Double Mutant Mice Research Articles

Disruption of hemochromatosis protein and transferrin receptor 2 causes iron-induced liver injury in mice

Mutations in hemochromatosis protein (HFE) or transferrin receptor 2 (TFR2) cause hereditary hemochromatosis (HH) by impeding production of the liver iron-regulatory hormone, hepcidin (HAMP). This study examined the effects of disruption of Hfe or Tfr2, either alone or together, on liver iron loading and injury in mouse models of HH. Iron status was determined in Hfe knockout (Hfe(-/-)), Tfr2 Y245X mutant (Tfr2(mut)), and double-mutant (Hfe(-/-) ×Tfr2(mut) ) mice by measuring plasma and liver iron levels. Plasma alanine transaminase (ALT) activity, liver histology, and collagen deposition were evaluated to assess liver injury. Hepatic oxidative stress was assessed by measuring superoxide dismutase (SOD) activity and F(2)-isoprostane levels. Gene expression was measured by real-time polymerase chain reaction. Hfe(-/-) ×Tfr2(mut) mice had elevated hepatic iron with a periportal distribution and increased plasma iron, transferrin saturation, and non-transferrin-bound iron, compared with Hfe(-/-), Tfr2(mut), and wild-type (WT) mice. Hamp1 expression was reduced to 40% (Hfe(-/-) and Tfr2(mut) ) and 1% (Hfe(-/-) ×Tfr2(mut)) of WT values. Hfe(-/-) ×Tfr2(mut) mice had elevated plasma ALT activity and mild hepatic inflammation with scattered aggregates of infiltrating inflammatory cluster of differentiation 45 (CD45)-positive cells. Increased hepatic hydoxyproline levels as well as Sirius red and Masson's Trichrome staining demonstrated advanced portal collagen deposition. Hfe(-/-) and Tfr2(mut) mice had less hepatic inflammation and collagen deposition. Liver F(2) -isoprostane levels were elevated, and copper/zinc and manganese SOD activities decreased in Hfe(-/-) ×Tfr2(mut), Tfr2(mut), and Hfe(-/-) mice, compared with WT mice. Disruption of both Hfe and Tfr2 caused more severe hepatic iron overload with more advanced lipid peroxidation, inflammation, and portal fibrosis than was observed with the disruption of either gene alone. The Hfe(-/-) ×Tfr2(mut) mouse model of iron-induced liver injury reflects the liver injury phenotype observed in human HH.

Loss of Angiotensin-Converting Enzyme-2 Exacerbates Diabetic Cardiovascular Complications and Leads to Systolic and Vascular Dysfunction

Rationale: Diabetic cardiovascular complications are reaching epidemic proportions. Angiotensin-converting enzyme-2 (ACE2) is a negative regulator of the renin-angiotensin system. We hypothesize that loss of ACE2 exacerbates cardiovascular complications induced by diabetes. Objective: To define the role of ACE2 in diabetic cardiovascular complications. Methods and Results: We used the well-validated Akita mice, a model of human diabetes, and generated double-mutant mice using the ACE2 knockout (KO) mice (Akita/ACE2 −/y ). Diabetic state was associated with increased ACE2 in Akita mice, whereas additional loss of ACE2 in these mice leads to increased plasma and tissue angiotensin II levels, resulting in systolic dysfunction on a background of impaired diastolic function. Downregulation of SERCA2 and lipotoxicity were equivalent in Akita and Akita/ACE2KO hearts and are likely mediators of the diastolic dysfunction. However, greater activation of protein kinase C and loss of Akt and endothelial nitric oxide synthase phosphorylation occurred in the Akita/ACE2KO hearts. Systolic dysfunction in Akita/ACE2KO mice was linked to enhanced activation of NADPH oxidase and metalloproteinases, resulting in greater oxidative stress and degradation of the extracellular matrix. Impaired flow-mediated dilation in vivo correlated with increased vascular oxidative stress in Akita/ACE2KO mice. Treatment with the AT1 receptor blocker, irbesartan rescued the systolic dysfunction, normalized altered signaling pathways, flow-mediated dilation, and the increased oxidative stress in the cardiovascular system. Conclusions: Loss of ACE2 disrupts the balance of the renin-angiotensin system in a diabetic state and leads to an angiotensin II/AT1 receptor-dependent systolic dysfunction and impaired vascular function. Our study demonstrates that ACE2 serves as a protective mechanism against diabetes-induced cardiovascular complications.

