RESUMO
BACKGROUND: Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice, Lsq-1 and Civq-1, that affected limb muscle survival and stroke volume after femoral artery or middle cerebral artery ligation, respectively. Here, we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hind-limb ischemia. METHODS: We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2-associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia. RESULTS: We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6-Lsq1-3). BALB/c mice treated with adeno-associated virus encoding the BL6 BAG3 variant (Ile81; n=25) displayed reduced limb-tissue necrosis and increased limb tissue perfusion compared with Met81- (n=25) or green fluorescent protein- (n=29) expressing animals. BAG3Ile81, but not BAG3Met81, improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of adeno-associated virus-BAG3Ile81 (n=9), but not BAG3Met81 (n=10) or green fluorescent protein (n=5), improved ischemic limb blood flow and limb muscle histology and restored muscle function (force production). Compared with BAG3Met81, BAG3Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. CONCLUSIONS: Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Variação Genética/genética , Membro Posterior/irrigação sanguínea , Isquemia/genética , Doenças Musculares/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Transformada , Membro Posterior/patologia , Isquemia/patologia , Isquemia/prevenção & controle , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Doenças Musculares/patologia , Doenças Musculares/prevenção & controle , Ligação Proteica/fisiologiaRESUMO
Empathy is the important capacity to recognize and share emotions with others. Recent evidence shows that rodents possess a remarkable affective sensitivity to the emotional state of others and that primitive forms of empathy exist in social lives of rodents. However, due to the ambiguous definitional boundaries between empathy, emotional contagion and other related terms, distinct components of empathic behaviors in rodents need to be clarified. Hence, we review recent experimental studies demonstrating that rodents are able to share emotions with others. Specifically, we highlight several behavioral models that examine different aspects of rodent empathic behaviors in response to the various distress of conspecifics. Experimental approaches using rodent behavioral models will help elucidate the neural circuitry of empathy and its neurochemical association. Integrating these findings with corresponding experiments in humans will ultimately provide novel insights into therapeutic interventions for mental disorders associated with empathy.
Assuntos
Comportamento Animal/fisiologia , Modelos Animais de Doenças , Empatia/fisiologia , Roedores/fisiologia , Comportamento Social , AnimaisRESUMO
During ischemic stroke, occlusion of the cerebrovasculature causes neuronal cell death (infarction), but naturally occurring genetic factors modulating infarction have been difficult to identify in human populations. In a surgically induced mouse model of ischemic stroke, we have previously mapped Civq1 to distal chromosome 7 as a quantitative trait locus determining infarct volume. In this study, genome-wide association mapping using 32 inbred mouse strains and an additional linkage scan for infarct volume confirmed that the size of the infarct is determined by ancestral alleles of the causative gene(s). The genetically isolated Civq1 locus in reciprocal recombinant congenic mice refined the critical interval and demonstrated that infarct size is determined by both vascular (collateral vessel anatomy) and non-vascular (neuroprotection) effects. Through the use of interval-specific SNP haplotype analysis, we further refined the Civq1 locus and identified integrin alpha L (Itgal) as one of the causative genes for Civq1. Itgal is the only gene that exhibits both strain-specific amino acid substitutions and expression differences. Coding SNPs, a 5-bp insertion in exon 30b, and increased mRNA and protein expression of a splice variant of the gene (Itgal-003, ENSMUST00000120857), all segregate with infarct volume. Mice lacking Itgal show increased neuronal cell death in both ex vivo brain slice and in vivo focal cerebral ischemia. Our data demonstrate that sequence variation in Itgal modulates ischemic brain injury, and that infarct volume is determined by both vascular and non-vascular mechanisms.
