SirT1 regulates energy metabolism and response to caloric restriction in mice

The yeast sir2 gene and its orthologues in Drosophila and C. elegans have well-established roles in lifespan determination and response to caloric restriction. We have studied mice carrying two null alleles for SirT1, the mammalian orthologue of sir2, and found that these animals inefficiently utilize ingested food. These mice are hypermetabolic, contain inefficient liver mitochondria, and have elevated rates of lipid oxidation. When challenged with a 40% reduction in caloric intake, normal mice maintained their metabolic rate and increased their physical activity while the metabolic rate of SirT1-null mice dropped and their activity did not increase. Moreover, CR did not extend lifespan of SirT1-null mice. Thus, SirT1 is an important regulator of energy metabolism and, like its orthologues from simpler eukaryotes, the SirT1 protein appears to be required for a normal response to caloric restriction.     

sirt1-null mice develop an autoimmune-like condition

The sirt1 gene encodes a protein deacetylase with a broad spectrum of reported substrates. Mice carrying null alleles for sirt1 are viable on outbred genetic backgrounds so we have examined them in detail to identify the biological processes that are dependent on SIRT1. Sera from adult sirt1-null mice contain antibodies that react with nuclear antigens and immune complexes become deposited in the livers and kidneys of these animals. Some of the sirt1-null animals develop a disease resembling diabetes insipidus when they approach 2 years of age although the relationship to the autoimmunity remains unclear. We interpret these observations as consistent with a role for SIRT1 in sustaining normal immune function and in this way delaying the onset of autoimmune disease.     

MEK kinase activity is not necessary for Raf-1 function

Raf-1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by RAS:GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a 'knockout' of the raf-1 gene in mice as well as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations. Raf-1(-/-) mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf-1 from cells derived from raf-1(FF/FF) mice has no detectable activity towards MEK in vitro, and yet raf-1(FF/FF) mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf-1(-/-) and raf-1(FF/FF) mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis.     

mIGF-1/JNK1/SirT1 signaling confers protection against oxidative stress in the heart

Oxidative stress contributes to the pathogenesis of aging-associated heart failure. Among various signaling pathways mediating oxidative stress, the NAD(+) -dependent protein deacetylase SirT1 has been implicated in the protection of heart muscle. Expression of a locally acting insulin-like growth factor-1 (IGF-1) propeptide (mIGF-1) helps the heart to recover from infarct and enhances SirT1 expression in cardiomyocytes (CM) in vitro, exerting protection from hypertrophic and oxidative stresses. To study the role of mIGF-1/SirT1 signaling in vivo, we generated cardiac-specific mIGF-1 transgenic mice in which SirT1 was depleted from adult CM in a tamoxifen-inducible and conditional fashion. Analysis of these mice confirmed that mIGF-1-induced SirT1 activity is necessary to protect the heart from paraquat (PQ)-induced oxidative stress and lethality. In cultured CM, mIGF-1 increases SirT1 expression through a c-Jun NH(2)-terminal protein kinase 1 (JNK1)-dependent signaling mechanism. Thus, mIGF-1 protects the heart from oxidative stress via SirT1/JNK1 activity, suggesting new avenues for cardiac therapy during aging and heart failure.     

Role of the N-terminal catalytic domain of angiotensin-converting enzyme investigated by targeted inactivation in mice

Angiotensin-converting enzyme (ACE) produces the vasoconstrictor angiotensin II. The ACE protein is composed of two homologous domains, each binding zinc and each independently catalytic. To assess the physiologic significance of the two ACE catalytic domains, we used gene targeting in mice to introduce two point mutations (H395K and H399K) that selectively inactivated the ACE N-terminal catalytic site. This modification does not affect C-terminal enzymatic activity or ACE protein expression. In addition, the testis ACE isozyme is not affected by the mutations. Analysis of homozygous mutant mice (termed ACE 7/7) showed normal plasma levels of angiotensin II but an elevation of plasma and urine N-acetyl-Ser-Asp-Lys-Pro, a peptide suggested to inhibit bone marrow maturation. Despite this, ACE 7/7 mice had blood pressure, renal function, and hematocrit that were indistinguishable from wild-type mice. We also studied compound heterozygous mice in which one ACE allele was null (no ACE expression) and the second allele encoded the mutations selectively inactivating the N-terminal catalytic domain. These mice produced approximately half the normal levels of ACE, with the ACE protein lacking N-terminal catalytic activity. Despite this, the mice have a phenotype indistinguishable from wild-type animals. This study shows that, in vivo, the presence of the C-terminal ACE catalytic domain is sufficient to maintain a functional renin-angiotensin system. It also strongly suggests that the anemia present in ACE null mice is not due to the accumulation of the peptide N-acetyl-Ser-Asp-Lys-Pro.     