Disease severity in a mouse model of ataxia telangiectasia is modulated by the DNA damage checkpoint gene Hus1

The human genomic instability syndrome ataxia telangiectasia (A-T), caused by mutations in the gene encoding the DNA damage checkpoint kinase ATM, is characterized by multisystem defects including neurodegeneration, immunodeficiency and increased cancer predisposition. ATM is central to a pathway that responds to double-strand DNA breaks, whereas the related kinase ATR leads a parallel signaling cascade that is activated by replication stress. To dissect the physiological relationship between the ATM and ATR pathways, we generated mice defective for both. Because complete ATR pathway inactivation causes embryonic lethality, we weakened the ATR mechanism to different degrees by impairing HUS1, a member of the 911 complex that is required for efficient ATR signaling. Notably, simultaneous ATM and HUS1 defects caused synthetic lethality. Atm/Hus1 double-mutant embryos showed widespread apoptosis and died mid-gestationally. Despite the underlying DNA damage checkpoint defects, increased DNA damage signaling was observed, as evidenced by H2AX phosphorylation and p53 accumulation. A less severe Hus1 defect together with Atm loss resulted in partial embryonic lethality, with the surviving double-mutant mice showing synergistic increases in genomic instability and specific developmental defects, including dwarfism, craniofacial abnormalities and brachymesophalangy, phenotypes that are observed in several human genomic instability disorders. In addition to identifying tissue-specific consequences of checkpoint dysfunction, these data highlight a robust, cooperative configuration for the mammalian DNA damage response network and further suggest HUS1 and related genes in the ATR pathway as candidate modifiers of disease severity in A-T patients.

Dystrophin and dysferlin double mutant mice: a novel model for rhabdomyosarcoma

Although researchers have yet to establish a link between muscular dystrophy (MD) and sarcomas in human patients, literature suggests that the MD genes dystrophin and dysferlin act as tumor suppressor genes in mouse models of MD. For instance, dystrophin-deficient mdx and dysferlin-deficient A/J mice, models of human Duchenne MD and limb-girdle MD type 2B, respectively, develop mixed sarcomas with variable penetrance and latency. To further establish the correlation between MD and sarcoma development, and to test whether a combined deletion of dystrophin and dysferlin exacerbates MD and augments the incidence of sarcomas, we generated dystrophin and dysferlin double mutant mice (STOCK-Dysf(prmd)Dmd(mdx-5Cv)). Not surprisingly, the double mutant mice develop severe MD symptoms and, moreover, develop rhabdomyosarcoma (RMS) at an average age of 12 months, with an incidence of >90%. Histological and immunohistochemical analyses, using a panel of antibodies against skeletal muscle cell proteins, electron microscopy, cytogenetics, and molecular analysis reveal that the double mutant mice develop RMS. The present finding bolsters the correlation between MD and sarcomas, and provides a model not only to examine the cellular origins but also to identify mechanisms and signal transduction pathways triggering development of RMS.

Abstract 85: Atherosclerosis Induces Thoracic and Abdominal Aortic Disease in Preaneurysmal Transgenic Fibulin-4 Mice

