RESUMEN
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by a progressive movement disorder, psychiatric symptoms, and cognitive impairments. HD is caused by a CAG repeat expansion encoding a stretch of polyglutamine residues in the N-terminus of mutant huntingtin (mHTT) protein. Proteolytic processing of mHTT yields toxic fragments, which cause neurotoxicity and massive neuronal cell death predominantly in the striatum and cortex. Inhibition of mHTT cleavage reduces neuronal toxicity suggesting mHTT proteolysis contributes to HD pathogenesis. A previously conducted unbiased siRNA screen in our lab for known human proteases identified matrix metalloproteinases (MMPs) as modifiers of mHTT proteolysis and toxicity. To further study MMP activation in HD, isogenic HD, and control corrected (C116) neural stem cells (NSCs) prepared from HD patient-derived induced pluripotent stem cells were used to examine the role of MMPs and their endogenous inhibitors in this highly relevant model system. We found altered expression of MMP-2 and MMP-9 (gelatinases), MMP-3/10, and MMP-14, activity in HD-NSCs when compared to control C116-NSCs. Dysregulation in MMP activity was accompanied with concomitant changes in levels of endogenous inhibitors of MMPs, called tissue inhibitors of matrix metalloproteinases (TIMPs). Specifically, we observed decreased levels of TIMP-1 and TIMP-2 in HD-NSCs, suggesting part of the altered expression and activity of MMPs is due to lower abundance of these endogenous inhibitors. Immunofluorescence analysis revealed increased MMP/TIMP localization in the nucleus or aggregates of HD-NSCs, suggesting potential interaction with mHTT. TIMP-1 was found to associate with mHTT aggregates in discrete punctate structures in HD-NSCs. These events collectively contribute to increased neurotoxicity in HD. Previous characterization of these NSCs revealed transforming growth factor beta (TGF-ß) pathway as the top dysregulated pathway in HD. TGF-ß was significantly upregulated in HD-NSCs and addition of TGF-ß to HD-NSCs was found to be neuroprotective. To determine if TGF-ß regulated MMP and TIMP activity, C116- and HD-NSCs were exogenously treated with recombinant TGF-ß. TIMP-1 levels were found to be elevated in response to TGF-ß treatment, representing a potential mechanism through which elevated TGF-ß levels confer neuroprotection in HD. Studying the mechanism of action of MMPs and TIMPs, and their interactions with mHTT in human isogenic patient-derived NSCs elucidates new mechanisms of HD neurotoxicity and will likely provide novel therapeutics for treatment of HD.
RESUMEN
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. The disease generally manifests in middle age with both physical and mental symptoms. There are no effective treatments or cures and death usually occurs 10-20 years after initial symptoms. Since the original identification of the Huntington disease associated gene, in 1993, a variety of models have been created and used to advance our understanding of HD. The most recent advances have utilized stem cell models derived from HD-patient induced pluripotent stem cells (iPSCs) offering a variety of screening and model options that were not previously available. The discovery and advancement of technology to make human iPSCs has allowed for a more thorough characterization of human HD on a cellular and developmental level. The interaction between the genome editing and the stem cell fields promises to further expand the variety of HD cellular models available for researchers. In this review, we will discuss the history of Huntington's disease models, common screening assays, currently available models and future directions for modeling HD using iPSCs-derived from HD patients. This article is part of a Special Issue entitled SI: PSC and the brain.
Asunto(s)
Enfermedad de Huntington/tratamiento farmacológico , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Animales , Línea Celular , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Células Madre Pluripotentes Inducidas/trasplanteRESUMEN
We utilized induced pluripotent stem cells (iPSCs) derived from Huntington's disease (HD) patients as a human model of HD and determined that the disease phenotypes only manifest in the differentiated neural stem cell (NSC) stage, not in iPSCs. To understand the molecular basis for the CAG repeat expansion-dependent disease phenotypes in NSCs, we performed transcriptomic analysis of HD iPSCs and HD NSCs compared to isogenic controls. Differential gene expression and pathway analysis pointed to transforming growth factor ß (TGF-ß) and netrin-1 as the top dysregulated pathways. Using data-driven gene coexpression network analysis, we identified seven distinct coexpression modules and focused on two that were correlated with changes in gene expression due to the CAG expansion. Our HD NSC model revealed the dysregulation of genes involved in neuronal development and the formation of the dorsal striatum. The striatal and neuronal networks disrupted could be modulated to correct HD phenotypes and provide therapeutic targets.