Genetic modification of the schisis phenotype in a mouse model of X-linked retinoschisis

X-linked retinoschisis (XLRS) is an inherited form of macular degeneration that is caused by mutations in the retinoschisin (RS1) gene. In addition to macular degeneration, other major characteristics of XLRS include splitting of the retina (schisis) and impaired synaptic transmission as indicated by a reduction in the electroretinogram b-wave. It has been known that patients carrying RS1 mutations show a broad range of phenotypic variability. Interestingly, phenotypic variation is observed even among family members with the same RS1 mutation, suggesting the existence of genetic or environmental factors that contribute to the severity of XLRS. However, in the human population, the cause of phenotypic variability and the contribution of genetic modifiers for this relatively rare disease are difficult to study and poorly understood. In this study, using a mouse model for XLRS, we show that genetic factors can contribute to the severity of the retinoschisis phenotype. We report evidence of a major genetic modifier of Rs1, which affects the disease severity in these animals. A quantitative trait locus (QTL), named modifier of Rs1 1 (Mor1), is mapped on chromosome (Chr) 7. When homozygous, the Mor1 allele from the inbred mouse strain AKR/J diminishes the severity of the schisis phenotype in Rs1(tmgc1)/Y male and Rs1(tmgc1)/Rs1(tmgc1) female mice. We also show that the penetrance of the disease phenotype is affected by additional genetic factor(s). Our study suggests that multiple genetic modifiers could potentially be responsible for the phenotypic variation in human XLRS.     

Loss of hairless confers susceptibility to UVB-induced tumorigenesis via disruption of NF-kappaB signaling

In order to model squamous cell carcinoma development in vivo, researchers have long preferred hairless mouse models such as SKH-1 mice that have traditionally been classified as 'wild-type' mice irrespective of the genetic factors underlying their hairless phenotype. The work presented here shows that mutations in the Hairless (Hr) gene not only result in the hairless phenotype of the SKH-1 and Hr(-/-) mouse lines but also cause aberrant activation of NFκB and its downstream effectors. We show that in the epidermis, Hr is an early UVB response gene that regulates NFκB activation and thereby controls cellular responses to irradiation. Therefore, when Hr expression is decreased in Hr mutant animals there is a corresponding increase in NFκB activity that is augmented by UVB irradiation. This constitutive activation of NFκB in the Hr mutant epidermis leads to the stimulation a large variety of downstream effectors including the cell cycle regulators cyclin D1 and cyclin E, the anti-apoptosis protein Bcl-2, and the pro-inflammatory protein Cox-2. Therefore, Hr loss results in a state of uncontrolled epidermal proliferation that promotes tumor development, and Hr mutant mice should no longer be considered merely hairless 'wild-type' mice. Instead, Hr is a crucial UVB response gene and its loss creates a permissive environment that potentiates increased tumorigenesis.     

Nijmegen breakage syndrome 1 protein hyperacetylation as a molecular mechanism underlying metabolic syndrome

Metabolic Syndrome (MetS) is characterized by visceral obesity, insulin resistance, hypertension, atherogenic dyslipidemia, and increased atherosclerotic plaque development and cardiovascular disease. It arises from a high-calorie “Western diet” and physical inactivity. MetS confers an elevated risk for type II diabetes, cancer, and cardiovascular disease, significantly shortening the affected individual's life. While many gene products affect the course of MetS, SirT1 and ataxia–telangiectasia mutated (ATM) protein activities ameliorate the pathophysiological effects of MetS in rodent models. SirT1 activity protects mice from the deleterious effects of a high-fat diet, promoting insulin sensitivity, fat mobilization, lowered blood pressure, and cell survival and genomic stability. ATM activation attenuates hypertension, diet-induced atherosclerotic plaque development, and glucose resistance in mice. ATM activity partially depends on Nijmegen breakage syndrome 1 protein (NBS1) activity. NBS1 can be acetylated, which inhibits its interactions with ATM, attenuating ATM function. Restoration of ATM activity requires NBS1 deacetylation by Sirt1. Interestingly, ATM activation increases SirT1 expression. Several studies show that a high-fat–sugar/high-calorie diet suppresses SirT1 expression in many tissues. Here we hypothesize that SirT1 suppression increases NBS1 acetylation, suppressing ATM activity, and finally attenuating ATM-mediated SirT1 expression. The resulting viscous cycle would promote MetS.     

