RESUMEN
Primary familial brain calcification (PFBC) is a genetic neurological disease, yet no effective treatment is currently available. Here, we identified five novel intronic variants in SLC20A2 gene from six PFBC families. Three of these variants increased aberrant SLC20A2 pre-mRNA splicing by altering the binding affinity of splicing machineries to newly characterized cryptic exons, ultimately causing premature termination of SLC20A2 translation. Inhibiting the cryptic-exon incorporation with splice-switching ASOs increased the expression levels of functional SLC20A2 in cells carrying SLC20A2 mutations. Moreover, by knocking in a humanized SLC20A2 intron 2 sequence carrying a PFBC-associated intronic variant, the SLC20A2-KI mice exhibited increased inorganic phosphate (Pi) levels in cerebrospinal fluid (CSF) and progressive brain calcification. Intracerebroventricular administration of ASOs to these SLC20A2-KI mice reduced CSF Pi levels and suppressed brain calcification. Together, our findings expand the genetic etiology of PFBC and demonstrate ASO-mediated splice modulation as a potential therapy for PFBC patients with SLC20A2 haploinsufficiency.
RESUMEN
Aberrant inorganic phosphate (Pi) homeostasis causes brain calcification and aggravates neurodegeneration, but the underlying mechanism remains unclear. Here, we found that primary familial brain calcification (PFBC)-associated Pi transporter genes Pit2 and Xpr1 were highly expressed in astrocytes, with importer PiT2 distributed over the entire astrocyte processes and exporter XPR1 localized to astrocyte end-feet on blood vessels. This polarized PiT2 and XPR1 distribution endowed astrocyte with Pi transport capacity competent for brain Pi homeostasis, which was disrupted in mice with astrocyte-specific knockout (KO) of either Pit2 or Xpr1. Moreover, we found that Pi uptake by PiT2, and its facilitation by PFBC-associated galactosidase MYORG, were required for the high Pi transport capacity of astrocytes. Finally, brain calcification was suppressed by astrocyte-specific PiT2 re-expression in Pit2-KO mice. Thus, astrocyte-mediated Pi transport is pivotal for brain Pi homeostasis, and elevating astrocytic Pi transporter function represents a potential therapeutic strategy for reducing brain calcification.
RESUMEN
OBJECTIVE: Most paroxysmal kinesigenic dyskinesia (PKD) cases are hereditary, yet approximately 60% of patients remain genetically undiagnosed. We undertook the present study to uncover the genetic basis for undiagnosed PKD patients. METHODS: Whole-exome sequencing was performed for 106 PRRT2-negative PKD probands. The functional impact of the genetic variants was investigated in HEK293T cells and Drosophila. RESULTS: Heterozygous variants in KCNJ10 were identified in 11 individuals from 8 unrelated families, which accounted for 7.5% (8/106) of the PRRT2-negative probands. Both co-segregation of the identified variants and the significantly higher frequency of rare KCNJ10 variants in PKD cases supported impacts from the detected KCNJ10 heterozygous variants on PKD pathogenesis. Moreover, a KCNJ10 mutation-carrying father from a typical EAST/SeSAME family was identified as a PKD patient. All patients manifested dystonia attacks triggered by sudden movement with a short episodic duration. Patch-clamp recordings in HEK293T cells revealed apparent reductions in K+ currents of the patient-derived variants, indicating a loss-of-function. In Drosophila, milder hyperexcitability phenotypes were observed in heterozygous Irk2 knock-in flies compared to homozygotes, supporting haploinsufficiency as the mechanism for the detected heterozygous variants. Electrophysiological recordings showed that excitatory neurons in Irk2 haploinsufficiency flies exhibited increased excitability, and glia-specific complementation with human Kir4.1 rescued the Irk2 mutant phenotypes. INTERPRETATION: Our study established haploinsufficiency resulting from heterozygous variants in KCNJ10 can be understood as a previously unrecognized genetic cause for PKD and provided evidence of glial involvement in the pathophysiology of PKD. ANN NEUROL 2024.
