Ubiquitination of the GluA1 Subunit of AMPA Receptors Is Required for Synaptic Plasticity, Memory, and Cognitive Flexibility.

Activity-dependent changes in the number of AMPA-type glutamate receptors (AMPARs) at the synapse underpin the expression of LTP and LTD, cellular correlates of learning and memory. Post-translational ubiquitination has emerged as a key regulator of the trafficking and surface expression of AMPARs, with ubiquitination of the GluA1 subunit at Lys-868 controlling the post-endocytic sorting of the receptors into the late endosome for degradation, thereby regulating their stability at synapses. However, the physiological significance of GluA1 ubiquitination remains unknown. In this study, we generated mice with a knock-in mutation in the major GluA1 ubiquitination site (K868R) to investigate the role of GluA1 ubiquitination in synaptic plasticity, learning, and memory. Our results reveal that these male mice have normal basal synaptic transmission but exhibit enhanced LTP and deficits in LTD. They also display deficits in short-term spatial memory and cognitive flexibility. These findings underscore the critical roles of GluA1 ubiquitination in bidirectional synaptic plasticity and cognition in male mice.SIGNIFICANCE STATEMENT Subcellular targeting and membrane trafficking determine the precise number of AMPA-type glutamate receptors at synapses, processes that are essential for synaptic plasticity, learning, and memory. Post-translational ubiquitination of the GluA1 subunit marks AMPARs for degradation, but its functional role in vivo remains unknown. Here we demonstrate that the GluA1 ubiquitin-deficient mice exhibit an altered threshold for synaptic plasticity accompanied by deficits in short-term memory and cognitive flexibility. Our findings suggest that activity-dependent ubiquitination of GluA1 fine-tunes the optimal number of synaptic AMPARs required for bidirectional synaptic plasticity and cognition in male mice. Given that increases in amyloid-ß cause excessive ubiquitination of GluA1, inhibiting that GluA1 ubiquitination may have the potential to ameliorate amyloid-ß-induced synaptic depression in Alzheimer's disease.

EHF is essential for epidermal and colonic epithelial homeostasis, and suppresses Apc-initiated colonic tumorigenesis.

Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf-/-) or specifically in the gut epithelium. Ehf-/- mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf-/- mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf-/-mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.

Synthesis of human amyloid restricted to liver results in an Alzheimer disease-like neurodegenerative phenotype.

Several lines of study suggest that peripheral metabolism of amyloid beta (Aß) is associated with risk for Alzheimer disease (AD). In blood, greater than 90% of Aß is complexed as an apolipoprotein, raising the possibility of a lipoprotein-mediated axis for AD risk. In this study, we report that genetic modification of C57BL/6J mice engineered to synthesise human Aß only in liver (hepatocyte-specific human amyloid (HSHA) strain) has marked neurodegeneration concomitant with capillary dysfunction, parenchymal extravasation of lipoprotein-Aß, and neurovascular inflammation. Moreover, the HSHA mice showed impaired performance in the passive avoidance test, suggesting impairment in hippocampal-dependent learning. Transmission electron microscopy shows marked neurovascular disruption in HSHA mice. This study provides causal evidence of a lipoprotein-Aß /capillary axis for onset and progression of a neurodegenerative process.

The testicular soma of Tsc22d3 knockout mice supports spermatogenesis and germline transmission from spermatogonial stem cell lines upon transplantation.

Spermatogonial stem cells (SSCs) are adult stem cells that are slowly cycling and self-renewing. The pool of SSCs generates very large numbers of male gametes throughout the life of the individual. SSCs can be cultured in vitro for long periods of time, and established SSC lines can be manipulated genetically. Upon transplantation into the testes of infertile mice, long-term cultured mouse SSCs can differentiate into fertile spermatozoa, which can give rise to live offspring. Here, we show that the testicular soma of mice with a conditional knockout (conKO) in the X-linked gene Tsc22d3 supports spermatogenesis and germline transmission from cultured mouse SSCs upon transplantation. Infertile males were produced by crossing homozygous Tsc22d3 floxed females with homozygous ROSA26-Cre males. We obtained 96 live offspring from six long-term cultured SSC lines with the aid of intracytoplasmic sperm injection. We advocate the further optimization of Tsc22d3-conKO males as recipients for testis transplantation of SSC lines.

The N-terminal SH2 domain of Syk is required for (hem)ITAM, but not integrin, signaling in mouse platelets.