Genetic mapping of a mouse modifier gene that can prevent ALS onset

Mutations in the cytoplasmic Cu/Zn superoxide dismutase (SOD1) gene on human chromosome 21q22.1 cause 10-20% of familial amyotrophic lateral sclerosis (ALS) cases. The expression of the ALS phenotype in mice carrying the murine G86R SOD1 mutation is highly dependent upon the mouse genetic background. This is similar to the phenotypic variation observed in ALS patients containing identical SOD1 mutations. In the FVB/N background, mice expressing mG86R SOD1 develop an ALS phenotype at approximately 100 days. However, when these mice were bred into a mixed background of C57Bl6/129Sv, the onset of the ALS phenotype was delayed (143 days to >2 years). Using 129 polymorphic autosomal markers in a whole genome scan, we have identified a major genetic modifier locus with a maximum lod score of 5.07 on mouse chromosome 13 between D13mit36 and D13mit76. This 5- to 8-cM interval contains the spinal muscular atrophy (SMA)-associated gene Smn (survival motor neuron) and seven copies of Naip (neuronal apoptosis inhibitory protein), suggesting a potential link between SMA and ALS.     

Functional redundancy of the DE-1 and alpha A-CRYBP1 regulatory sites of the mouse alpha A-crystallin promoter.

Previous studies have implicated the DE-1 (-111/-106) and alpha A-CRYBP1 (-66/-57) sites for activity of the mouse alpha A-crystallin promoter in transiently transfected lens cells. Here we have used the bacterial chloramphenicol acetyltransferase (CAT) reporter gene to test the functional importance of the putative DE-1 and alpha A-CRYBP1 regulatory elements by site-specific and deletion mutagenesis in stably transformed alpha TN4-1 lens cells and in transgenic mice. FVB/N and C57BL/6 x SJL F2 hybrid transgenic mice were assayed for CAT activity in the lens, heart, lung, kidney, spleen, liver, cerebrum, and muscle. F0, F1, and F2 mice from multiple lines carrying single mutations of the DE-1 or alpha A-CRYBP1 sites showed high levels of CAT activity in the lens, but not in any of the non-lens tissues. By contrast, despite activity of the wild-type promoter, none of the mutant promoter/CAT constructs were active in the transiently transfected and stably transformed lens cells. The mice carrying transgenes with either site-specific mutations in both the DE-1 and alpha A-CRYBP1 sites or a deletion of the entire DE-1 and part of the alpha A-CRYBP1 site (-60/+46) fused to the CAT gene did not exhibit CAT activity above background in any of the tissues examined, including the lens. Our results thus indicate that the DE-1 and alpha A-CRYBP1 sites are functionally redundant in transgenic mice. Moreover, the present data coupled with previous transfection and transgenic mouse experiments suggest that this functional redundancy is confined to lens expression within the mouse and is not evident in transiently transfected and stably transformed lens cells, making the cultured lens cells sensitive indicators of functional elements of crystallin genes.     

Cdk2 knockout mice are viable

BACKGROUND: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be required because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself is essential for cell proliferation during early embryonic development. RESULTS: In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable, and therefore Cdk2 is not an essential gene in the mouse. However, Cdk2 is required for germ cell development; both male and female Cdk2(-/-) mice are sterile. Immunoprecipitates of cyclin E1 complexes from Cdk2(-/-) spleen extracts displayed no activity toward histone H1. Cyclin A2 complexes were active in primary mouse embryonic fibroblasts (MEFs), embryo extracts and in spleen extracts from young animals. In contrast, there was little cyclin A2 kinase activity in immortalized MEFs and spleen extracts from adult animals. Cdk2(-/-) MEFs proliferate but enter delayed into S phase. Ectopic expression of Cdk2 in Cdk2(-/-) MEFs rescued the delayed entry into S phase. CONCLUSIONS: Although Cdk2 is not an essential gene in the mouse, it is required for germ cell development and meiosis. Loss of Cdk2 affects the timing of S phase, suggesting that Cdk2 is involved in regulating progression through the mitotic cell cycle.     

