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1.
EMBO J ; 39(17): e105696, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32716134

RESUMO

Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Núcleo Celular/genética , Retículo Endoplasmático/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/genética , Camundongos , Oryzias
2.
Genet Med ; 26(3): 101034, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38054405

RESUMO

PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.


Assuntos
Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Animais , Humanos , Camundongos , Bicarbonatos/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Deficiência Intelectual/genética , Proteínas de Membrana Transportadoras , Camundongos Knockout , Transtornos do Neurodesenvolvimento/genética , Sódio/metabolismo , Bicarbonato de Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/genética
3.
Brain ; 146(11): 4547-4561, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37459438

RESUMO

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.


Assuntos
Convulsões , Simportadores de Sódio-Bicarbonato , Criança , Camundongos , Humanos , Animais , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Convulsões/genética , Mutação/genética , Neurotransmissores , Ácido gama-Aminobutírico/genética , Mamíferos/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo
4.
Am J Hum Genet ; 107(2): 364-373, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32707086

RESUMO

We report bi-allelic pathogenic HPDL variants as a cause of a progressive, pediatric-onset spastic movement disorder with variable clinical presentation. The single-exon gene HPDL encodes a protein of unknown function with sequence similarity to 4-hydroxyphenylpyruvate dioxygenase. Exome sequencing studies in 13 families revealed bi-allelic HPDL variants in each of the 17 individuals affected with this clinically heterogeneous autosomal-recessive neurological disorder. HPDL levels were significantly reduced in fibroblast cell lines derived from more severely affected individuals, indicating the identified HPDL variants resulted in the loss of HPDL protein. Clinical presentation ranged from severe, neonatal-onset neurodevelopmental delay with neuroimaging findings resembling mitochondrial encephalopathy to milder manifestation of adolescent-onset, isolated hereditary spastic paraplegia. All affected individuals developed spasticity predominantly of the lower limbs over the course of the disease. We demonstrated through bioinformatic and cellular studies that HPDL has a mitochondrial localization signal and consequently localizes to mitochondria suggesting a putative role in mitochondrial metabolism. Taken together, these genetic, bioinformatic, and functional studies demonstrate HPDL is a mitochondrial protein, the loss of which causes a clinically variable form of pediatric-onset spastic movement disorder.


Assuntos
Encefalopatias/genética , Proteínas Mitocondriais/genética , Doenças Neurodegenerativas/genética , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto , Alelos , Sequência de Aminoácidos , Criança , Feminino , Humanos , Masculino , Mitocôndrias/genética , Linhagem , Fenótipo , Adulto Jovem
5.
EMBO Rep ; 22(9): e52289, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34338405

RESUMO

Degradation of the endoplasmic reticulum (ER) via selective autophagy (ER-phagy) is vital for cellular homeostasis. We identify FAM134A/RETREG2 and FAM134C/RETREG3 as ER-phagy receptors, which predominantly exist in an inactive state under basal conditions. Upon autophagy induction and ER stress signal, they can induce significant ER fragmentation and subsequent lysosomal degradation. FAM134A, FAM134B/RETREG1, and FAM134C are essential for maintaining ER morphology in a LC3-interacting region (LIR)-dependent manner. Overexpression of any FAM134 paralogue has the capacity to significantly augment the general ER-phagy flux upon starvation or ER-stress. Global proteomic analysis of FAM134 overexpressing and knockout cell lines reveals several protein clusters that are distinctly regulated by each of the FAM134 paralogues as well as a cluster of commonly regulated ER-resident proteins. Utilizing pro-Collagen I, as a shared ER-phagy substrate, we observe that FAM134A acts in a LIR-independent manner and compensates for the loss of FAM134B and FAM134C, respectively. FAM134C instead is unable to compensate for the loss of its paralogues. Taken together, our data show that FAM134 paralogues contribute to common and unique ER-phagy pathways.


Assuntos
Proteínas de Membrana , Proteômica , Autofagia/genética , Colágeno , Retículo Endoplasmático/genética , Proteínas de Membrana/genética , Controle de Qualidade
6.
Nature ; 522(7556): 354-8, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26040720

RESUMO

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.


Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Biomarcadores/metabolismo , Linhagem Celular , Retículo Endoplasmático/química , Feminino , Deleção de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Fagossomos/metabolismo , Ligação Proteica , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia
7.
PLoS Genet ; 11(8): e1005454, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26284655

RESUMO

Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.


Assuntos
Autofagia , Lisossomos/fisiologia , Proteínas/genética , Paraplegia Espástica Hereditária/patologia , Animais , Células Cultivadas , Cerebelo/patologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Córtex Motor/patologia , Células de Purkinje/patologia , Paraplegia Espástica Hereditária/genética
8.
J Am Soc Nephrol ; 28(1): 209-217, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27335120

RESUMO

Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.


Assuntos
Proteínas de Transporte de Ânions/fisiologia , Canais de Cloreto/fisiologia , Néfrons/metabolismo , Cloreto de Sódio/metabolismo , Animais , Diuréticos/farmacologia , Furosemida/farmacologia , Camundongos , Camundongos Knockout , Néfrons/efeitos dos fármacos , Inibidores de Simportadores de Cloreto de Sódio/farmacologia
9.
J Am Soc Nephrol ; 28(5): 1507-1520, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27932475

RESUMO

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3- cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.