We have used a novel knockin mouse to investigate the effect of disruption of phosphotyrosine binding of the N-terminal SH2 domain of Syk on platelet activation by GPVI, CLEC-2, and integrin αIIbß3. The Syk(R41Afl/fl) mouse was crossed to a PF4-Cre(+) mouse to induce expression of the Syk mutant in the megakaryocyte/platelet lineage. Syk(R41Afl/fl;PF4-Cre) mice are born at approximately 50% of the expected frequency and have a similar phenotype to Syk(fl/fl;PF4-Cre) mice, including blood-lymphatic mixing and chyloascites. Anastomosis of the venous and lymphatic vasculatures can be seen in the mesenteric circulation accounting for rapid and continuous mixing of the 2 vasculatures. Platelet activation by CLEC-2 and GPVI is abolished in Syk(R41Afl/fl;PF4-Cre) platelets. Syk phosphorylation on Tyr519/20 is blocked in CLEC-2-stimulated platelets, suggesting a model in which binding of Syk via its N-terminal SH2 domain regulates autophosphorylation. In contrast, outside-in signaling by integrin αIIbß3 is not altered, but it is inhibited in the presence of inhibitors of Src and Syk tyrosine kinases. These results demonstrate that αIIbß3 regulates Syk through an ITAM-independent pathway in mice and provide novel insight into the course of events underlying Syk activation and hemITAM phosphorylation by CLEC-2.

Exclusive transmission of the embryonic stem cell-derived genome through the mouse germline.

Gene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell-derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26-Cre males. This cross produces males that are sterile due to a complete cell-autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell-derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene-targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non-ES cell-derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326-333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

Progranulin overexpression in sensory neurons attenuates neuropathic pain in mice: Role of autophagy.

Peripheral or central nerve injury is a frequent cause of chronic pain and the mechanisms are not fully understood. Using newly generated transgenic mice we show that progranulin overexpression in sensory neurons attenuates neuropathic pain after sciatic nerve injury and accelerates nerve healing. A yeast-2-hybrid screen revealed putative interactions of progranulin with autophagy-related proteins, ATG12 and ATG4b. This was supported by colocalization and proteomic studies showing regulations of ATG13 and ATG4b and other members of the autophagy network, lysosomal proteins and proteins involved in endocytosis. The association of progranulin with the autophagic pathway was functionally confirmed in primary sensory neurons. Autophagy and survival were impaired in progranulin-deficient neurons and improved in progranulin overexpressing neurons. Nerve injury in vivo caused an accumulation of LC3b-EGFP positive bodies in neurons of the dorsal root ganglia and nerves suggesting an impairment of autophagic flux. Overexpression of progranulin in these neurons was associated with a reduction of the stress marker ATF3, fewer protein aggregates in the injured nerve and enhanced stump healing. At the behavioral level, further inhibition of the autophagic flux by hydroxychloroquine intensified cold and heat nociception after sciatic nerve injury and offset the pain protection provided by progranulin. We infer that progranulin may assist in removal of protein waste and thereby helps to resolve neuropathic pain after nerve injury.

Borrelia-induced cytokine production is mediated by spleen tyrosine kinase (Syk) but is Dectin-1 and Dectin-2 independent.

Although it is known that Borrelia species express sugar-like structures on their outer surface, not much is known about the role of these structures in immune recognition by host cells. Fungi, like Candida albicans, are mainly recognized by C-type lectin receptors, in specific Dectin-1 and Dectin-2. In this study we assessed the role of Dectin-1 and Dectin-2 in the recognition process of Borrelia spirochetes. Using specific inhibitors against these receptors on human cells did not influenced cytokine production. Individuals carrying a SNP leading to an early stop codon in the DECTIN-1 gene also did not lead to differential induction of Borrelia-dependent cytokines. After injection of live Borrelia into knee joints of Dectin-2 deficient mice a trend towards lower inflammation was observed. Inhibition of Syk in human cells resulted in lower cytokine production after Borrelia stimulation. In conclusion, Dectin-1 and Dectin-2 seem not to play a major role in Borrelia recognition or Borrelia-induced inflammation. However, Syk seems to be involved in Borrelia-induced cytokine production.

A human sleep homeostasis phenotype in mice expressing a primate-specific PER3 variable-number tandem-repeat coding-region polymorphism.

In humans, a primate-specific variable-number tandem-repeat (VNTR) polymorphism (4 or 5 repeats 54 nt in length) in the circadian gene PER3 is associated with differences in sleep timing and homeostatic responses to sleep loss. We investigated the effects of this polymorphism on circadian rhythmicity and sleep homeostasis by introducing the polymorphism into mice and assessing circadian and sleep parameters at baseline and during and after 12 h of sleep deprivation (SD). Microarray analysis was used to measure hypothalamic and cortical gene expression. Circadian behavior and sleep were normal at baseline. The response to SD of 2 electrophysiological markers of sleep homeostasis, electroencephalography (EEG) θ power during wakefulness and δ power during sleep, were greater in the Per3(5/5) mice. During recovery, the Per3(5/5) mice fully compensated for the SD-induced deficit in δ power, but the Per3(4/4) and wild-type mice did not. Sleep homeostasis-related transcripts (e.g., Homer1, Ptgs2, and Kcna2) were differentially expressed between the humanized mice, but circadian clock genes were not. These data are in accordance with the hypothesis derived from human data that the PER3 VNTR polymorphism modifies the sleep homeostatic response without significantly influencing circadian parameters.