RESUMEN
Primary familial brain calcification (PFBC) is an inherited neurodegenerative disorder mainly characterized by progressive calcium deposition bilaterally in the brain, accompanied by various symptoms, such as dystonia, ataxia, parkinsonism, dementia, depression, headaches, and epilepsy. Currently, the etiology of PFBC is largely unknown, and no specific prevention or treatment is available. During the past 10 years, six causative genes (SLC20A2, PDGFRB, PDGFB, XPR1, MYORG, and JAM2) have been identified in PFBC. In this review, considering mechanistic studies of these genes at the cellular level and in animals, we summarize the pathogenesis and potential preventive and therapeutic strategies for PFBC patients. Our systematic analysis suggests a classification for PFBC genetic etiology based on several characteristics, provides a summary of the known composition of brain calcification, and identifies some potential therapeutic targets for PFBC.
Asunto(s)
Encefalopatías , Animales , Encefalopatías/genética , Encefalopatías/terapia , Receptor de Retrovirus Xenotrópico y Politrópico , Encéfalo/patologíaRESUMEN
Brain calcification is a critical aging-associated pathology and can cause multifaceted neurological symptoms. Cerebral phosphate homeostasis dysregulation, blood-brain barrier defects, and immune dysregulation have been implicated as major pathological processes in familial brain calcification (FBC). Here, we analyzed two brain calcification families and identified calcification co-segregated biallelic variants in the CMPK2 gene that disrupt mitochondrial functions. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) isolated from these patients showed impaired mitochondria-associated metabolism pathways. In situ hybridization and single-cell RNA sequencing revealed robust Cmpk2 expression in neurons and vascular endothelial cells (vECs), two cell types with high energy expenditure in the brain. The neurons in Cmpk2-knockout (KO) mice have fewer mitochondrial DNA copies, down-regulated mitochondrial proteins, reduced ATP production, and elevated intracellular inorganic phosphate (Pi) level, recapitulating the mitochondrial dysfunction observed in the PBMCs isolated from the FBC patients. Morphologically, the cristae architecture of the Cmpk2-KO murine neurons was also impaired. Notably, calcification developed in a progressive manner in the homozygous Cmpk2-KO mice thalamus region as well as in the Cmpk2-knock-in mice bearing the patient mutation, thus phenocopying the calcification pathology observed in the patients. Together, our study identifies biallelic variants of CMPK2 as novel genetic factors for FBC; and demonstrates how CMPK2 deficiency alters mitochondrial structures and functions, thereby highlighting the mitochondria dysregulation as a critical pathogenic mechanism underlying brain calcification.
RESUMEN
A novel bacteria with heterotrophic nitrification and aerobic denitrification ability was obtained from a membrane bioreactor (MBR) and identified as Acinetobacter sp. TSH1. The nitrogen removal characteristics, nitrogen balance analysis, kinetic characteristics, and enhanced biological treatment in MBR of the novel isolated strain TSH1 were determined. Results showed that strain TSH1 could remove approximately 96.6% of NH4+-N, 82.9% of NO2--N and 98.7% of NO3--N in 24 h, and the corresponding maximum removal rates were 3.64 mg-N/(L·h), 1.77 mg-N/(L·h) and 3.94 mg-N/(L·h). The nitrogen balance analysis indicated that most of NH4+-N (62.6%) and NO3--N (71.9%) were transformed to gaseous nitrogen. The kinetic experiments showed that strain TSH1 had a high Km of 151.64 mg-NH4+-N/L and 203.25 mg-NO3--N/L. The enhanced biological treatment of synthetic wastewater in MBR showed that the strain TSH1 can significantly improve the nitrogen removal efficiency.
Asunto(s)
Desnitrificación , Nitrificación , Aerobiosis , Bacterias , Reactores Biológicos , Procesos Heterotróficos , Nitritos , Nitrógeno , Aguas ResidualesRESUMEN
This study investigated an Iron-carbon (Fe-C) micro-electrolysis method to enhance nitrogen removal of Sesuvium portulacastrum constructed wetlands (CWs) when treating mariculture effluents. The main objective was to investigate the effects of Fe-C on nitrogen purification performance and microbial characteristics of Sesuvium portulacastrum CWs. Results showed that the presence of Fe-C and Sesuvium portulacastrum could improve nitrogen removal efficiency by 20-30% and 15-30%, respectively. CWs with 33% v/v Fe-C addition performed well on nitrogen removal: TAN, 41.49 ± 13.64%; NO2--N, 13.32%; NO3--N, 60.02 ± 6.17%; TIN, 63.40 ± 12.11%. Microbial analysis revealed that Fe-C altered the microbial communities, and improved the abundance of denitrification related genera. Based on microbial enzyme activities and genes abundance, the anammox and denitrification processes were promoted by Fe-C in CWs. These findings indicate that Sesuvium portulacastrum CWs with 33% v/v Fe-C represents an effective nitrogen removal for mariculture wastewater with insufficient carbon source.