Extracellular matrix degradation in the aortic wall plays an important role in the formation and progression of aneurysms. In mice, a deficiency of the extracellular matrix protein fibulin-4, induces matrix metalloproteinases (MMP) upregulation and transforming growth factor-β dysregulation. This results in the formation of aneurysms in the thoracic aorta of homozygous Fibulin-4 R/R mice. In humans, aneurysms usually develop in the abdominal aorta during aging and are most often associated with atherosclerosis. To investigate the molecular mechanisms underlying the interaction of aneurysmatic and atherosclerotic aortic disease we developed a new mouse model. Fibulin-4 knockdown (Fibulin-4 +/R ) mice, which display mild extracellular matrix changes, were crossbred with atherosclerotic ApoE -/- mice. The double mutant mice were fed a high fat diet for 10 or 20 weeks starting at 9 weeks after birth. Thoracic and abdominal aortic architecture were assessed in vivo using high-resolution ultrasound. MMP activity in the aorta was determined using fluorescent imaging with protease-activatable near-infrared fluorescence probes. Following sacrifice, plaque burden was evaluated through quantitative oil-red-o staining and histological analysis. Already after 10 weeks of high fat diet, ApoE -/- Fibulin-4 +/R mice displayed increased matrix metalloproteinase activity in the abdominal aorta and increased plaque formation at the level of the visceral arteries as compared with ApoE -/- Fibulin-4 +/+ mice, with further increases observed after 20 weeks of high fat diet. Surprisingly, after 20 weeks high fat diet the aortic diameter increased up to 60% in the aortic arch and 20% in the abdominal aorta in the ApoE -/- Fibulin-4 +/R mice as compared with ApoE -/- Fibulin-4 +/= mice. This novel mutant mouse model is the first viable murine model which mimics the clinical situation of coexistent aneurysm formation and atherogenesis. It provides the opportunity to unravel the biological processes underlying aortic wall degeneration and to identify markers that elucidate key events in the early stages of the pathogenic sequence that culminates in an aneurysm. We identified that partial degradation of the extracellular matrix under the influence of atherosclerosis leads to aortic dilatation. In addition, we show that protease-activatable fluorescent sensors are promising new tools to monitor non-invasively the enhanced matrix metalloproteinases activity in the aortic wall that precedes aortic wall degeneration.

Deletion of NF-κB/p50 influences the outcome of Notch3-dependent T cell leukemia (63.4)

Abstract The deregulation of Notch3 signaling inside T-cell compartment of transgenic (N3-tg) mice, induces an aggressive form of T-cell acute lymphoblastic lymphoma (T-ALL), sustained by a constitutive activation of the NF-κB canonical pathway, mainly represented by the p50/p65 heterodimer. To clarify the Notch/NF-κB relationships in the development of T-ALL, we generated double mutant mice, deleted of the NF-κB/p50 subunit in a Notch3 transgenic background (N3-tg/p50-/-). The follow-up of N3-tg/p50-/- versus N3-tg mice revealed that p50 deletion strongly inhibits the development of Notch3-dependent T-ALL. Surprisingly, double mutant succumb earlier than N3-tg mice, displaying the trait of a myeloproliferative disease, with an aberrant accumulation of Mac1+Gr1+ myeloid cells in both spleen and peripheral blood, as well as of granulocyte/monocyte progenitors in the bone marrow. Our preliminary results suggest that Notch3 overexpression in T-cell compartment is able to influence, possibly in trans, the equilibrium of the myeloid compartment and that the ablation of NF-κB canonical pathway may impact on the outcomes of a T-cell specific deregulation of Notch signaling. We provide a useful experimental model to extend our understanding of Notch/NF-κB interplay in hemopoietic system and to unravel novel relationships between lymphoid and myeloid differentiation.

Mo1821 Normalization of CFTR Brush Border Membrane Trafficking Defects in the Intestines of Myosin 1A/6 Double Mutant Mice

Mo1821 Normalization of CFTR Brush Border Membrane Trafficking Defects in the Intestines of Myosin 1A/6 Double Mutant Mice

Abstract 4182: Overexpression of Mig-6 suppresses endometrial cancer development and progression in ablation of Pten mice

Abstract Endometrial cancer is the most common gynecological cancer. Estrogen-dependent endometrioid carcinoma is the most common type of endometrial cancer. In previous studies, we have identified mitogen-inducible gene 6 (Mig-6) as a downstream target of Progesterone Receptor (Pgr) action in the uterus. Ablation of Mig-6 in the murine uterus leads to the development of endometrial hyperplasia and estrogen-induced endometrial cancer. These results suggest that Mig-6 act as a tumor suppressor in endometrial cancer. To assess the effects of the overexpression Mig-6 and the PTEN/PI3K/AKT signaling pathways on uterine tumorigenesis, mice with Pten floxed (Ptenf/f) and LoxP-STOP-LoxP Mig-6 (R26Mig-6LSL) were bred to the PRCre mouse model to generate ablation of Pten and overexpression of Mig-6 in the uterus (PRcre+ R26Mig-6LSL Ptenf/f). PRcre+ R26Mig-6LSL Ptenf/f showed significantly increased survival time compared to PRcre/+ Ptenf/f mice. Double mutant mice showed significantly decreased uterine weight compared to PRcre/+Ptenf/f at 3 months of age. Gross morphology at 3 months of age showed that double mutant mice dramatically retarded the development of endometrial cancer compared to ablation of Pten alone. Histological analysis showed that endometrial adenocarcinoma with invasion into the myometrium was observed in the PRcre/+Ptenf/f mice at 3 months of age. However, double mutant mice did not develop endometrial cancer. Immunohistochemstry analysis showed significantly increased apoptosis and decreased proliferation at epithelial cells in PRcre+ R26Mig-6LSL Ptenf/f mice compared to PRcre/+ Ptenf/f mice at 3 months of age. The expression of pERK1/2 was significantly decreased in PRcre+ R26Mig-6LSL Ptenf/f mice compared to PRcre/+ Ptenf/f mice at 2 and 3 months of age. The expression of Pgr and its tartget genes was significantly increased in PRcre+ R26Mig-6LSL Ptenf/f mice compare to PRcre/+ Ptenf/f mice. However, the expression of Estrogen Receptor ≤ (Esr1) and its target genes was significantly decreased. The ability of Mig-6 to repress cancer development indicates that it exerts a tumor suppressor function in endometrial cancer. These results will contribute to the understanding of the molecular mechanism of tumorigenesis and to the development of therapeutic approaches for endometrial cancer.(Supported by NIH, R01 HD057873) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4182. doi:1538-7445.AM2012-4182