Role of Dectin-2 for host defense against systemic infection with Candida glabrata.

Although Candida glabrata is an important pathogenic Candida species, relatively little is known about its innate immune recognition. Here, we explore the potential role of Dectin-2 for host defense against C. glabrata. Dectin-2-deficient (Dectin-2(-/-)) mice were found to be more susceptible to C. glabrata infections, showing a defective fungal clearance in kidneys but not in the liver. The increased susceptibility to infection was accompanied by lower production of T helper 1 (Th1) and Th17-derived cytokines by splenocytes of Dectin-2(-/-) mice, while macrophage-derived cytokines were less affected. These defects were associated with a moderate yet significant decrease in phagocytosis of the fungus by the Dectin-2(-/-) macrophages and neutrophils. Neutrophils of Dectin-2(-/-) mice also displayed lower production of reactive oxygen species (ROS) upon challenge with opsonized C. glabrata or C. albicans. This study suggests that Dectin-2 is important in host defense against C. glabrata and provides new insights into the host defense mechanisms against this important fungal pathogen.

Distinct APCs explain the cytokine bias of α-galactosylceramide variants in vivo.

α-Galactosylceramide represents a new class of vaccine adjuvants and immunomodulators that stimulate NKT cells to secrete Th1 and Th2 cytokines. Synthetic variants with short or unsaturated acyl chains exhibit a striking Th2 bias in vivo but no evidence of defect in TCR signaling or stimulation of NKT cells in vitro. Using cd1d1(fl/fl) mice, we demonstrated that distinct APC types explained the cytokine bias in vivo. Whereas NKT stimulation by α-Galactosylceramide required CD1d expression by dendritic cells (DCs), presentation of the Th2 variants was promiscuous and unaffected by DC-specific ablation of CD1d. This DC-independent stimulation failed to activate the feedback loop between DC IL-12 and NK cell IFN-γ, explaining the Th2 bias. Conversely, forced presentation of the Th2 variants by DC induced high IL-12. Thus, lipid structural variations that do not alter TCR recognition can activate distinct Th1 or Th2 cellular networks by changing APC targeting in vivo.

Genetic deletion of cell division autoantigen 1 retards diabetes-associated renal injury.

Cell division autoantigen 1 (CDA1) enhances TGF-ß signaling in renal and vascular cells, and renal expression of CDA1 is elevated in animal models of diabetes. In this study, we investigated the genetic deletion of Tspyl2, the gene encoding CDA1, in C57BL6 and ApoE knockout mice. The increased renal expression of TGF-ß1, TGF-ß type I and II receptors, and phosphorylated Smad3 associated with diabetes in wild-type mice was attenuated in diabetic CDA1 knockout mice. Notably, CDA1 deletion significantly reduced diabetes-associated renal matrix accumulation and immunohistochemical staining for collagens III and IV and attenuated glomerular and tubulointerstitial injury indices, despite the presence of persistent hyperglycemia, polyuria, renal hypertrophy, and hyperfiltration. Furthermore, CDA1 deletion reduced gene expression of TGF-ß1 receptors in the kidney, resulting in a functionally attenuated response to exogenous TGF-ß, including reduced levels of phosphorylated Smad3 and ERK1/2, in primary kidney cells from CDA1 knockout animals. Taken together, these data suggest that CDA1 deletion reduces but does not block renal TGF-ß signaling. Because direct antagonism of TGF-ß or its receptors has unwanted effects, CDA1 may be a potential therapeutic target for retarding DN and perhaps, other kidney diseases associated with TGF-ß-mediated fibrogenesis.

Ki67 deficiency impedes chromatin accessibility and BCR gene rearrangement.

The proliferation marker Ki67 has been attributed critical functions in maintaining mitotic chromosome morphology and heterochromatin organization during the cell cycle, indicating a potential role in developmental processes requiring rigid cell-cycle control. Here, we discovered that despite normal fecundity and organogenesis, germline deficiency in Ki67 resulted in substantial defects specifically in peripheral B and T lymphocytes. This was not due to impaired cell proliferation but rather to early lymphopoiesis at specific stages where antigen-receptor gene rearrangements occurred. We identified that Ki67 was required for normal global chromatin accessibility involving regulatory regions of genes critical for checkpoint stages in B cell lymphopoiesis. In line with this, mRNA expression of Rag1 was diminished and gene rearrangement was less efficient in the absence of Ki67. Transgenes encoding productively rearranged immunoglobulin heavy and light chains complemented Ki67 deficiency, completely rescuing early B cell development. Collectively, these results identify a unique contribution from Ki67 to somatic antigen-receptor gene rearrangement during lymphopoiesis.