Asunto(s)
Aizoaceae , Humedales , Carbono , Desnitrificación , Hierro , Nitrógeno , Eliminación de Residuos LíquidosRESUMEN
OBJECTIVE: Mutations of the cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders characterized by intractable epilepsy, intellectual disability, and autism. Multiple mouse models generated for mechanistic studies have exhibited phenotypes similar to some human pathological features, but none of the models has developed one of the major symptoms affecting CDKL5 deficiency disorder (CDD) patients: intractable recurrent seizures. As disrupted neuronal excitation/inhibition balance is closely associated with the activity of glutamatergic and γ-aminobutyric acidergic (GABAergic) neurons, our aim was to study the effect of the loss of CDKL5 in different types of neurons on epilepsy. METHODS: Using the Cre-LoxP system, we generated conditional knockout (cKO) mouse lines allowing CDKL5 deficiency in glutamatergic or GABAergic neurons. We employed noninvasive video recording and in vivo electrophysiological approaches to study seizure activity in these Cdkl5 cKO mice. Furthermore, we conducted Timm staining to confirm a morphological alteration, mossy fiber sprouting, which occurs with limbic epilepsy in both human and mouse brains. Finally, we performed whole-cell patch clamp in dentate granule cells to investigate cell-intrinsic properties and synaptic excitatory activity. RESULTS: We demonstrate that Emx1- or CamK2α-derived Cdkl5 cKO mice manifest high-frequency spontaneous seizure activities recapitulating the epilepsy of CDD patients, which ultimately led to sudden death in mice. However, Cdkl5 deficiency in GABAergic neurons does not generate such seizures. The seizures were accompanied by typical epileptic features including higher amplitude spikes for epileptiform discharges and abnormal hippocampal mossy fiber sprouting. We also found an increase in spontaneous and miniature excitatory postsynaptic current frequencies but no change in amplitudes in the dentate granule cells of Emx1-cKO mice, indicating enhanced excitatory synaptic activity. SIGNIFICANCE: Our study demonstrates that Cdkl5 cKO mice, serving as an animal model to study recurrent spontaneous seizures, have potential value for the pathological study of CDD-related seizures and for therapeutic innovation.
Asunto(s)
Síndromes Epilépticos/genética , Neuronas GABAérgicas/metabolismo , Ácido Glutámico/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Convulsiones/genética , Espasmos Infantiles/genética , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Giro Dentado/citología , Giro Dentado/metabolismo , Modelos Animales de Enfermedad , Electroencefalografía , Síndromes Epilépticos/metabolismo , Síndromes Epilépticos/fisiopatología , Potenciales Postsinápticos Excitadores/fisiología , Neuronas GABAérgicas/patología , Proteínas de Homeodominio , Ratones , Ratones Noqueados , Fibras Musgosas del Hipocampo/patología , Neuronas/metabolismo , Neuronas/patología , Técnicas de Placa-Clamp , Prosencéfalo , Convulsiones/metabolismo , Convulsiones/fisiopatología , Espasmos Infantiles/metabolismo , Espasmos Infantiles/fisiopatología , Factores de TranscripciónRESUMEN
Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am-/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11-13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am-/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am-/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.
Asunto(s)
Espinas Dendríticas/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Proteína Fosfatasa 2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Trastorno del Espectro Autista/metabolismo , Encéfalo/enzimología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Ratones , Ratones Transgénicos , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteolisis , Transmisión Sináptica , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Primary familial brain calcification (PFBC) is a genetically heterogeneous disorder characterized by bilateral calcifications in the basal ganglia and other brain regions. The genetic basis of this disorder remains unknown in a significant portion of familial cases. Here, we reported a recessive causal gene, MYORG, for PFBC. Compound heterozygous or homozygous mutations of MYORG co-segregated completely with PFBC in six families, with logarithm of odds (LOD) score of 4.91 at the zero recombination fraction. In mice, Myorg mRNA was expressed specifically in S100ß-positive astrocytes, and knockout of Myorg induced the formation of brain calcification at 9 months of age. Our findings provide strong evidence that loss-of-function mutations of MYORG cause brain calcification in humans and mice.