Asunto(s)
Enfermedad de Huntington/patología , Células Madre Pluripotentes Inducidas/patología , Células-Madre Neurales/patología , Transcriptoma , Línea Celular , Redes Reguladoras de Genes , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/fisiopatología , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Netrina-1 , Células-Madre Neurales/metabolismo , Neurogénesis , Factor de Crecimiento Transformador beta/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The cascade of events that lead to cognitive decline, motor deficits, and psychiatric symptoms in patients with Huntington disease (HD) is triggered by a polyglutamine expansion in the N-terminal region of the huntingtin (HTT) protein. A significant mechanism in HD is the generation of mutant HTT fragments, which are generally more toxic than the full-length HTT. The protein fragments observed in human HD tissue and mouse models of HD are formed by proteolysis or aberrant splicing of HTT. To systematically investigate the relative contribution of the various HTT protein proteolysis events observed in vivo, we generated transgenic mouse models of HD representing five distinct proteolysis fragments ending at amino acids 171, 463, 536, 552, and 586 with a polyglutamine length of 148. All lines contain a single integration at the ROSA26 locus, with expression of the fragments driven by the chicken ß-actin promoter at nearly identical levels. The transgenic mice N171-Q148 and N552-Q148 display significantly accelerated phenotypes and a shortened life span when compared with N463-Q148, N536-Q148, and N586-Q148 transgenic mice. We hypothesized that the accelerated phenotype was due to altered HTT protein interactions/complexes that accumulate with age. We found evidence for altered HTT complexes in caspase-2 fragment transgenic mice (N552-Q148) and a stronger interaction with the endogenous HTT protein. These findings correlate with an altered HTT molecular complex and distinct proteins in the HTT interactome set identified by mass spectrometry. In particular, we identified HSP90AA1 (HSP86) as a potential modulator of the distinct neurotoxicity of the caspase-2 fragment mice (N552-Q148) when compared with the caspase-6 transgenic mice (N586-Q148).
Asunto(s)
Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Animales , Encéfalo/patología , Codón sin Sentido , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Proteína Huntingtina , Enfermedad de Huntington/fisiopatología , Longevidad , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Mapeo de Interacción de Proteínas , ProteolisisRESUMEN
Tauopathies represent a group of neurodegenerative disorders characterized by the accumulation of pathological TAU protein in brains. We report a human neuronal model of tauopathy derived from induced pluripotent stem cells (iPSCs) carrying a TAU-A152T mutation. Using zinc-finger nuclease-mediated gene editing, we generated two isogenic iPSC lines: one with the mutation corrected, and another with the homozygous mutation engineered. The A152T mutation increased TAU fragmentation and phosphorylation, leading to neurodegeneration and especially axonal degeneration. These cellular phenotypes were consistent with those observed in a patient with TAU-A152T. Upon mutation correction, normal neuronal and axonal morphologies were restored, accompanied by decreases in TAU fragmentation and phosphorylation, whereas the severity of tauopathy was intensified in neurons with the homozygous mutation. These isogenic TAU-iPSC lines represent a critical advancement toward the accurate modeling and mechanistic study of tauopathies with human neurons and will be invaluable for drug-screening efforts and future cell-based therapies.