Assuntos
Estudo de Associação Genômica Ampla , Cadeias alfa de Integrinas/genética , Acidente Vascular Cerebral/genética , Alelos , Animais , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Ligação Genética , Haplótipos , Humanos , Cadeias alfa de Integrinas/metabolismo , Camundongos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Acidente Vascular Cerebral/fisiopatologiaRESUMO
In the mouse model of permanent, middle cerebral artery occlusion, infarct volume varies widely across inbred strains but generally is inversely correlated with collateral vessel number. However, we also observed certain mouse strains that share similar collateral vessel anatomy but exhibit significantly different infarct volume. To identify genetic factors determining infarct volume in a collateral vessel-independent manner, we performed quantitative trait locus analysis on a F2 cross between C57BL/6J and C3H/HeJ strains. We mapped four novel loci (Civq4 through Civq7) that modulate infarct volume. Civq4, on chromosome 8, is the strongest locus (logarithm of the odds 9.8) that contributes 21% of the phenotypic variance of infarct volume in the cross. The Civq4 and Civq6 loci represent transgressive B6 alleles that render animals susceptible to larger infarcts. Based on genomic sequence and microarray analyses, we propose candidate genes for the Civq4 locus. By selecting inbred strains with similar collateral vessel anatomy but that vary significantly in infarct volume, we have mapped four loci determining infarct volume in a mouse model of ischemic stroke. Two of the loci appear to modulate infarct volume through a collateral vessel-independent mechanism. Based on strain-specific sequence variants and differences in transcript levels, Msr1 and Mtmr7 appear to be strong candidate genes for Civq4. Identifying the underlying genetic factors of these loci will elucidate the genetic architecture response to cerebral ischemia, shed new light on disease mechanisms of ischemic stroke, and identify potential therapeutic targets for clinical applications.
Assuntos
Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Circulação Colateral/genética , Locos de Características Quantitativas , Animais , Infarto Encefálico/genética , Infarto Encefálico/patologia , Mapeamento Cromossômico , Modelos Animais de Doenças , Ligação Genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único , Proteínas Tirosina Fosfatases não Receptoras/genética , Receptores Depuradores Classe A/genéticaRESUMO
Genetics plays an important role in determining peripheral arterial disease (PAD) pathology, which causes a spectrum of clinical disorders that range from clinically silent reductions in blood flow to limb-threatening ischemia. The cell-type specificity of PAD pathology, however, has received little attention. To determine whether strain-dependent differences in skeletal muscle cells might account for the differential responses to ischemia observed in C57BL/6 and BALB/c mice, endothelial and skeletal muscle cells were subjected to hypoxia and nutrient deprivation (HND) in vitro, to mimic ischemia. Muscle cells were more susceptible to HND than were endothelial cells. In vivo, C57BL/6 and BALB/c mice displayed strain-specific differences in myofiber responses after hindlimb ischemia, with significantly greater myofiber atrophy, greater apoptosis, and attenuated myogenic regulatory gene expression and stress-responsive signaling in BALB/c mice. Strain-specific deficits were recapitulated in vitro in primary muscle cells from both strains after HND. Muscle cells from BALB/c mice congenic for the C57BL/6 Lsq-1 quantitative trait locus were protected from HND-induced atrophy, and gene expression of vascular growth factors and their receptors was significantly greater in C57BL/6 primary muscle cells. Our results indicate that the previously identified specific genetic locus regulating strain-dependent collateral vessel density has a nonvascular or muscle cell-autonomous role involving both the myogenic program and traditional vascular growth factor receptor expression.
Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/genética , Músculo Esquelético/irrigação sanguínea , Animais , Apoptose/genética , Hipóxia Celular/fisiologia , Células Cultivadas , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais/fisiologia , Especificidade da EspécieRESUMO
Although it has recently been shown that A/J mice are highly susceptible to Staphylococcus aureus sepsis as compared to C57BL/6J, the specific genes responsible for this differential phenotype are unknown. Using chromosome substitution strains (CSS), we found that loci on chromosomes 8, 11, and 18 influence susceptibility to S. aureus sepsis in A/J mice. We then used two candidate gene selection strategies to identify genes on these three chromosomes associated with S. aureus susceptibility, and targeted genes identified by both gene selection strategies. First, we used whole genome transcription profiling to identify 191 (56 on chr. 8, 100 on chr. 11, and 35 on chr. 18) genes on our three chromosomes of interest that are differentially expressed between S. aureus-infected A/J and C57BL/6J. Second, we identified two significant quantitative trait loci (QTL) for survival post-infection on chr. 18 using N(2) backcross mice (F(1) [C18A]xC57BL/6J). Ten genes on chr. 18 (March3, Cep120, Chmp1b, Dcp2, Dtwd2, Isoc1, Lman1, Spire1, Tnfaip8, and Seh1l) mapped to the two significant QTL regions and were also identified by the expression array selection strategy. Using real-time PCR, 6 of these 10 genes (Chmp1b, Dtwd2, Isoc1, Lman1, Tnfaip8, and Seh1l) showed significantly different expression levels between S. aureus-infected A/J and C57BL/6J. For two (Tnfaip8 and Seh1l) of these 6 genes, siRNA-mediated knockdown of gene expression in S. aureus-challenged RAW264.7 macrophages induced significant changes in the cytokine response (IL-1 beta and GM-CSF) compared to negative controls. These cytokine response changes were consistent with those seen in S. aureus-challenged peritoneal macrophages from CSS 18 mice (which contain A/J chromosome 18 but are otherwise C57BL/6J), but not C57BL/6J mice. These findings suggest that two genes, Tnfaip8 and Seh1l, may contribute to susceptibility to S. aureus in A/J mice, and represent promising candidates for human genetic susceptibility studies.
Assuntos
Cromossomos de Mamíferos/genética , Predisposição Genética para Doença , Locos de Características Quantitativas/genética , Sepse/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores/metabolismo , Western Blotting , Quimiocinas/metabolismo , Mapeamento Cromossômico , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/microbiologia , Sepse/patologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidadeRESUMO
Optogenetic approaches for controlling Ca2+ channels provide powerful means for modulating diverse Ca2+-specific biological events in space and time. However, blue light-responsive photoreceptors are, in principle, considered inadequate for deep tissue stimulation unless accompanied by optic fiber insertion. Here, we present an ultra-light-sensitive optogenetic Ca2+ modulator, named monSTIM1 encompassing engineered cryptochrome2 for manipulating Ca2+ signaling in the brain of awake mice through non-invasive light delivery. Activation of monSTIM1 in either excitatory neurons or astrocytes of mice brain is able to induce Ca2+-dependent gene expression without any mechanical damage in the brain. Furthermore, we demonstrate that non-invasive Ca2+ modulation in neurons can be sufficiently and effectively translated into changes in behavioral phenotypes of awake mice.
Assuntos
Canais de Cálcio/metabolismo , Criptocromos/metabolismo , Tecnologia de Fibra Óptica , Optogenética , Molécula 1 de Interação Estromal/metabolismo , Animais , Astrócitos , Encéfalo/metabolismo , Cálcio/metabolismo , Criptocromos/química , Criptocromos/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Neurônios/metabolismo , Alinhamento de Sequência , Molécula 1 de Interação Estromal/química , VigíliaRESUMO
BACKGROUND: Peripheral arterial disease (PAD) caused by occlusive atherosclerosis of the lower extremity has 2 major clinical manifestations. Critical limb ischemia is characterized by rest pain and/or tissue loss and has a > or = 40% risk of death and major amputation. Intermittent claudication causes pain on walking, has no tissue loss, and has amputation plus mortality rates of 2% to 4% per year. Progression from claudication to limb ischemia is infrequent. Risk factors in most PAD patients overlap. Thus, we hypothesized that genetic variations may be linked to presence or absence of tissue loss in PAD. METHODS AND RESULTS: Hindlimb ischemia (murine model of PAD) was induced in C57BL/6, BALB/c, C57BL/6 x BALB/c (F1), F1 x BALB/c (N2), A/J, and C57BL/6J-Chr7(A/J)/NaJ chromosome substitution strains. Mice were monitored for perfusion recovery and tissue necrosis. Genome-wide scanning with polymorphic markers across the 19 murine autosomes was performed on the N2 mice. Greater tissue loss and poorer perfusion recovery occurred in BALB/c than in the C57BL/6 strain. Analysis of 105 N2 progeny identified a single quantitative trait locus on chromosome 7 that exhibited significant linkage to both tissue necrosis and extent of perfusion recovery. Using the appropriate chromosome substitution strain, we demonstrate that C57BL/6-derived chromosome 7 is required for tissue preservation. CONCLUSIONS: We have identified a quantitative trait locus on murine chromosome 7 (LSq-1) that is associated with the absence of tissue loss in a preclinical model of PAD and may be useful in identifying gene(s) that influence PAD in humans.