Abstract 1302: Effect of intestinal-specific Kaiso overexpression on the APC MIN/+ mouse colon cancer model

Abstract The canonical Wnt signaling pathway is a crucial regulator of various cellular processes during embryogenesis but when aberrantly activated, it leads to tumorigenesis. The key downstream effector of the Wnt pathway is the Armadillo protein, β-catenin, which was first characterized as a cytoplasmic cofactor for the transmembrane, cell-cell adhesion tumor suppressor, E-cadherin. Recent evidence from our lab and others indicates that another E-cadherin cofactor, p120ctn and its binding partner, the POZ-ZF transcription factor Kaiso, co-regulate a subset of Wnt/β-catenin target genes and antagonize Wnt signaling. Specifically, using a Xenopus model system, we and others found that Kaiso overexpression rescued the β-catenin-induced duplicate axis phenotype; this implicated Kaiso as a negative regulator of Wnt signaling. However, it remains to be determined whether an analogous antagonistic role for Kaiso is conserved in mammals. Indeed, when Kaiso-null mice (which were viable and had no overt developmental defects or tumors) were crossed with the APCMIN/+ mouse model of colon cancer, the progeny had delayed tumor onset and reduced polyp size. This suggested that Kaiso played a positive role in tumor initiation and possibly cell proliferation in mammals. To resolve this discrepancy, we generated intestine-specific Kaiso overexpressing transgenic mice (KaisoTg/+) for subsequent mating with the APCMIN/+ mouse model of colon cancer. Histological analysis of small and large intestines from KaisoTg/+ and non-Tg mice revealed no gross morphological differences. Immunohistochemical (IHC) and immunofluorescence (IF) staining revealed strong nuclear Kaiso staining in intestinal tissues from KaisoTg/+ mice whereas intestinal tissues from non-Tg mice displayed weak cytoplasmic or no Kaiso staining. We detected no difference in β-Catenin expression levels in tissues from KaisoTg/+ mice and their wildtype littermates. This suggests that Kaiso's regulation of WNT signaling is downstream of its main effector β-Catenin. KaisoTg-APCMIN/+ double mutant mice have been generated and surprisingly they display decreased body size and weight, and have a decreased life span when compared to the parentals (i.e. KaisoTg or APCMIN/+). Current studies are aimed at assessing polyp formation and elucidating the expression levels of p120ctn, β-catenin, E-cadherin and cyclinD1 in the KaisoTg-APCMIN/+ intestines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1302. doi:1538-7445.AM2012-1302

Reduction of BDNF expression in Fmr1 knockout mice worsens cognitive deficits but improves hyperactivity and sensorimotor deficits

