKs and their natural regulator LEKTI.

A viable mouse model for Netherton syndrome based on mosaic inactivation of the Spink5 gene.

Netherton syndrome (NS) is caused by mutations in the SPINK5 gene. Several Spink5-deficient mouse models were generated to understand the mechanisms of NS in vivo. However, Spink5-deficiency in mice is associated with postnatal lethality that hampers further analysis. Here we present a viable mouse model for NS generated by mosaic inactivation of the Spink5 gene. We propose that these mice are a valuable experimental tool to study NS, especially for long-term studies evaluating potential therapeutic compounds. Furthermore, we show that mosaic inactivation of a gene using TALENs or CRISPR/Cas9 systems can be used to study lethal phenotypes in adult mice.

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains.

A key aspect of nutrient absorption is the exquisite division of labour across the length of the small intestine, with individual nutrients taken up at different proximal:distal positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum and ileum. By examining the fine-scale longitudinal transcriptional patterns that span the mouse and human small intestine, we instead identified five domains of nutrient absorption that mount distinct responses to dietary changes, and three regional stem cell populations. Molecular domain identity can be detected with machine learning, which provides a systematic method to computationally identify intestinal domains in mice. We generated a predictive model of transcriptional control of domain identity and validated the roles of Ppar-δ and Cdx1 in patterning lipid metabolism-associated genes. These findings represent a foundational framework for the zonation of absorption across the mammalian small intestine.

Ablation of Gabra5 Influences Corticosterone Levels and Anxiety-like Behavior in Mice.

Stress responses are activated by the hypothalamic-pituitary-adrenal axis (HPA axis), culminating in the release of glucocorticoids. During prolonged periods of secretion of glucocorticoids or inappropriate behavioral responses to a stressor, pathologic conditions may occur. Increased glucocorticoid concentration is linked to generalized anxiety, and there are knowledge gaps regarding its regulation. It is known that the HPA axis is under GABAergic control, but the contribution of the individual subunits of the GABA receptor is largely unknown. In this study, we investigated the relationship between the α5 subunit and corticosterone levels in a new mouse model deficient for Gabra5, which is known to be linked to anxiety disorders in humans and phenologs observed in mice. We observed decreased rearing behavior, suggesting lower anxiety in the Gabra5-/- animals; however, such a phenotype was absent in the open field and elevated plus maze tests. In addition to decreased rearing behavior, we also found decreased levels of fecal corticosterone metabolites in Gabra5-/- mice indicating a lowered stress response. Moreover, based on the electrophysiological recordings where we observed a hyperpolarized state of hippocampal neurons, we hypothesize that the constitutive ablation of the Gabra5 gene leads to functional compensation with other channels or GABA receptor subunits in this model.