Asunto(s)
Astrocitos/metabolismo , Encefalopatías/genética , Calcinosis/genética , Glicósido Hidrolasas/genética , Mutación con Pérdida de Función , ARN Mensajero/metabolismo , Adulto , Anciano , Alelos , Animales , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Mutación , LinajeRESUMEN
Mutations in the proline-rich transmembrane protein 2 (PRRT2) are associated with paroxysmal kinesigenic dyskinesia (PKD) and several other paroxysmal neurological diseases, but the PRRT2 function and pathogenic mechanisms remain largely obscure. Here we show that PRRT2 is a presynaptic protein that interacts with components of the SNARE complex and downregulates its formation. Loss-of-function mutant mice showed PKD-like phenotypes triggered by generalized seizures, hyperthermia, or optogenetic stimulation of the cerebellum. Mutant mice with specific PRRT2 deletion in cerebellar granule cells (GCs) recapitulate the behavioral phenotypes seen in Prrt2-null mice. Furthermore, recording made in cerebellar slices showed that optogenetic stimulation of GCs results in transient elevation followed by suppression of Purkinje cell firing. The anticonvulsant drug carbamazepine used in PKD treatment also relieved PKD-like behaviors in mutant mice. Together, our findings identify PRRT2 as a novel regulator of the SNARE complex and provide a circuit mechanism underlying the PRRT2-related behaviors.
Asunto(s)
Cerebelo/fisiopatología , Distonía/genética , Proteínas de la Membrana/fisiología , Proteínas SNARE/metabolismo , Transmisión Sináptica/genética , Animales , Carbamazepina/farmacología , Carbamazepina/uso terapéutico , Cerebelo/metabolismo , Distonía/tratamiento farmacológico , Proteínas de la Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Mutación , Células de Purkinje/metabolismoRESUMEN
The etiology of the highly myopic condition has been unclear for decades. We investigated the genetic contributions to early-onset high myopia (EOHM), which is defined as having a refraction of less than or equal to -6 diopters before the age of 6, when children are less likely to be exposed to high educational pressures. Trios (two nonmyopic parents and one child) were examined to uncover pathogenic mutations using whole-exome sequencing. We identified parent-transmitted biallelic mutations or de novo mutations in as-yet-unknown or reported genes in 16 probands. Interestingly, an increased rate of de novo mutations was identified in the EOHM patients. Among the newly identified candidate genes, a BSG mutation was identified in one EOHM proband. Expanded screening of 1,040 patients found an additional four mutations in the same gene. Then, we generated Bsg mutant mice to further elucidate the functional impact of this gene and observed typical myopic phenotypes, including an elongated axial length. Using a trio-based exonic screening study in EOHM, we deciphered a prominent role for de novo mutations in EOHM patients without myopic parents. The discovery of a disease gene, BSG, provides insights into myopic development and its etiology, which expands our current understanding of high myopia and might be useful for future treatment and prevention.
Asunto(s)
Basigina/genética , Exoma , Predisposición Genética a la Enfermedad , Mutación , Miopía/genética , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Miopía/patología , Linaje , Fenotipo , Análisis de Secuencia de ADNRESUMEN
High myopia (HM) is a leading cause of mid-way blindness with a high heritability in East Asia. Although only a few disease genes have been reported, a small proportion of patients could be identified with genetic predispositions. In order to expand the mutation spectrum of the causative genes in Chinese adult population, we investigated three genes, SLC39A5, LEPREL1 and LRPAP1, in a cohort of 187 independent Chinese patients with high myopia. Sanger sequencing was used to find possible pathogenic mutations, which were further screened in normal controls. After a pipeline of database and predictive assessments filtering, we, thereby, identified totally seven heterozygous mutations in the three genes. Among them, three novel missense mutations, c.860C > T, p.Pro287Leu and c.956G > C, p.Arg319Thr in SLC39A5, c.1982A > G, p.Lys661Arg in LEPREL1, were identified as potentially causative mutations. Additionally, the two heterozygous mutations (c.1582G > A, p.Ala528Thr; c.1982A > G, p.Lys661Arg) in one patient in LEPREL1 gene were reported in this study. Our findings will not only augment the mutation spectrum of these three genes, but also provide insights of the contribution of these genes to adult high myopia in Chinese. However, further studies are still needed to address the pathogenicity of each of the mutations reported in this study.