Assuntos
Acidose Tubular Renal/enzimologia , Proteína 1 de Troca de Ânion do Eritrócito/fisiologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/enzimologia , ATPases Vacuolares Próton-Translocadoras/fisiologia , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Masculino , Camundongos , Modelos Biológicos
10.
EMBO J ; 32(16): 2275-86, 2013 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-23881097

RESUMO

Brain carbonic anhydrases (CAs) are known to modulate neuronal signalling. Using a novel CA VII (Car7) knockout (KO) mouse as well as a CA II (Car2) KO and a CA II/VII double KO, we show that mature hippocampal pyramidal neurons are endowed with two cytosolic isoforms. CA VII is predominantly expressed by neurons starting around postnatal day 10 (P10). The ubiquitous isoform II is expressed in neurons at P20. Both isoforms enhance bicarbonate-driven GABAergic excitation during intense GABAA-receptor activation. P13-14 CA VII KO mice show behavioural manifestations atypical of experimental febrile seizures (eFS) and a complete absence of electrographic seizures. A low dose of diazepam promotes eFS in P13-P14 rat pups, whereas seizures are blocked at higher concentrations that suppress breathing. Thus, the respiratory alkalosis-dependent eFS are exacerbated by GABAergic excitation. We found that CA VII mRNA is expressed in the human cerebral cortex before the age when febrile seizures (FS) occur in children. Our data indicate that CA VII is a key molecule in age-dependent neuronal pH regulation with consequent effects on generation of FS.


Assuntos
Anidrase Carbônica II/metabolismo , Anidrases Carbônicas/metabolismo , Córtex Cerebral/citologia , Neurônios GABAérgicos/metabolismo , Convulsões Febris/enzimologia , Fatores Etários , Análise de Variância , Animais , Northern Blotting , Western Blotting , Anidrase Carbônica II/genética , Anidrases Carbônicas/genética , Córtex Cerebral/metabolismo , Diazepam/toxicidade , Eletroencefalografia , Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Knockout , Ratos , Convulsões Febris/induzido quimicamente , Convulsões Febris/metabolismo
11.
Am J Hum Genet ; 93(4): 727-34, 2013 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-24035193

RESUMO

In guanosine diphosphate (GDP)-mannose pyrophosphorylase A (GMPPA), we identified a homozygous nonsense mutation that segregated with achalasia and alacrima, delayed developmental milestones, and gait abnormalities in a consanguineous Pakistani pedigree. Mutations in GMPPA were subsequently found in ten additional individuals from eight independent families affected by the combination of achalasia, alacrima, and neurological deficits. This autosomal-recessive disorder shows many similarities with triple A syndrome, which is characterized by achalasia, alacrima, and variable neurological deficits in combination with adrenal insufficiency. GMPPA is a largely uncharacterized homolog of GMPPB. GMPPB catalyzes the formation of GDP-mannose, which is an essential precursor of glycan moieties of glycoproteins and glycolipids and is associated with congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-dystroglycan. Surprisingly, GDP-mannose pyrophosphorylase activity was unchanged and GDP-mannose levels were strongly increased in lymphoblasts of individuals with GMPPA mutations. This suggests that GMPPA might serve as a GMPPB regulatory subunit mediating feedback inhibition of GMPPB instead of displaying catalytic enzyme activity itself. Thus, a triple-A-like syndrome can be added to the growing list of congenital disorders of glycosylation, in which dysregulation rather than mere enzyme deficiency is the basal pathophysiological mechanism.


Assuntos
Códon sem Sentido , Genes Recessivos/genética , Guanosina Difosfato Manose/genética , Deficiência Intelectual/genética , Nucleotidiltransferases/genética , Adolescente , Insuficiência Adrenal/genética , Adulto , Criança , Consanguinidade , Acalasia Esofágica/genética , Oftalmopatias Hereditárias/genética , Glicosilação , Guanosina Difosfato Manose/metabolismo , Homozigoto , Humanos , Deficiência Intelectual/enzimologia , Doenças do Aparelho Lacrimal/genética , Doenças do Sistema Nervoso/genética , Nucleotidiltransferases/metabolismo , Linhagem , Adulto Jovem
12.
PLoS Genet ; 9(12): e1003988, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24367272

RESUMO

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.


