Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients

Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder characteristics of this syndrome. So far, 3 Rab27a missense mutations have been identified. They open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the alpha 4 (Ala152Pro) or beta 5 (Leu130Pro) loop, observed in 2 of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within an invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding and GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.     

Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex

Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1-dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.     

Studies on a new variant of the Hermansky-Pudlak syndrome: Qualitative,ultrastructural, and functional abnormalities of the platelet-dense bodies associated with a phospholipase a defect

The structure and functions of platelets from a patient in whom albinism and hemorrhagic diathesis were associated have been investigated. Electron microscope studies showed a large reduction in the number of dense bodies and this was confirmed by an examination of fluorescent platelets loaded with mepacrine. The rare dense bodies were much bigger than normally observed; their density was diminished and was localized in a peripheral ring. Other platelet constituents were found to be normal. Platelet peroxidase activity was normal in the canaliculi of the dense tubular system; catalase-positive granules were also present. Serotonin uptake by the patient's platelets was much decreased and reserpine, a potent inhibitor of serotonin accumulation by normal human platelets, did not further decrease this incorporation. The uptake of free ¹⁴C-arachidonic acid by the platelets was greatly diminished, as was its thrombin-induced liberation from phosphatidylcholine and phosphatidyl inositol. Moreover, platelet phospholipase A₁ activity was much reduced and phospholipase A₂ activity was undetectable.     

Toll-like receptor modulation of murine cerebral malaria is dependent on the genetic background of the host

Infection with Plasmodium berghei ANKA is a well-established model of human cerebral malaria (CM). We show herein that Toll-like receptor (TLR) signaling influences the development of lethal CM in P. berghei ANKA-infected mice. Modulation of outcome was dependent on genetic background, such that deletion of myeloid differentiation factor (MyD) 88 on the susceptible C57BL/6 background resulted in resistance to CM, whereas deletion of MyD88 on the resistant BALB/c background led to increased mortality. Our data show that MyD88 influenced the production of T helper-polarizing cytokines, including interferon (IFN)- gamma, interleukin (IL)-4, and IL-17, as well as the total number of Foxp3(+) regulatory T (T(reg)) cells in a manner dependent on host genetic background. In addition, mRNA levels of IFN- gamma, CXCL10, and CXCL9 were strongly up-regulated in the brains of susceptible wild-type but not MyD88(-/-) infected mice. These results suggest that TLR signaling and host genetic background influences the pathogenesis of CM via modulation of cytokine production and T(reg) cell numbers.     

Cappuccino,a mouse model of Hermansky-Pudlak syndrome,encodes a novel protein that is part of the pallidin-muted complex (BLOC-1)

Hermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis affecting 3 related organelles-melanosomes, platelet dense bodies, and lysosomes. Four genes causing HPS in humans (HPS1-HPS4) are known, and at least 15 nonallelic mutations cause HPS in the mouse. Where their functions are known, the HPS-associated proteins are involved in some aspect of intracellular vesicular trafficking, that is, protein sorting and vesicle docking and fusion. Biochemical and genetic evidence indicates that the HPS-associated genes encode components of at least 3 distinct protein complexes: the adaptor complex AP-3; the HPS1/HPS4 complex; and BLOC-1 (biogenesis of lysosome-related organelles complex-1), consisting of the proteins encoded at 2 mouse HPS loci, pallid (pa) and muted (mu), and at least 3 other unidentified proteins. Here, we report the cloning of the mouse HPS mutation cappuccino (cno). We show that the wild-type cno gene encodes a novel, ubiquitously expressed cytoplasmic protein that coassembles with pallidin and the muted protein in the BLOC-1 complex. Further, we identify a frameshift mutation in mutant cno/cno mice. The C-terminal 81 amino acids are replaced with 72 different amino acids in the mutant CNO protein, and its ability to interact in BLOC-1 is abolished. We performed mutation screening of patients with HPS and failed to identify any CNO defects. Notably, although defects in components of the HPS1/HPS4 and the AP-3 complexes are associated with HPS in humans, no defects in the known components of BLOC-1 have been identified in 142 patients with HPS screened to date, suggesting that BLOC-1 function may be critical in humans.     

Effect of the genetic background and specific mutation on adenylate cyclase activity in obesity syndromes

Adenylate cyclase activity and its modulation by guanine nucleotides and isoproterenol were assessed in adipocyte membranes of mice with mutations causing different genetic obesity syndromes. The object was to determine whether the defect in inhibitory modulation observed in the obese (ob/ob) mouse was also present in the diabetes (db/db) mouse. The data show that adipocyte adenylate cyclase in both the ob/ob and the db/db mouse is resistant to activation by isoproterenol. The response to guanosine triphosphate (GTP) differed between the two mutants, such that an inhibitory phase was visible in the db/db but not in the ob/ob membranes. Moreover, pertussis toxin attenuated the inhibitory effect of GTP and significantly stimulated cyclase activity in the db/db but not in the ob/ob membranes. The data show that the two mutations affect the expression of adenylate cyclase activity via different mechanisms.     