Fragile X syndrome (FXS) is a common cause of inherited intellectual disability and a well-characterized form of autism spectrum disorder. As brain-derived neurotrophic factor (BDNF) is implicated in the pathophysiology of FXS we examined the effects of reduced BDNF expression on the behavioral phenotype of an animal model of FXS, Fmr1 knockout (KO) mice, crossed with mice carrying a deletion of one copy of the Bdnf gene (Bdnf(+/-)). Fmr1 KO mice showed age-dependent alterations in hippocampal BDNF expression that declined after the age of 4 months compared to wild-type controls. Mild deficits in water maze learning in Bdnf(+/-) and Fmr1 KO mice were exaggerated and contextual fear learning significantly impaired in double transgenics. Reduced BDNF expression did not alter basal nociceptive responses or central hypersensitivity in Fmr1 KO mice. Paradoxically, the locomotor hyperactivity and deficits in sensorimotor learning and startle responses characteristic of Fmr1 KO mice were ameliorated by reducing BNDF, suggesting changes in simultaneously and in parallel working hippocampus-dependent and striatum-dependent systems. Furthermore, the obesity normally seen in Bdnf(+/-) mice was eliminated by the absence of fragile X mental retardation protein 1 (FMRP). Reduced BDNF decreased the survival of newborn cells in the ventral part of the hippocampus both in the presence and absence of FMRP. Since a short neurite phenotype characteristic of newborn cells lacking FMRP was not found in cells derived from double mutant mice, changes in neuronal maturation likely contributed to the behavioral phenotype. Our results show that the absence of FMRP modifies the diverse effects of BDNF on the FXS phenotype.

Loss of XLαs (extra-large αs) imprinting results in early postnatal hypoglycemia and lethality in a mouse model of pseudohypoparathyroidism Ib

Maternal deletion of the NESP55 differentially methylated region (DMR) (delNESP55/ASdel3-4(m), delNAS(m)) from the GNAS locus in humans causes autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-Ib(delNASm)), a disorder of proximal tubular parathyroid hormone (PTH) resistance associated with loss of maternal GNAS methylation imprints. Mice carrying a similar, maternally inherited deletion of the Nesp55 DMR (ΔNesp55(m)) replicate these Gnas epigenetic abnormalities and show evidence for PTH resistance, yet these mice demonstrate 100% mortality during the early postnatal period. We investigated whether the loss of extralarge αs (XLαs) imprinting and the resultant biallelic expression of XLαs are responsible for the early postnatal lethality in ΔNesp55(m) mice. First, we found that ΔNesp55(m) mice are hypoglycemic and have reduced stomach-to-body weight ratio. We then generated mice having the same epigenetic abnormalities as the ΔNesp55(m) mice but with normalized XLαs expression due to the paternal disruption of the exon giving rise to this Gnas product. These mice (ΔNesp55(m)/Gnasxl(m+/p-)) showed nearly 100% survival up to postnatal day 10, and a substantial number of them lived to adulthood. The hypoglycemia and reduced stomach-to-body weight ratio observed in 2-d-old ΔNesp55(m) mice were rescued in the ΔNesp55(m)/Gnasxl(m+/p-) mice. Surviving double-mutant animals had significantly reduced Gαs mRNA levels and showed hypocalcemia, hyperphosphatemia, and elevated PTH levels, thus providing a viable model of human AD-PHP-Ib. Our findings show that the hypoglycemia and early postnatal lethality caused by the maternal deletion of the Nesp55 DMR result from biallelic XLαs expression. The double-mutant mice will help elucidate the pathophysiological mechanisms underlying AD-PHP-Ib.

Mediator Subunits MED1 and MED24 Cooperatively Contribute to Pubertal Mammary Gland Development and Growth of Breast Carcinoma Cells

The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G(1)/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.

Roles of Heparan Sulfate Sulfation in Dentinogenesis

Cell surface heparan sulfate (HS) is an essential regulator of cell signaling and development. HS traps signaling molecules, like Wnt in the glycosaminoglycan side chains of HS proteoglycans (HSPGs), and regulates their functions. Endosulfatases Sulf1 and Sulf2 are secreted at the cell surface to selectively remove 6-O-sulfate groups from HSPGs, thereby modifying the affinity of cell surface HSPGs for its ligands. This study provides molecular evidence for the functional roles of HSPG sulfation and desulfation in dentinogenesis. We show that odontogenic cells are highly sulfated on the cell surface and become desulfated during their differentiation to odontoblasts, which produce tooth dentin. Sulf1/Sulf2 double null mutant mice exhibit a thin dentin matrix and short roots combined with reduced expression of dentin sialophosphoprotein (Dspp) mRNA, encoding a dentin-specific extracellular matrix precursor protein, whereas single Sulf mutants do not show such defective phenotypes. In odontoblast cell lines, Dspp mRNA expression is potentiated by the activation of the Wnt canonical signaling pathway. In addition, pharmacological interference with HS sulfation promotes Dspp mRNA expression through activation of Wnt signaling. On the contrary, the silencing of Sulf suppresses the Wnt signaling pathway and subsequently Dspp mRNA expression. We also show that Wnt10a protein binds to cell surface HSPGs in odontoblasts, and interference with HS sulfation decreases the binding affinity of Wnt10a for HSPGs, which facilitates the binding of Wnt10a to its receptor and potentiates the Wnt signaling pathway, thereby up-regulating Dspp mRNA expression. These results demonstrate that Sulf-mediated desulfation of cellular HSPGs is an important modification that is critical for the activation of the Wnt signaling in odontoblasts and for production of the dentin matrix.