A Redox-resistant Sirtuin-1 Mutant Protects against Hepatic Metabolic and Oxidant Stress

Sirtuin-1 (SirT1), a member of the NAD⁺-dependent class III histone deacetylase family, is inactivated in vitro by oxidation of critical cysteine thiols. In a model of metabolic syndrome, SirT1 activation attenuated apoptosis of hepatocytes and improved liver function including lipid metabolism. We show in SirT1-overexpressing HepG2 cells that oxidants (nitrosocysteine and hydrogen peroxide) or metabolic stress (high palmitate and high glucose) inactivated SirT1 by reversible oxidative post-translational modifications (OPTMs) on three cysteines. Mutating these oxidation-sensitive cysteines to serine preserved SirT1 activity and abolished reversible OPTMs. Overexpressed mutant SirT1 maintained deacetylase activity and attenuated proapoptotic signaling, whereas overexpressed wild type SirT1 was less protective in metabolically or oxidant-stressed cells. To prove that OPTMs of SirT1 are glutathione (GSH) adducts, glutaredoxin-1 was overexpressed to remove this modification. Glutaredoxin-1 overexpression maintained endogenous SirT1 activity and prevented proapoptotic signaling in metabolically stressed HepG2 cells. The in vivo significance of oxidative inactivation of SirT1 was investigated in livers of high fat diet-fed C57/B6J mice. SirT1 deacetylase activity was decreased in the absence of changes in SirT1 expression and associated with a marked increase in OPTMs. These results indicate that glutathione adducts on specific SirT1 thiols may be responsible for dysfunctional SirT1 associated with liver disease in metabolic syndrome.     

Chaperone-mediated gene therapy with recombinant AAV-PPCA in a new mouse model of type I sialidosis

The lysosomal storage disease sialidosis is caused by a primary deficiency of the sialidase N-acetyl-α-neuraminidase-1 (NEU1). Patients with type I sialidosis develop an attenuated, non-neuropathic form of the disease also named cherry red spot myoclonus syndrome, with symptoms arising during juvenile/ adult age. NEU1 requires binding to its chaperone, protective protein/cathepsin A (PPCA), for lysosomal compartmentalization, stability and catalytic activation. We have generated a new mouse model of type I sialidosis that ubiquitously expresses a NEU1 variant carrying a V54M amino acid substitution identified in an adult patient with type I sialidosis. Mutant mice developed signs of lysosomal disease after 1 year of age, predominantly in the kidney, albeit low residual NEU1 activity was detected in most organs and cell types. We demonstrate that the activity of the mutant enzyme could be effectively increased in all systemic tissues by chaperone-mediated gene therapy with a liver-tropic recombinant AAV2/8 vector expressing PPCA. This resulted in clear amelioration of the disease phenotype. These results suggest that at least some of the NEU1 mutations associated with type I sialidosis may respond to PPCA-chaperone-mediated gene therapy.     

Contrasting effects of ENU induced embryonic lethal mutations of the quaking gene.

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qkl-1, qkkt1, qkk2, and qkkt3. The mutation in qkl-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains in qkk2 and qkkt3, respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qkv differ. Compound heterozygous qkv animals carrying qkkt1, qkk2, and qkkt3 all exhibit a permanent quaking phenotype similar to that of qkv/qkv animals, whereas qkv/qkl-1 animals exhibit only a transient quaking phenotype. The qkl-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed in qkv/qkl-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.     

Genetic analysis of the mammalian K+ channel beta subunit Kvbeta 2 (Kcnab2)

Kvbeta2 binds to K(+) channel alpha subunits from at least two different families (Kv1 and Kv4) and is a member of the aldo-ketoreductase (AKR) superfamily. Proposed functions for this protein in vivo include a chaperone-like role in Kv1 alpha subunit biogenesis and catalytic activity as an AKR oxidoreductase. To investigate the in vivo function of Kvbeta2, Kvbeta2-null and point mutant (Y90F) mice were generated through gene targeting in embryonic stem cells. In Kvbeta2-null mice, Kv1.1 and Kv1.2 localize normally in cerebellar basket cell terminals and the juxtaparanodal region of myelinated nerves. Moreover, normal glycosylation patterns are observed for Kv1.1 and Kv1.2 in whole brain lysates. Thus, loss of the chaperone-like activity does not appear to account for the phenotype of Kvbeta2-null mice, which include reduced life spans, occasional seizures, and cold swim-induced tremors similar to that observed in Kv1.1-null mice. Mice expressing Kvbeta2, mutated at a site (Y90F) that abolishes AKR-like catalytic activity in other family members, have no overt phenotype. We conclude that Kvbeta2 contributes to regulation of excitability in vivo, although not directly through either chaperone-like or typical AKR catalytic activity. Rather, Kvbeta2 relies upon as yet unidentified mechanisms in the regulation of K(+) channel and/or oxidoreductive functions.     