Asunto(s)
Proteínas de Transporte de Catión/genética , Predisposición Genética a la Enfermedad , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/genética , Mutación , Miopía/genética , Procolágeno-Prolina Dioxigenasa/genética , Pueblo Asiatico , China , Pruebas Genéticas , Humanos , Análisis de Secuencia de ADNRESUMEN
Proper neuronal migration is orchestrated by combined membrane signal paradigms, whereas the role and mechanism of regulated intramembrane proteolysis (RIP) remain to be illustrated. We show here that the disintegrin and metalloprotease-domain containing protein 10 (ADAM10) regulates cortical neurons migration by initiating the RIP of Notch. We found that Notch intracellular domain (NICD) significantly rescued the migration defect of ADAM10-deficient neurons. Moreover, ADAM10 deficiency led to reduced neuronal motility and disrupted microtubule (MT) structure, which were associated with downregulated expression of acetylated tubulin and MT-associated proteins. Specifically, the NICD/RBPJ complex bound directly to the promoter, and regulated the neuronal expression level of doublecortin (DCX), a modulator of the MT cytoskeleton. Functionally, DCX overexpression largely restored neuron motility and reversed migration defect caused by ADAM10 knockout. Taken together, these findings demonstrate the direct requirement of ADAM10 in cortical radial migration and reveal the underlying mechanism by linking ADAM10-initiated RIP of Notch to the regulation of MT cytoskeleton through transcriptional control of Dcx expression.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Movimiento Celular/fisiología , Corteza Cerebral/crecimiento & desarrollo , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Neuronas/fisiología , Receptores Notch/metabolismo , Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Células Cultivadas , Corteza Cerebral/metabolismo , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de la Membrana/genética , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Dominios Proteicos , ProteolisisRESUMEN
Purpose: Accumulating evidence has demonstrated that excessive immunoreaction plays a prominent role in the pathogenesis of dry AMD. Toll-like receptor 3 (TLR3) can be activated by double-stranded (ds)RNA in retinal pigment epithelia and trigger an innate immunity-mediated inflammatory response. However, its role in photoreceptor cells, the effectors of AMD geographic atrophy, remains unclear. Methods: The expression of TLR3 was examined in mouse retina and in a murine photoreceptor cell line (661W). Retinal structure, function, and cell death in the polyinosine-polycytidylic acid (poly I:C)-treated retina were investigated by optical coherence tomography, electroretinography (ERG), and immunostaining. Cytokine and chemokine expression as well as cell death were measured in poly I:C-exposed 661W cells and explant retinas. By comparing the RNA sequencing (seq) data of 661W cells and murine retina, we comprehensively investigated the contribution of photoreceptor in poly I:C-induced retinal immune response. Results: Toll-like receptor 3 was highly expressed in the inner segment of the photoreceptor and in 661W cells. We found poly I:C induced significant retinal structural damages and impairment of ERG responses. Focal ERG demonstrated that injected and parainjected zones were functionally damaged by poly I:C. In addition, poly I:C acted on cultured photoreceptor cells directly and evoked an inflammatory response that exhibited similarities with the immune response in mouse retina. Moreover, TLR3 activation initiated cell death in murine photoreceptor cells in vivo and in vitro. Additionally, poly I:C initiated immune response in explant retinas. Conclusions: We deciphered the TLR3-mediated inflammatory response in photoreceptor cells. Our findings suggested TLR3-mediated inflammatory response in photoreceptor cells may play an important role in dry AMD, offering new insights of potential treatments targeting photoreceptor immunity.