Asunto(s)
Terapia Genética , Células Madre Pluripotentes Inducidas/metabolismo , Tauopatías/genética , Tauopatías/terapia , Proteínas tau/genética , Axones/metabolismo , Axones/patología , Axones/fisiología , Diferenciación Celular/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Mutación , Neuronas/metabolismo , Neuronas/patología , Neuronas/fisiología , Fenotipo , Tauopatías/patología , Proteínas tau/metabolismoRESUMEN
BACKGROUND: Although extensive research has demonstrated the importance of excitatory granule neurons in the dentate gyrus of the hippocampus in normal learning and memory and in the pathogenesis of amnesia in Alzheimer's disease (AD), the role of hilar GABAergic inhibitory interneurons, which control the granule neuron activity, remains unclear. METHODOLOGY AND PRINCIPAL FINDINGS: We explored the function of hilar GABAergic interneurons in spatial learning and memory by inhibiting their activity through Cre-dependent viral expression of enhanced halorhodopsin (eNpHR3.0)--a light-driven chloride pump. Hilar GABAergic interneuron-specific expression of eNpHR3.0 was achieved by bilaterally injecting adeno-associated virus containing a double-floxed inverted open-reading frame encoding eNpHR3.0 into the hilus of the dentate gyrus of mice expressing Cre recombinase under the control of an enhancer specific for GABAergic interneurons. In vitro and in vivo illumination with a yellow laser elicited inhibition of hilar GABAergic interneurons and consequent activation of dentate granule neurons, without affecting pyramidal neurons in the CA3 and CA1 regions of the hippocampus. We found that optogenetic inhibition of hilar GABAergic interneuron activity impaired spatial learning and memory retrieval, without affecting memory retention, as determined in the Morris water maze test. Importantly, optogenetic inhibition of hilar GABAergic interneuron activity did not alter short-term working memory, motor coordination, or exploratory activity. CONCLUSIONS AND SIGNIFICANCE: Our findings establish a critical role for hilar GABAergic interneuron activity in controlling spatial learning and memory retrieval and provide evidence for the potential contribution of GABAergic interneuron impairment to the pathogenesis of amnesia in AD.
Asunto(s)
Giro Dentado/metabolismo , Neuronas GABAérgicas/metabolismo , Interneuronas/metabolismo , Aprendizaje/fisiología , Memoria/fisiología , Animales , Giro Dentado/citología , Dependovirus/genética , Genes Reporteros , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Memoria a Corto Plazo/fisiología , Ratones , Ratones Transgénicos , Desempeño PsicomotorRESUMEN
The generation of induced pluripotent stem cells (iPSCs) and induced neuronal cells (iNCs) from somatic cells provides new avenues for basic research and potential transplantation therapies for neurological diseases. However, clinical applications must consider the risk of tumor formation by iPSCs and the inability of iNCs to self-renew in culture. Here we report the generation of induced neural stem cells (iNSCs) from mouse and human fibroblasts by direct reprogramming with a single factor, Sox2. iNSCs express NSC markers and resemble wild-type NSCs in their morphology, self-renewal, ability to form neurospheres, and gene expression profiles. Cloned iNSCs differentiate into several types of mature neurons, as well as astrocytes and oligodendrocytes, indicating multipotency. Implanted iNSCs can survive and integrate in mouse brains and, unlike iPSC-derived NSCs, do not generate tumors. Thus, self-renewable and multipotent iNSCs without tumorigenic potential can be generated directly from fibroblasts by reprogramming.