The Psen1‐L166P ‐knock‐in mutation leads to amyloid deposition in human wild‐type amyloid precursor protein YAC transgenic mice

Genetically engineered mice have been generated to model cerebral β-amyloidosis, one of the hallmarks of Alzheimer disease (AD) pathology, based on the overexpression of a mutated cDNA of the amyloid-β precursor protein (AβPP) or by knock-in of the murine Aβpp gene alone or with presenilin1 mutations. Here we describe the generation and initial characterization of a new mouse line based on the presence of 2 copies of the human genomic region encoding the wild-type AβPP and the L166P presenilin 1 mutation. At ∼6 mo of age, double-mutant mice develop amyloid pathology, with signs of neuritic dystrophy, intracellular Aβ accumulation, and glial inflammation, an increase in AβPP C-terminal fragments, and an 8 times increase in Aβ42 levels with a 40% decrease in Aβ40 levels, leading to a significant increase (14 times) of Aβ42/Aβ40 ratios, with minimal effects on presenilin or the Notch1 pathway in the brain. We conclude that in mice, neither mutations in AβPP nor overexpression of an AβPP isoform are a prerequisite for Aβ pathology. This model will allow the study of AD pathogenesis and testing of therapeutic strategies in a more relevant environment without experimental artifacts due to the overexpression of a single-mutant AβPP isoform using exogenous promoters.

Sex-specific and region-specific changes in BDNF–TrkB signalling in the hippocampus of 5-HT1A receptor and BDNF single and double mutant mice

Brain-derived neurotrophic factor (BDNF) and serotonin 5-HT1A receptors are implicated in the pathophysiology of depression and the mechanism of action of antidepressant drugs. Here, we explore possible reciprocal interactions of 5-HT1A receptor knockout and the expression of BDNF, its receptor TrkB and downstream mitogen-activated protein kinase (MAPK) in the ventral (VHP) and dorsal hippocampus (DHP). We compared female and male double mutant mice (5-HT1A−/−/BDNF+/−) with single mutant mice (5-HT1A−/−, BDNF+/−) and wildtype (WT) controls. Protein expression of BDNF, TrkB, phosphorylation of TrkB (pTrkB) and MAPKs (ERK1, ERK2) was examined using Western blot analysis (n=5–7). As expected, the BDNF+/− mice showed ~50% BDNF reduction. Loss of 5-HT1A receptors induced a significant decrease in BDNF levels in the VHP in female mice. The pTrkB/TrkB ratio was also significantly decreased in female 5-HT1A−/− mice and 5-HT1A−/−/BDNF+/− mice but not in males. Despite markedly reduced BDNF levels in BDNF+/− mice and double mutants, ERK1 activation was unchanged in the female mice. In contrast, ERK2 activation was significantly elevated in the VHP of female BDNF+/− mice and double mutants. Given the greater vulnerability of women to develop depression and the role of the VHP in stress responses and anxiety-related behaviours, our results may shed more light on sex differences in depression and other psychiatric disorders with BDNF and 5-HT1A receptor dysfunction.