Asunto(s)
Muerte Celular/fisiología , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Receptor Toll-Like 3/metabolismo , Análisis de Varianza , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Inductores de Interferón/farmacología , Ratones , Ratones Endogámicos C57BL , Neuroglía/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/inmunología , Poli I-C/farmacología , Retina/efectos de los fármacos , Retina/fisiopatología , Análisis de Secuencia de ARN , Tomografía de Coherencia ÓpticaRESUMEN
To date, at least 18 causative genes have been identified in amyotrophic lateral sclerosis (ALS). Because of the clinical and genetic heterogeneity, molecular diagnosis for ALS faces great challenges. HaloPlex target enrichment system is a new targeted sequencing approach, which can detect already known mutations or candidate genes. We performed this approach to screen 18 causative genes of ALS, including SOD1, SETX, FUS, ANG, TARDBP, ALS2, FIG4, VAPB, OPTN, DAO, VCP, UBQLN2, SPG11, SIGMAR1, DCTN1, SQSTM1, PFN1, and CHMP2B in 8 ALS probands. Using this approach, we got an average of 9.5 synonymous or missense mutations per sample. After validation by Sanger sequencing, we identified 3 documented SOD1 mutations (p.F21C, p.G148D, and p.C147R) and 1 novel DCTN1 p.G59R mutation in 4 probands. The novel DCTN1 mutation appeared to segregate with the disease in the pedigree and was absent in 200 control subjects. The high throughput and efficiency of this approach indicated that it could be applied to diagnose ALS and other inherited diseases with multiple causative genes in clinical practice.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Estudios de Asociación Genética/métodos , Predisposición Genética a la Enfermedad/genética , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas Asociadas a Microtúbulos/genética , Mutación Missense , Superóxido Dismutasa/genética , Adulto , Complejo Dinactina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Superóxido Dismutasa-1 , Adulto JovenRESUMEN
Serum inducible kinase (SNK), also known as polo-like kinase 2 (PLK2), is a known regulator of mitosis, synaptogenesis and synaptic homeostasis. However, its role in early cortical development is unknown. Herein, we show that snk is expressed in the cortical plate from embryonic day 14, but not in the ventricular/subventricular zones (VZ/SVZ), and SNK protein localizes to the soma and dendrites of cultured immature cortical neurons. Loss of SNK impaired dendritic but not axonal arborization in a dose-dependent manner and overexpression had opposite effects, both in vitro and in vivo. Overexpression of SNK also caused abnormal branching of the leading process of migrating cortical neurons in electroporated cortices. The kinase activity was necessary for these effects. Extracellular signal-regulated kinase (ERK) pathway activity downstream of brain-derived neurotrophic factor (BDNF) stimulation led to increases in SNK protein expression via transcriptional regulation, and this upregulation was necessary for the growth-promoting effect of BDNF on dendritic arborization. Taken together, our results indicate that SNK is essential for dendrite morphogenesis in cortical neurons.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Dendritas/fisiología , Proteínas Quinasas/metabolismo , Animales , Células Cultivadas , Dendritas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Transducción de Señal , Transcripción GenéticaRESUMEN
PURPOSE: The ketogenic diet (KD) has been used as an effective antiepileptic treatment for nearly a century. Inhibition of glycolysis and increased levels of ketone bodies are both known to contribute to the antiepileptic effects of the KD. Neuron-restrictive silencer factor (NRSF), also known as RE-1 silencing transcription factor (REST), is implicated in the antiepileptic effects of the glycolytic inhibitor 2-deoxy-d-glucose (2DG). Glycolytic inhibition is a common feature of the KD and 2DG treatment, leading to the hypothesis that NRSF might also be involved in the antiepileptic effect of the KD. To test this hypothesis, the present study was designed to investigate the role of NRSF in the antiepileptic effect of 2DG, the KD, and acetone in vivo. METHODS: Kindling was used as a model to test the antiepileptic effects of 2DG, the KD, and acetone on control and NRSF conditional knockout mice (NRSF-cKO; from the intercross of CamKIIα-iCre and NRSF exon 2 floxed mice). After recovery from electrode implantation, adult mice were stimulated twice a day at afterdischarge threshold (ADT) current intensity. In the 2DG- (500 mg/kg) and acetone- (10 mmol/kg) treated groups, drugs were injected intraperitoneally 20 min before each stimulus. In the 2DG group, mice were pretreated with intraperitoneal injections for 3 days in addition to the injections administered before the regular kindling stimulation. In the KD group, mice were fed the KD instead of a control diet until the end of stimulations. KEY FINDINGS: Compared with control mice, the antiepileptic effect of 2DG was abolished in NRSF-cKO mice, indicating that NRSF is required for the antiepileptic effect of 2DG. In the KD-fed group, kindling development was retarded in both control and NRSF-cKO mice. In the acetone-treated group, inhibition of kindling-induced epileptogenesis was observed in both control and NRSF-cKO mice, similar to the action of the KD. SIGNIFICANCE: These findings imply that NRSF repression complex is not essential for the antiepileptic effect of the ketogenic diet.