Decreased ethanol sensitivity and tolerance development in gamma-protein kinase C null mutant mice is dependent on genetic background

Initial sensitivity and tolerance development to the sedative-hypnotic and hypothermic effects of ethanol were investigated in gamma-protein kinase C (PKC) null mutant mice. Null mutants from a C57BL/6J x 129/SvJ mixed genetic background demonstrated decreased ethanol sensitivity and failed to develop chronic tolerance after 10 days of ethanol liquid diet. However, when the null mutation was introgressed onto a C57BL/6J background for six generations, the "no tolerance" phenotype for sedative-hypnotic and hypothermic effects of ethanol was no longer apparent Outcrossing the gamma-PKC null mutation to a C57BL/6J x 129/SvEvTac mixed background restored the "no tolerance" phenotype to ethanol-induced sedation after chronic ethanol diet; however, as measured by hypothermia, tolerance was still evident in the null mutant mice. These observations and the results of tests of chronic tolerance in the C57BL/6J, 129/SvJ, and 129/SvEvTac background inbred strains indicate that gamma-PKC plays an important role in initial sensitivity and tolerance to ethanol. However, the impact of gamma-PKC is modulated by the background genotype. These results stress the importance of including the effect of genetic background when evaluating the effects of single gene mutations on quantitative behavioral traits.     

Dissecting the genetic complexity of human 6p deletion syndromes by using a region-specific, phenotype-driven mouse screen

Monosomy of the human chromosome 6p terminal region results in a variety of congenital malformations that include brain, craniofacial, and organogenesis abnormalities. To examine the genetic basis of these phenotypes, we have carried out an unbiased functional analysis of the syntenic region of the mouse genome (proximal Mmu13). A genetic screen for recessive mutations in this region recovered thirteen lines with phenotypes relevant to a variety of clinical conditions. These include two loci that cause holoprosencephaly, two that underlie anophthalmia, one of which also contributes to other craniofacial abnormalities such as microcephaly, agnathia, and palatogenesis defects, and one locus responsible for developmental heart and kidney defects. Analysis of heterozygous carriers of these mutations shows that a high proportion of these loci manifest with behavioral activity and sensorimotor deficits in the heterozygous state. This finding argues for the systematic, reciprocal phenotypic assessment of dominant and recessive mouse mutants. In addition to providing a resource of single gene mutants that model 6p-associated disorders, the work reveals unsuspected genetic complexity at this region. In particular, many of the phenotypes associated with 6p deletions can be elicited by mutation in one of a number of genes. This finding implies that phenotypes associated with contiguous gene deletion syndromes can result not only from dosage sensitivity of one gene in the region but also from the combined effect of monosomy for multiple genes that function within the same biological process.     

Chronic creatine kinase deficiency eventually leads to congestive heart failure, but severity is dependent on genetic background, gender and age

The creatine kinase (CK) energy transport and buffering system supports cardiac function at times of high demand and is impaired in the failing heart. Mice deficient in muscle- and mitochondrial-CK (M/Mt-CK(-/-)) have previously been described, but exhibit an unexpectedly mild phenotype of compensated left ventricular (LV) hypertrophy. We hypothesised that heart failure would develop with age and performed echocardiography and LV haemodynamics at 1?year. Since all previous studies have utilised mice with a mixed genetic background, we backcrossed for >10 generations on to C57BL/6, and repeated the in vivo investigations. Male M/Mt-CK(-/-) mice on the mixed genetic background developed congestive heart failure as evidenced by significantly elevated end-diastolic pressure, impaired contractility, LV dilatation, hypertrophy and pulmonary congestion. Female mice were less severely affected, only showing trends for these parameters. After backcrossing, M/Mt-CK(-/-) mice had LV dysfunction consisting of impaired isovolumetric pressure changes and reduced contractile reserve, but did not develop congestive heart failure. Body weight was lower in knockout mice as a consequence of reduced total body fat. LV weight was not significantly elevated in relation to other internal organs and gene expression of LVH markers was normal, suggesting an absence of hypertrophy. In conclusion, the consequences of CK deficiency are highly dependent on genetic modifiers, gender and age. However, the observation that a primary defect in CK can, under the right conditions, result in heart failure suggests that impaired CK activity in the failing heart could contribute to disease progression.     

Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex

Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.     

Influence of the genetic background on platelet function,microparticle and thrombin generation in the common laboratory mouse

Genetic background has been shown to affect phenotype in transgenic mice with hemostatic defects. We present comparative data on various platelet function tests, as well as on microparticle and thrombin generation capacity in three mouse strains most commonly used in transgenic applications. Normal, inbred 129Sv ( n = 24), C57BL/6 ( n = 14) and BALB/c ( n = 22) mice were used. BALB/c mice showed statistically significantly longer tail-bleeding times (158 - 42 s, P <0.02) than 129Sv (113 - 37) and C57BL/6 (122 - 29) and paradoxically increased velocity of platelet aggregation ( P <0.01) with ADP, collagen, ionophore and thrombin. ATP-release did not differ between strains, it was weakest with ADP and strongest with thrombin. 129Sv platelets were less responsive to thrombin. Generation of platelet microparticles did not differ between strains either and could efficiently be inhibited by EDTA but not by aspirin or alprostadil. Two anti-GPIIIa monoclonal antibodies did not inhibit microparticles either, although they could block aggregation. Thrombin generation was equivalent in C57BL/6 and BALB/c, but slower in 129Sv. Interestingly, the reaction in all strains was immediate and different from the human model in that no lag-phase was seen after triggering. In summary these mouse strains show similar patterns of ex vivo platelet aggregation, bleeding times as well as microparticle and thrombin generation. Subtle differences were found, which should be taken into consideration when interpreting data from wild-type or transgenic models.     

Effects of dietary sodium and genetic background on angiotensinogen and Renin in mouse

Elements of a renin-angiotensin system expressed along the entire nephron, including angiotensinogen secreted by proximal tubule and renin expressed in connecting tubule, may participate in the regulation of sodium reabsorption at multiple sites of the nephron. The response of this tubular renin-angiotensin system to stepwise changes in dietary sodium was investigated in 2 mouse strains, the sodium-sensitive inbred C57BL/6 and the sodium-resistant CD1 outbred. Plasma angiotensinogen was not affected by sodium regimen, whereas plasma renin increased 2-fold under low sodium. In both strains, the variation in urinary parameters did not parallel the changes observed in plasma. Angiotensinogen and renin excretion were significantly higher under high sodium than under low sodium. Water deprivation, by contrast, induced significant activation in the tubular expression of angiotensinogen and renin. C57BL/6 exhibited significantly higher urinary excretion of angiotensinogen than did CD1 animals under both conditions of sodium intake. The extent to which these urinary parameters reflect systemic or tubular responses to challenges of sodium homeostasis may depend on the relative contribution of sodium restriction and volume depletion.     

The influence of genetic background on the induction of oxidative stress and impaired insulin secretion in mouse islets

Aims/hypothesis We determined whether high-glucose-induced beta cell dysfunction is associated with oxidative stress in the DBA/2 mouse, a mouse strain susceptible to islet failure.Materials and methods Glucose- and non-glucose-mediated insulin secretion from the islets of DBA/2 and control C57BL/6 mice was determined following a 48-h exposure to high glucose. Flux via the hexosamine biosynthesis pathway was assessed by determining O-glycosylated protein levels. Oxidative stress was determined by measuring hydrogen peroxide levels and the expression of anti-oxidant enzymes.Results Exposure to high glucose levels impaired glucose-stimulated insulin secretion in DBA/2 islets but not C57BL/6 islets, and this was associated with reduced islet insulin content and lower ATP levels than in C57BL/6 islets. Exposure of islets to glucosamine for 48 h mimicked the effects of high glucose on insulin secretion in the DBA/2 islets. High glucose exposure elevated O-glycosylated proteins; however, this occurred in islets from both strains, excluding a role for O-glycosylation in the impairment of DBA/2 insulin secretion. Additionally, both glucosamine and high glucose caused an increase in hydrogen peroxide in DBA/2 islets but not in C57BL/6 islets, an effect prevented by the antioxidant N-acetyl-l-cysteine. Interestingly, while glutathione peroxidase and catalase expression was comparable between the two strains, the antioxidant enzyme manganese superoxide dismutase, which converts superoxide to hydrogen peroxide, was increased in DBA/2 islets, possibly explaining the increase in hydrogen peroxide levels.Conclusions/interpretation Chronic high glucose culture caused an impairment in glucose-stimulated insulin secretion in DBA/2 islets, which have a genetic predisposition to failure, and this may be the result of oxidative stress.     