Alcohol and aldehyde dehydrogenases: retinoid metabolic effects in mouse knockout models.

Retinoic acid (RA) is the active metabolite of vitamin A (retinol) that controls growth and development. The first step of RA synthesis is controlled by enzymes of the alcohol dehydrogenase (ADH) and retinol dehydrogenase (RDH) families that catalyze oxidation of retinol to retinaldehyde. The second step of RA synthesis is controlled by members of the aldehyde dehydrogenase (ALDH) family also known as retinaldehyde dehydrogenase (RALDH) that further oxidize retinaldehyde to produce RA. RA functions as a ligand for DNA-binding RA receptors that directly regulate transcription of specific target genes. Elucidation of the vitamin A metabolic pathway and investigation of the endogenous function of vitamin A metabolites has been greatly improved by development of mouse ADH, RDH, and RALDH loss-of-function models. ADH knockouts have demonstrated a postnatal role for this enzyme family in clearance of excess retinol to prevent vitamin A toxicity and in generation of RA for postnatal survival during vitamin A deficiency. A point mutation in Rdh10 generated by ethylnitrosourea has demonstrated that RDH10 generates much of the retinaldehyde needed for RA synthesis during embryonic development. Raldh1, Raldh2, and Raldh3 knockouts have demonstrated that RALDH1, RALDH2, and RALDH3 generate most of the RA needed during embryogenesis. These mouse models serve as instrumental tools for providing new insight into retinoid function. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.

Lung-specific loss of the laminin α3 subunit confers resistance to mechanical injury.

Laminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of α3 laminin (Lama3(fl/fl)), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3(fl/fl) mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of α3 laminin in whole lung homogenates was more than 50% lower than that in control-treated mice, suggesting a relatively long half-life for the protein in the lung. Upon exposure to an injurious ventilation strategy (tidal volume of 35 ml per kg of body weight for 2 hours), the mice with a knockdown of the α3 laminin subunit had less severe injury, as shown by lung mechanics, histology, alveolar capillary permeability and survival when compared with Ad-Null-treated mice. Knockdown of the α3 laminin subunit resulted in evidence of lung inflammation. However, this did not account for their resistance to mechanical ventilation. Rather, the loss of α3 laminin was associated with a significant increase in the collagen content of the lungs. We conclude that the loss of α3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury.

Deletion of cIAP1 and cIAP2 in murine B lymphocytes constitutively activates cell survival pathways and inactivates the germinal center response.

B cells require signals delivered through B-cell activating factor of the TNF family receptor (BAFF-R) and CD40 to survive and produce antibody responses in vivo. In vitro data indicate that these signals are controlled by the homologous RING finger proteins cIAP1 and cIAP2, in collaboration with TRAF2 and TRAF3. There is also mounting evidence that all 4 of these signaling molecules can act as tumor suppressors in human B-lineage malignancies. However, it has not been possible to identify the roles of cIAP1 and cIAP2 in controlling B-cell physiology because of the absence of an appropriate in vivo model. Here we describe a unique genetically modified mouse in which the linked cIap1 and cIap2 genes can be independently inactivated. Deletion of cIAP1 plus cIAP2 (but not either protein alone) rendered primary B cells independent of BAFF-R for their survival and led to their uncontrolled accumulation in vivo. B cells deficient in cIAP1 and cIAP2 were also incapable of forming germinal centers, a key step in antibody-mediated immunity. These data define a fundamental role for cIAP1/cIAP2 in regulating B-cell survival and responsiveness, show this requires direct binding to TRAF2, and suggest how mutations of TRAF2, TRAF3, and cIAP1/cIAP2 contribute to B-lineage malignancies, such as multiple myeloma.

Evidence that Cd101 is an autoimmune diabetes gene in nonobese diabetic mice.

We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.

Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice.

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) and KO mice were fed ³H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested ³H-SM in the intestinal content increased by 6-fold and the formation of ³H-ceramide decreased markedly, resulting in 98% reduction of ³H-ceramide/³H-SM ratio 1 h after gavage. The absorbed ³H-palmitic acid portion was decreased by 95%. After 3 h, a small increase in ³H-ceramide was identified in distal intestine in KO mice. In feces, ³H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.

c-Cbl promotes T cell receptor-induced thymocyte apoptosis by activating the phosphatidylinositol 3-kinase/Akt pathway.

The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.