PTEN: a new player controlling structural and functional synaptic plasticity

Phosphoinositide 3-kinase (PI 3-kinase) is a ubiquitous enzyme that catalyses the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI 3-kinase driven generation of PIP3 activates various downstream signalling cascades which in turn regulate numerous biological processes including growth, metabolism, proliferation, migration and cell size. PTEN (phosphatase and tensin homologue deleted on chromosome 10), which was originally identified as a tumour promoter, is a constitutively active lipid phosphatase that opposes PI 3-kinase-dependent signalling by promoting dephosphorylation of PIP3 into PIP2. PTEN is highly expressed in neurons and several lines of evidence support a role for PTEN in regulating important neuronal functions. Indeed, familial mutations that result in PTEN inactivation have been linked to neurological disorders such as ataxia, mental retardation and seizures (Backman et al. 2001). Moreover, loss of PTEN function at early stages of development results in widespread deficits in neuronal growth, synaptogenesis and synaptic plasticity suggesting additional roles for PTEN in these processes. However, it is not clear if PTEN-linked synaptic impairments are a direct consequence of loss of PTEN function or are secondary to PTEN-driven alterations in neuronal morphology. A recent insightful study by Sperow et al. (2012) in The Journal of Physiology aims to address this issue. This study demonstrates that the structural and functional properties of hippocampal synapses are independently controlled by PTEN and that PTEN plays a direct role in activity-dependent hippocampal synaptic plasticity. Several studies have observed that a key feature of deleting PTEN during CNS development is progressive hypertrophy of neuronal dendrites and spines as well as a marked increase in the size of neurons and synapses. PTEN deletion or down-regulation in hippocampal neurons also results in impairments in two forms of activity-dependent synaptic plasticity, namely long-term potentiation (LTP) and long-term depression (LTD). In order to determine if the structural and functional effects of PTEN on hippocampal synapses are related, Sperow and colleagues generated transgenic mice with conditional knockout of PTEN in forebrain excitatory neurons. As PTEN deletion was apparent after postnatal day 14 (P14) in these mice, the impact of PTEN deletion on hippocampal synaptic function could be examined prior to its hypertrophic effects. Postnatal deletion of PTEN had no significant effect on excitatory synaptic transmission at hippocampal CA1 synapses, as basal synaptic transmission and paired pulse facilitation was normal in PTEN−/− mice compared to wild-type littermates. In contrast, deficits in both hippocampal LTP and LTD were observed in PTEN−/− mice. Postnatal deletion of PTEN also resulted in hippocampal-specific memory deficits as significant impairments in spatial memory tasks performed in the Morris water maze were observed in PTEN−/− mice. However, no differences in soma size or dendritic morphology were detected between PTEN+ and PTEN− neurons in PTEN−/− mice, suggesting that structural abnormalities do not contribute to the deficits in synaptic plasticity following postnatal PTEN deletion. As PTEN negatively regulates PI 3-kinase driven signalling, deletion or inhibition of PTEN results in uncontrolled stimulation of downstream components of the PI 3-kinase cascade, including the protein kinase PDK1. Consequently reducing PDK1 expression can reverse some neuronal abnormalities linked to PTEN deletion (Chalhoub et al. 2009). In this study, Sperow et al. examined whether postnatal deletion of PDK1 rescued the impairments in hippocampal synaptic plasticity in PTEN−/− mice. Postnatal deletion of PDK1 had no effect on hippocampal synaptic plasticity or neuronal morphology per se. However, deletion of PDK1 rescued the synaptic plasticity deficits, as the magnitude of hippocampal LTP and LTD was comparable in double mutant mice (PTEN−/−; PDK1−/−) and wild-type littermates. In contrast, however, significant impairments in spatial memory were still observed in the double mutant mice compared to wild-type littermates, suggesting that PDK1 deletion is able to counterbalance the effects of postnatal PTEN deletion on synaptic plasticity but not on spatial memory. In conclusion this study indicates that hippocampal synaptic plasticity and neuronal morphology are independently regulated by PTEN in vivo. It also highlights an important role for PTEN in hippocampal LTP and LTD. Although the precise cellular mechanisms underlying PTEN modulation of synaptic plasticity are not fully known, recent studies suggest involvement of a postsynaptic mechanism as PTEN inhibition promotes AMPA receptor trafficking to synapses leading to a persistent increase in excitatory synaptic strength in adult hippocampal slices (Moult et al. 2010). Conversely enhanced PTEN lipid phosphatase activity has been reported to depress excitatory synaptic transmission, which is in turn required for NMDA receptor-dependent LTD (Jurado et al. 2011). Together these findings provide important new insights into the role of PTEN in regulating hippocampal synaptic function.

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1.