The influence of genetic background on the expression of the obese (ob) gene in the mouse

Summary A new congenic strain of obese mice, C57BK/KsJ-ob, has been developed for comparison with the C57BL/6J-ob congenic strain. While obese mice of both strains are characterized by obesity, hyperphagia, and hyperglycemia, the C57BL/Ks obese mice have severe diabetes, marked hyperglycemia, temporarily elevated plasma insulin concentrations, and typical degenerative changes in the islets of Langerhans. In contrast, the C57BL/6J obese mice have mild hyperglycemia and marked hyperinsulinemia coupled with hypertrophy and hyperplasia of the islets of Langerhans. The severe diabetic condition produced by obese (ob) on the C57BL/KsJ background is similar, if not identical, to that produced by the diabetes (db) gene on the same background. The metabolic disorder produced by these mutations is associated with the capacity of the islets to respond to an increased demand for insulin. The islet response, whether atrophy or hypertrophy, appears to be due to the interaction of the obese and diabetes genes with modifiers in the genetic background rather than the specific consequences of the particular gene. The markedly different diabetic syndromes that result when the obese mutation is on different genetic backgrounds emphasize the importance of strict genetic control in studies with obese-hyperglycemic mutants.Supported in part by NIH Research Grants AM 14461 from the National Institute of Arthritis, Metabolism, and Digestive Diseases; CA 05873 from the National Cancer Institute and allocations from the South Waite Foundation and the Virginia and D.K. Ludwig Foundation.The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Microbial,metabolomic, and immunologic dynamics in a relapsing genetic mouse model of colitis induced by T-synthase deficiency

Intestinal dysbiosis is thought to confer susceptibility to inflammatory bowel disease (IBD), but it is unknown whether dynamic changes in the microbiome contribute to fluctuations in disease activity. We explored this question using mice with intestine-specific deletion of C1galt1 (also known as T-synthase) (Tsyn mice). These mice develop spontaneous microbiota-dependent colitis with a remitting/relapsing course due to loss of mucin core-1 derived O-glycans. 16S rRNA sequencing and untargeted metabolomics demonstrated age-specific perturbations in the intestinal microbiome and metabolome of Tsyn mice compare with littermate controls at weeks 3 (disease onset), 5 (during remission), and 9 (after relapse). Colitis remission corresponded to increased levels of FoxP3+RORγt+CD4+ T cells in the colonic lamina propria that were positively correlated with operational taxonomic units (OTUs) in the S24–7 family and negatively correlated with OTUs in the Clostridiales order. Relapse was characterized by marked expansion of FoxP3-RORγt+CD4+ T cells expressing IFNγ and IL17A, which were associated with Clostridiales OTUs distinct from those negatively correlated with FoxP3+RORγt+CD4+ T cells. Our findings suggest that colitis remission and relapse in the Tsyn model may reflect alterations in the microbiome due to reduced core-1 O-glycosylation that shift the balance of regulatory and pro-inflammatory T cell subsets. We investigated whether genetic variation in C1galt1 correlated with the microbiome in a cohort of 78 Crohn's disease patients and 101 healthy controls. Polymorphisms near C1galt1 (rs10486157) and its molecular chaperone, Cosmc (rs4825729), were associated with altered composition of the colonic mucosal microbiota, supporting the relevance of core-1 O-glycosylation to host regulation of the microbiome.     

Behavioral,neurochemical, and electrophysiological characterization of a genetic mouse model of depression

Depression is a multifactorial illness and genetic factors play a role in its etiology. The understanding of its physiopathology relies on the availability of experimental models potentially mimicking the disease. Here we describe a model built up by selective breeding of mice with strikingly different responses in the tail suspension test, a stress paradigm aimed at screening potential antidepressants. Indeed, "helpless" mice are essentially immobile in the tail suspension test, as well as the Porsolt forced-swim test, and they show reduced consumption of a palatable 2% sucrose solution. In addition, helpless mice exhibit sleep-wakefulness alterations resembling those classically observed in depressed patients, notably a lighter and more fragmented sleep, with an increased pressure of rapid eye movement sleep. Compared with "nonhelpless" mice, they display higher basal seric corticosterone levels and lower serotonin metabolism index in the hippocampus. Remarkably, serotonin(1A) autoreceptor stimulation induces larger hypothermia and inhibition of serotoninergic neuronal firing in the nucleus raphe dorsalis in helpless than in nonhelpless mice. Thus, helpless mice exhibit a decrease in serotoninergic tone, which evokes that associated with endogenous depression in humans. Finally, both the behavioral impairments and the serotoninergic dysfunction can be improved by chronic treatment with the antidepressant fluoxetine. The helpless line of mice may provide an opportunity to approach genes influencing susceptibility to depression and to investigate neurophysiological and neurochemical substrates underlying antidepressant effects.