Asunto(s)
Reprogramación Celular/genética , Fibroblastos/citología , Células Madre Multipotentes/citología , Células-Madre Neurales/citología , Factores de Transcripción SOXB1/metabolismo , Animales , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Embrión de Mamíferos/citología , Feto/citología , Fibroblastos/metabolismo , Humanos , Ratones , Células Madre Multipotentes/metabolismo , Neoplasias/patología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismoRESUMEN
Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease (AD). ApoE4 has sex-dependent effects, whereby the risk of developing AD is higher in apoE4-expressing females than males. However, the mechanism underlying the sex difference, in relation to apoE4, is unknown. Previous findings indicate that apoE4 causes age-dependent impairments of hilar GABAergic interneurons in female mice, leading to learning and memory deficits. Here, we investigate whether the detrimental effects of apoE4 on hilar GABAergic interneurons are sex-dependent using apoE knock-in (KI) mice across different ages. We found that in female apoE-KI mice, there was an age-dependent depletion of hilar GABAergic interneurons, whereby GAD67- or somatostatin-positive--but not NPY- or parvalbumin-positive-interneuron loss was exacerbated by apoE4. Loss of these neuronal populations was correlated with the severity of spatial learning deficits at 16 months of age in female apoE4-KI mice; however, this effect was not observed in female apoE3-KI mice. In contrast, we found an increase in the numbers of hilar GABAergic interneurons with advancing age in male apoE-KI mice, regardless of apoE genotype. Moreover, male apoE-KI mice showed a consistent ratio of hilar inhibitory GABAergic interneurons to excitatory mossy cells approximating 1.5 that is independent of apoE genotype and age, whereas female apoE-KI mice exhibited an age-dependent decrease in this ratio, which was exacerbated by apoE4. Interestingly, there are no apoE genotype effects on GABAergic interneurons in the CA1 and CA3 subregions of the hippocampus as well as the entorhinal and auditory cortexes. These findings suggest that the sex-dependent effects of apoE4 on developing AD is in part attributable to inherent sex-based differences in the numbers of hilar GABAergic interneurons, which is further modulated by apoE genotype.
Asunto(s)
Apolipoproteína E4/genética , Neuronas GABAérgicas/metabolismo , Hipocampo/metabolismo , Interneuronas/metabolismo , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/genética , Factores de Edad , Animales , Apolipoproteína E4/metabolismo , Técnicas de Sustitución del Gen , Trastornos de la Memoria/metabolismo , Ratones , Ratones Transgénicos , Factores SexualesRESUMEN
PURPOSE: Comparison of two different methods for management of second stage of labor: immediate pushing at complete cervical dilation of 10 cm and delayed pushing 90 minutes after complete cervical dilation. STUDY DESIGN AND METHODS: This study was a randomized clinical trial in a labor and delivery unit of a not-for-profit community hospital. A sample of 44 nulliparous mothers with continuous epidural anesthesia were studied after random assignment to treatment groups. Subjects were managed with either immediate or delayed pushing during the second stage of labor at the time cervical dilation was complete. The primary outcome measure was the length of pushing during second stage of labor. Secondary outcomes included length of second stage of labor, maternal fatigue and perineal injuries, and fetal heart rate decelerations. Two-tailed, unpaired Student's t-tests and Chi-square analysis were used for data analysis. Level of significance was set at p < .01 following a Bonferroni correction for multiple t-tests. RESULTS: A total of 44 subjects received the study intervention (N = 28 immediate pushing; N = 16 delayed pushing). The delayed pushing group had significantly shorter amount of time spent in pushing compared with the immediate pushing group (38.9 +/- 6.9 vs. 78.7 +/- 7.9 minutes, respectively, p = .002). Maternal fatigue scores, perineal injuries, and fetal heart rate decelerations were similar for both groups. CLINICAL IMPLICATIONS: Delaying pushing for up to 90 minutes after complete cervical dilation resulted in a significant decrease in the time mothers spent pushing without a significant increase in total time in second stage of labor.In clinical practice, healthcare providers sometimes resist delaying the onset of pushing after second stage of labor has begun because of a belief it will increase labor time. This study's finding of a 51% reduction in pushing time when mothers delay pushing for up to 90 minutes, with no significant increase in overall time for second stage of labor, disputes that concern.