Excess α-synuclein worsens disease in mice lacking ubiquitin carboxy-terminal hydrolase L1

Mutations in α-synuclein (αSN) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been linked to familial Parkinson's disease (PD). Physical and functional interactions between these two proteins have been described. Whether they act additively in vivo to influence disease has remained controversial. αSN is a presynaptic protein and the major constituent of Lewy inclusions, histopathological hallmarks of PD. UCH-L1 regulates ubiquitin stability in the nervous system and its loss results in neurodegeneration in peripheral and central neurons. Here, we used genetics to show that UCH-L1-deficiency together with excess αSN worsen disease. Double mutant mice show earlier-onset motor deficits, a shorter lifespan and forebrain astrogliosis but the additive disease-worsening effects of UCH-L1-deficiency and excess αSN are not accompanied by microgliosis, ubiquitin pathology or changes in pathological αSN protein levels and species.

Brn3a/Pou4f1 regulates dorsal root ganglion sensory neuron specification and axonal projection into the spinal cord

The sensory neurons of the dorsal root ganglia (DRG) must project accurately to their central targets to convey proprioceptive, nociceptive and mechanoreceptive information to the spinal cord. How these different sensory modalities and central connectivities are specified and coordinated still remains unclear. Given the expression of the POU homeodomain transcription factors Brn3a/Pou4f1 and Brn3b/Pou4f2 in DRG and spinal cord sensory neurons, we determined the subtype specification of DRG and spinal cord sensory neurons as well as DRG central projections in Brn3a and Brn3b single and double mutant mice. Inactivation of either or both genes causes no gross abnormalities in early spinal cord neurogenesis; however, in Brn3a single and Brn3a;Brn3b double mutant mice, sensory afferent axons from the DRG fail to form normal trajectories in the spinal cord. The TrkA+ afferents remain outside the dorsal horn and fail to extend into the spinal cord, while the projections of TrkC+ proprioceptive afferents into the ventral horn are also impaired. Moreover, Brn3a mutant DRGs are defective in sensory neuron specification, as marked by the excessive generation of TrkB+ and TrkC+ neurons as well as TrkA+/TrkB+ and TrkA+/TrkC+ double positive cells at early embryonic stages. At later stages in the mutant, TrkB+, TrkC+ and parvalbumin+ neurons diminish while there is a significant increase of CGRP+ and c-ret+ neurons. In addition, Brn3a mutant DRGs display a dramatic down-regulation of Runx1 expression, suggesting that the regulation of DRG sensory neuron specification by Brn3a is mediated in part by Runx1. Our results together demonstrate a critical role for Brn3a in generating DRG sensory neuron diversity and regulating sensory afferent projections to the central targets.

Severe microcytic anemia but increased erythropoiesis in mice lacking Hfe or Tfr2 and Tmprss6

Cell surface proteins Hfe, Tfr2, hemojuvelin and Tmprss6 play key roles in iron homeostasis. Hfe and Tfr2 induce transcription of hepcidin, a small peptide that promotes the degradation of the iron transporter ferroportin. Hemojuvelin, a co-receptor for bone morphogenic proteins, induces hepcidin transcription through a Smad signaling pathway. Tmprss6 (also known as matriptase-2), a membrane serine protease that has been found to bind and degrade hemojuvelin in vitro, is a potent suppressor of hepcidin expression. In order to examine if Hfe and Tfr2 are substrates for Tmprss6, we generated mice lacking functional Hfe or Tfr2 and Tmprss6. We found that double mutant mice lacking functional Hfe or Tfr2 and Tmprss6 exhibited a severe iron deficiency microcytic anemia phenotype mimicking the phenotype of single mutant mice lacking functional Tmprss6 (Tmprss6msk/msk mutant) demonstrating that Hfe and Tfr2 are not substrates for Tmprss6. Nevertheless, the phenotype of the mice lacking Hfe or Tfr2 and Tmprss6 differed from Tmprss6 deficient mice alone, in that the double mutant mice exhibited much greater erythropoiesis. Hfe and Tfr2 have been shown to play important roles in the erythron, independent of their role in regulating liver hepcidin transcription. We demonstrate that lack of functional Tfr2 and Hfe allows for increased erythropoiesis even in the presence of high hepcidin expression, but the high levels of hepcidin levels significantly limit the availability of iron to the erythron, resulting in ineffective erythropoiesis. Furthermore, repression of hepcidin expression by hypoxia was unaffected by the loss of functional Hfe, Tfr2 and Tmprss6.