Tensor-based morphometry and stereology reveal brain pathology in the complexin1 knockout mouse

Complexins (Cplxs) are small, soluble, regulatory proteins that bind reversibly to the SNARE complex and modulate synaptic vesicle release. Cplx1 knockout mice (Cplx1(-/-)) have the earliest known onset of ataxia seen in a mouse model, although hitherto no histopathology has been described in these mice. Nevertheless, the profound neurological phenotype displayed by Cplx1(-/-) mutants suggests that significant functional abnormalities must be present in these animals. In this study, MRI was used to automatically detect regions where structural differences were not obvious when using a traditional histological approach. Tensor-based morphometry of Cplx1(-/-) mouse brains showed selective volume loss from the thalamus and cerebellum. Stereological analysis of Cplx1(-/-) and Cplx1(+/+) mice brain slices confirmed the volume loss in the thalamus as well as loss in some lobules of the cerebellum. Finally, stereology was used to show that there was loss of cerebellar granule cells in Cplx1(-/-) mice when compared to Cplx1(+/+) animals. Our study is the first to describe pathological changes in Cplx1(-/-) mouse brain. We suggest that the ataxia in Cplx1(-/-) mice is likely to be due to pathological changes in both cerebellum and thalamus. Reduced levels of Cplx proteins have been reported in brains of patients with neurodegenerative diseases. Therefore, understanding the effects of Cplx depletion in brains from Cplx1(-/-) mice may also shed light on the mechanisms underlying pathophysiology in disorders in which loss of Cplx1 occurs.     

Mouse neurovirulence determinants of poliovirus type 1 strain LS-a map to the coding regions of capsid protein VP1 and proteinase 2Apro.

Poliovirus type 1 strain LS-a [PV1(LS-a)] is a OV variant adapted to mice by multiple passages through mouse and monkey tissues. To investigate the molecular basis underlying mouse neurovirulence of PV1(LS-a), a cDNA of the viral genome containing nucleotides 112 to 7441 was cloned, and the nucleotide sequence was determined. Compared with that of the mouse avirulent progenitor PV1(Mahoney), 54 nucleotide changes were found in the genome of the PV1(LS-a) virus, resulting in 20 amino acid substitutions in the virus polyprotein. Whereas the nucleotide changes were scattered throughout the genome, the amino acid substitutions were largely clustered in the capsid proteins and, to a certain extent, in the virus proteinase 2Apro. By in vitro mutagenesis, PV1(LS-a)-specific capsid mutations were introduced into a cDNA clone of PV1(Mahoney). We show that neither the individual amino acid mutations nor combinations of mutations in the region encoding VP1 conferred to PV1(Mahoney) the mouse-adapted phenotype of PV1(LS-a). Chimeric cDNA studies demonstrated that a recombinant type 1 virus containing the PV1(LS-a) sequence from nucleotide 2470 to nucleotide 3625 displayed a neurovirulent phenotype in mice. Further dissection of this region revealed that mouse neurovirulence of PV1(LS-a) was determined by multiple mutations in regions encoding both viral proteinase 2Apro and capsid protein VP1. The mouse neurovirulent viruses, PV1(LS-a), W1-M/LS-Pf [nucleotides 496 to 3625 from PV1(LS-a)], and W1-M/LS-NP [nucleotides 2470 to 3625 from PV1(LS-a)], showed increased sensitivity to heat treatment at 45 degrees C for 1 h. Surprisingly, the thermolabile phenotype was also displayed by a recombinant of PV1(Mahoney) carrying a PV1(LS-a) DNA fragment encoding the N-terminal portion of 2Apro. This suggests that base substitutions in the region encoding 2Apro affected capsid stability, thereby contributing to the neurovirulence of the virus in mice.