Asunto(s)
Anticonvulsivantes/administración & dosificación , Dieta Cetogénica/métodos , Epilepsia/terapia , Excitación Neurológica/efectos de los fármacos , Proteínas Represoras/metabolismo , Acetona/uso terapéutico , Animales , Antimetabolitos/administración & dosificación , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Desoxiglucosa/administración & dosificación , Estimulación Eléctrica/efectos adversos , Epilepsia/etiología , Epilepsia/genética , Excitación Neurológica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Represoras/deficienciaRESUMEN
Neuron-restrictive silencer factor (NRSF), also known as repressor element-1 silencing transcription factor, is a transcriptional repressor that plays important roles in embryonic development and neurogenesis. Recent findings show that NRSF is upregulated after seizures activity however, the link between NRSF and epileptogenesis remains poorly understood. To investigate the role of NRSF in epilepsy, we employed a Cre-loxp system to specifically delete NRSF in excitatory neurons of the postnatal mouse forebrain. In the kindling model of epileptogenesis, conditional NRSF knockout (NRSF-cKO) mice exhibited dramatically accelerated seizure progression and prolonged afterdischarge duration compared with control mice. Moreover, seizures activity-induced mossy fiber sprouting was enhanced in the NRSF-cKO mice. The degree of upregulation of Fibroblast growth factor 14 and Brain-derived neurotrophic factor (BDNF) following kainic acid-induced status epilepticus was significantly increased in the cortex of NRSF-cKO mice compared with control mice. Furthermore, the derepression of BDNF was associated by activation of PLCγ and PI(3)K signaling pathways. These findings indicate that NRSF functions as an intrinsic repressor of limbic epileptogenesis.
Asunto(s)
Epilepsia/fisiopatología , Excitación Neurológica/fisiología , Neuronas/fisiología , Prosencéfalo/citología , Prosencéfalo/fisiología , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Animales , Conducta Animal/fisiología , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Fenómenos Electrofisiológicos , Activación Enzimática/fisiología , Factores de Crecimiento de Fibroblastos/genética , Eliminación de Gen , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Fibras Musgosas del Hipocampo/fisiología , Proteína Oncogénica v-akt/fisiología , Fosfatidilinositol 3-Quinasas/genética , Fosfolipasa C gamma/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Transducción de Señal/fisiología , Estado Epiléptico/genética , Estado Epiléptico/fisiopatologíaRESUMEN
Research over the past decades has demonstrated that adult brain produces neural progenitor cells which proliferate and differentiate to newborn neurons that integrate into the existing circuit. However, detailed differentiation processes and underlying mechanisms of newly generated neurons are largely unknown due to the limitation of available methods for labeling and manipulating neural progenitor cells and newborn neurons. In this study, we designed a tightly controlled, noninvasive system based on Cre/loxP recombination to achieve long-term tracing and genetic manipulation of adult neurons in vivo. In this system, tamoxifen-inducible recombinase, CreER(T2), was driven by BAC-based promoter of doublecortin (DCX, a marker of newborn neurons). By crossing this Cre line with reporter mouse, we found that newborn neurons in the dentate gyrus (DG) could be selectively pulse-labeled by tamoxifen-induced expression of yellow fluorescent protein (YFP). YFP-positive neurons were identified by coimmunostaining with cell type-specific markers and characterized by electrophysiological recording. Furthermore, analysis of the migration of these neurons showed that the majority of these labeled neurons migrated to the inner part of granule cell layer. Moreover, spine growth of inner molecular layer of newborn granule neurons takes a dynamic pattern of invert U-shape, in contrast to the wedge-shaped change in the outer molecular layer. Our transgenic tool provides an efficient way to selectively label and manipulate newborn neuron in adult mouse DG.