Asunto(s)
Parto Obstétrico/métodos , Segundo Periodo del Trabajo de Parto/fisiología , Esfuerzo Físico , Resultado del Embarazo , Contracción Uterina/fisiología , Adulto , Analgesia Epidural/métodos , Analgesia Obstétrica/métodos , Parto Obstétrico/enfermería , Femenino , Humanos , Recién Nacido , Dimensión del Dolor/métodos , Satisfacción del Paciente , Embarazo , Presión , Valores de Referencia , Adulto JovenRESUMEN
Apolipoprotein (apo) E, a polymorphic protein with three isoforms (apoE2, apoE3, and apoE4), is essential for lipid homeostasis. Carriers of apoE4 are at higher risk for developing Alzheimer's disease. We have investigated adult neurogenesis in mice with knockout (KO) for apoE or with knockin (KI) alleles for human apoE3 or apoE4, and we report that neurogenesis is reduced in both apoE-KO and apoE4-KI mice. In apoE-KO mice, increased BMP signaling promoted glial differentiation at the expense of neurogenesis. In contrast, in apoE4-KI mice, presynaptic GABAergic input-mediated maturation of newborn neurons was diminished. Tau phosphorylation, an Alzheimer's disease characteristic, and levels of neurotoxic apoE fragments were both elevated in apoE4-KI hippocampal neurons concomitant with decreased GABAergic interneuron survival. Potentiating GABAergic signaling restored neuronal maturation and neurogenesis in apoE4-KI mice to normal levels. These findings suggest that GABAergic signaling can be targeted to mitigate the deleterious effects of apoE4 on neurogenesis.
Asunto(s)
Células Madre Adultas/metabolismo , Enfermedad de Alzheimer/metabolismo , Apolipoproteínas/metabolismo , Neuroglía/metabolismo , Células Madre Adultas/efectos de los fármacos , Células Madre Adultas/patología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Animales Recién Nacidos , Apolipoproteínas/genética , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Agonistas del GABA/administración & dosificación , Técnicas de Sustitución del Gen , Hipocampo/patología , Humanos , Interneuronas/metabolismo , Interneuronas/patología , Ratones , Ratones Noqueados , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Neuroglía/efectos de los fármacos , Neuroglía/patología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas tau/metabolismoRESUMEN
We used the recently sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia to analyze the codon usage patterns in both organisms; we have analyzed codon usage bias, Gln codon usage, GC content and the nucleotide contexts of initiation and termination codons in Tetrahymena and Paramecium. We also studied how these trends change along the length of the genes and in a subset of highly expressed genes. Our results corroborate some of the trends previously described in Tetrahymena, but also negate some specific observations. In both genomes we found a strong bias toward codons with low GC content; however, in highly expressed genes this bias is smaller and codons ending in GC tend to be more frequent. We also found that codon bias increases along gene segments and in highly expressed genes and that the context surrounding initiation and termination codons are always AT rich. Our results also suggest differences in the efficiency of translation of the reassigned stop codons between the two species and between the reassigned codons. Finally, we discuss some of the possible causes for such translational efficiency differences.
Asunto(s)
Codón/genética , Paramecium tetraurelia/genética , Tetrahymena thermophila/genética , Aminoácidos/genética , Animales , Composición de Base , Secuencia de Bases , Codón/análisis , Codón Iniciador/análisis , Codón Iniciador/genética , Codón de Terminación/análisis , Codón de Terminación/genética , Expresión Génica , Genes ProtozoariosRESUMEN
Tetrahymena thermophila and Paramecium tetraurelia are ciliates that reassign TAA and TAG from stop codons to glutamine codons. Because of the lack of full genome sequences, few studies have concentrated on analyzing the effects of codon reassignment in protein evolution. We used the recently sequenced genome of these species to analyze the patterns of amino acid substitution in ciliates that reassign the code. We show that, as expected, the codon reassignment has a large impact on amino acid substitutions in closely related proteins; however, contrary to expectations, these effects also hold for very diverged proteins. Previous studies have used amino acid substitution data to calculate the minimization of the genetic code; our results show that because of the lasting influence of the code in the patterns of substitution, such studies are tautological. These different substitution patterns might affect alignment of ciliate proteins, as alignment programs use scoring matrices based on substitution patterns of organisms that use the standard code. We also show that glutamine is used more frequently in ciliates than in other species, as often as expected based on the presence of the 2 new reassigned codons, indicating that the frequencies of amino acids in proteomes is mostly determined by neutral processes based on their number of codons.