Assuntos
Cromossomos de Mamíferos/genética , Membro Posterior/irrigação sanguínea , Membro Posterior/cirurgia , Isquemia/genética , Isquemia/cirurgia , Locos de Características Quantitativas/genética , Animais , Feminino , Membro Posterior/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Especificidade da EspécieRESUMO
The neurocognitive ability to recognize and share the mental states of others is crucial for our emotional experience and social interaction. Extensive human studies have informed our understanding of the psychobehavioral and neurochemical bases of empathy. Recent evidence shows that simple forms of empathy are conserved from rodents to humans, and rodent models have become particularly useful for understanding the neurobiological correlates of empathy. In this review, we first summarize aspects of empathy at the behavioral and neural circuit levels, and describe recent developments in rodent model behavioral paradigms. We then highlight different neurobiological pathways involved in empathic abilities, with special emphasis on genetic polymorphisms associated with individual differences in empathy. By directly assessing various neurochemical correlates at molecular and neural circuit levels using relevant animal models, we conclude with the suggestion that rodent research can significantly advance our understanding of the neural basis of empathy. This article is part of the Special Issue entitled 'The neuropharmacology of social behavior: from bench to bedside'.
Assuntos
Empatia/genética , Variação Genética/genética , Polimorfismo de Nucleotídeo Único/genética , Animais , Emoções/fisiologia , Humanos , Relações Interpessoais , CamundongosRESUMO
Observational fear learning in rodents is a type of context-dependent fear conditioning in which an unconditioned stimulus (US) is provided vicariously by observing conspecific others receiving foot shocks. This suggests the involvement of affective empathy, with several recent studies showing many similarities between this behavior and human empathy. Neurobiologically, it is important to understand the neural mechanisms by which the vicarious US activates the fear circuit via the affective pain system, obviating the sensory pain pathway and eventually leading to fear memory formation. This paper reviews current studies on the neural mechanisms underlying observational fear learning and provides a perspective on future research on this subject.
Assuntos
Afeto , Condicionamento Clássico/fisiologia , Empatia , Medo , Giro do Cíngulo/fisiologia , Dor/fisiopatologia , Animais , Humanos , Aprendizagem/fisiologia , Observação , Dor/psicologiaRESUMO
Empathy enables social mammals to recognize and share emotion with others and is well-documented in non-human primates. During the past few years, systematic observations have showed that a primal form of empathy also exists in rodents, indicating that empathy has an evolutionary continuity. Now, using rodents exhibiting emotional empathy, the molecular and cellular study of empathy in animals has begun in earnest. In this article, we will review recent reports that indicate that rodents can share states of fear with others, and will try to highlight new understandings of the neural circuitry, biochemistry and genetics of empathic fear. We hope that the use of rodent models will enhance understanding of the mechanisms of human empathy and provide insights into how to treat social deficits in neuropsychiatric disorders characterized by empathy impairment.
Assuntos
Encéfalo/metabolismo , Empatia , Medo , Animais , Encéfalo/fisiologia , Interação Gene-Ambiente , Roedores , Meio SocialRESUMO
Empathy is crucial for our emotional experience and social interactions, and its abnormalities manifest in various psychiatric disorders. Observational fear is a useful behavioral paradigm for assessing affective empathy in rodents. However, specific genes that regulate observational fear remain unknown. Here we showed that 129S1/SvImJ mice carrying a unique missense variant in neurexin 3 (Nrxn3) exhibited a profound and selective enhancement in observational fear. Using the CRISPR/Cas9 system, the arginine-to-tryptophan (R498W) change in Nrxn3 was confirmed to be the causative variant. Selective deletion of Nrxn3 in somatostatin-expressing (SST+) interneurons in the anterior cingulate cortex (ACC) markedly increased observational fear and impaired inhibitory synaptic transmission from SST+ neurons. Concordantly, optogenetic manipulation revealed that SST+ neurons in the ACC bidirectionally controlled the degree of socially transmitted fear. Together, these results provide insights into the genetic basis of behavioral variability and the neurophysiological mechanism controlling empathy in mammalian brains.
Assuntos
Empatia/fisiologia , Medo/fisiologia , Loci Gênicos/fisiologia , Variação Genética/fisiologia , Mutação de Sentido Incorreto/fisiologia , Proteínas do Tecido Nervoso/genética , Animais , Medo/psicologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos ICR , Especificidade da EspécieRESUMO
Risk for ischemic stroke has a strong genetic basis, but heritable factors also contribute to the extent of damage after a stroke has occurred. We previously identified a locus on distal mouse chromosome 7 that contributes over 50% of the variation in postischemic cerebral infarct volume observed between inbred strains. Here, we used ancestral haplotype analysis to fine-map this locus to 12 candidate genes. The gene encoding the IL-21 receptor (Il21r) showed a marked difference in strain-specific transcription levels and coding variants in neonatal and adult cortical tissue. Collateral vessel connections were moderately reduced in Il21r-deficient mice, and cerebral infarct volume increased 2.3-fold, suggesting that Il21r modulates both collateral vessel anatomy and innate neuroprotection. In brain slice explants, oxygen deprivation (OD) activated apoptotic pathways and increased neuronal cell death in IL-21 receptor-deficient (IL-21R-deficient) mice compared with control animals. We determined that the neuroprotective effects of IL-21R arose from signaling through JAK/STAT pathways and upregulation of caspase 3. Thus, natural genetic variation in murine Il21r influences neuronal cell viability after ischemia by modulating receptor function and downstream signal transduction. The identification of neuroprotective genes based on naturally occurring allelic variations has the potential to inform the development of drug targets for ischemic stroke treatment.
Assuntos
Alelos , Isquemia Encefálica/imunologia , Subunidade alfa de Receptor de Interleucina-21/genética , Neurônios/metabolismo , Animais , Apoptose , Encéfalo/embriologia , Encéfalo/patologia , Infarto Encefálico/metabolismo , Sobrevivência Celular , Mapeamento Cromossômico , Feminino , Variação Genética , Haplótipos , Subunidade alfa de Receptor de Interleucina-21/metabolismo , Escore Lod , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Neuroproteção , Oxigênio/química , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Transdução de SinaisRESUMO
BACKGROUND: In a mouse model of focal cerebral ischemia, infarct volume is highly variable and strain dependent, but the natural genetic determinants responsible for this difference remain unknown. To identify genetic determinants regulating ischemic neuronal damage and to dissect apart the role of individual genes and physiological mechanisms in infarction in mice, we performed quantitative trait locus analysis of surgically induced cerebral infarct volume. METHODS AND RESULTS: After permanent occlusion of the distal middle cerebral artery, infarct volume was determined for 16 inbred strains of mice, chromosome substitution strains, and for 2 intercross cohorts, F2 (B6xBALB/c) and F2 (B6xSWR/J). Genome-wide linkage analysis was performed for infarct volume as a quantitative trait. Infarct volume varied up to 30-fold between strains, with heritability estimated at 0.88. Overall, 3 quantitative trait locus were identified that modulate infarct volume, with a major locus (Civq1) on chromosome 7 accounting for >50% of the variation, with a combined LOD score of 21.7. Interval-specific single nucleotide polymorphism haplotype analysis for Civq1 results in 12 candidate genes. CONCLUSIONS: The extent of ischemic tissue damage after distal middle cerebral artery occlusion in inbred strains of mice is modulated by genetic variation mapping to at least 3 different loci. A single locus on chromosome 7 determines the majority of the observed variation in the trait. This locus seems to be identical to LSq1, a locus conferring limb salvage and reperfusion in a mouse model of hindlimb ischemia. The identification of the genes underlying these loci may uncover novel genetic and physiological pathways that modulate cerebral infarction and provide new targets for therapeutic intervention in ischemic stroke, and possibly other ischemic diseases.