Assuntos
Proteínas de Transporte/genética , Endossomos/metabolismo , Lisossomos/metabolismo , Degeneração Retiniana/genética , Paraplegia Espástica Hereditária/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Transporte/metabolismo , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Modelos Animais de Doenças , Endossomos/patologia , Humanos , Lisossomos/genética , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Mutação , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Paraplegia Espástica Hereditária/metabolismo , Paraplegia Espástica Hereditária/patologia
13.
Am J Hum Genet ; 88(5): 621-7, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21549336

RESUMO

The fact that hereditary hearing loss is the most common sensory disorder in humans is reflected by, among other things, an extraordinary allelic and nonallelic genetic heterogeneity. X-chromosomal hearing impairment represents only a minor fraction of all cases. In a study of a Spanish family the locus for one of the X-chromosomal forms was assigned to Xp22 (DFNX4). We mapped the disease locus in the same chromosomal region in a large German pedigree with X-chromosomal nonsyndromic hearing impairment by using genome-wide linkage analysis. Males presented with postlingual hearing loss and onset at ages 3-7, whereas onset in female carriers was in the second to third decades. Targeted DNA capture with high-throughput sequencing detected a nonsense mutation in the small muscle protein, X-linked (SMPX) of affected individuals. We identified another nonsense mutation in SMPX in patients from the Spanish family who were previously analyzed to map DFNX4. SMPX encodes an 88 amino acid, cytoskeleton-associated protein that is responsive to mechanical stress. The presence of Smpx in hair cells and supporting cells of the murine cochlea indicates its role in the inner ear. The nonsense mutations detected in the two families suggest a loss-of-function mechanism underlying this form of hearing impairment. Results obtained after heterologous overexpression of SMPX proteins were compatible with this assumption. Because responsivity to physical force is a characteristic feature of the protein, we propose that long-term maintenance of mechanically stressed inner-ear cells critically depends on SMPX function.


Assuntos
Cromossomos Humanos X/genética , Códon sem Sentido , Perda Auditiva/genética , Proteínas Musculares/genética , Adolescente , Idade de Início , Alelos , Animais , Criança , Pré-Escolar , Cóclea , Orelha Interna/embriologia , Orelha Interna/metabolismo , Feminino , Ligação Genética , Estudo de Associação Genômica Ampla , Células Ciliadas Auditivas/metabolismo , Haplótipos , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linhagem
14.
J Assoc Res Otolaryngol ; 20(3): 233-245, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31001720

RESUMO

The unique composition of the endolymph with a high extracellular K+ concentration is essential for sensory transduction in the inner ear. It is secreted by a specialized epithelium, the stria vascularis, that is connected to the fibrocyte meshwork of the spiral ligament in the lateral wall of the cochlea via gap junctions. In this study, we show that in mice the expression of the bicarbonate transporter Slc4a10/Ncbe/Nbcn2 in spiral ligament fibrocytes starts shortly before hearing onset. Its disruption in a C57BL/6 background results in early onset progressive hearing loss. This hearing loss is characterized by a reduced endocochlear potential from hearing onset onward and progressive degeneration of outer hair cells. Notably, the expression of a related bicarbonate transporter, i.e., Slc4a7/Nbcn1, is also lost in spiral ligament fibrocytes of Slc4a10 knockout mice. The histological analysis of the spiral ligament of Slc4a10 knockout mice does not reveal overt fibrocyte loss as reported for Slc4a7 knockout mice. The ultrastructural analysis, however, shows mitochondrial alterations in fibrocytes of Slc4a10 knockout mice. Our data suggest that Slc4a10 and Slc4a7 are functionally related and essential for inner ear homeostasis.


Assuntos
Antiportadores de Cloreto-Bicarbonato/fisiologia , Audição/fisiologia , Simportadores de Sódio-Bicarbonato/metabolismo , Simportadores de Sódio-Bicarbonato/fisiologia , Ligamento Espiral da Cóclea/metabolismo , Animais , Conexina 26 , Conexina 30/metabolismo , Conexinas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura
15.
Arch Biochem Biophys ; 473(2): 210-7, 2008 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18307972

RESUMO

The Calca gene encodes two polypeptides, calcitonin (CT) and alpha-calcitonin gene-related peptide (alpha-CGRP), generated through alternative splicing. While CT, a hormone mainly produced by thyroidal C cells, has been described as a major regulator of bone resorption, alpha-CGRP, a neuropeptide expressed in the cells of the central and peripheral nervous system, is mostly known as a regulator of vascular tone. Surprisingly, the generation and skeletal analyses of two mouse deficiency models has recently uncovered a physiological function for both peptides in the regulation of bone formation. In the first model, where the replacement of exons 2-5 of the Calca gene resulted in the combined deficiency of CT and alpha-CGRP, an increased bone formation rate (BFR) was observed, whereas decreased BFR was found in the second model, where the introduction of a translational termination codon into exon 5 of the Calca gene resulted in the specific absence of alpha-CGRP.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Calcitonina/fisiologia , Osteogênese/fisiologia , Processamento Alternativo , Animais , Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/genética , Camundongos , Camundongos Knockout , Mutação
16.
J Bone Miner Res ; 21(12): 1924-34, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17002587

RESUMO

UNLABELLED: Although the pharmacological action of calcitonin (CT) as an inhibitor of bone resorption is well established, there is still some controversy regarding its physiological function. Unexpectedly, Calca-deficient mice lacking CT and alpha-calcitonin gene-related peptide (alphaCGRP) were described to have a high bone mass phenotype caused by increased bone formation with normal bone resorption. Here we show that these mice develop a phenotype of high bone turnover with age, suggesting that CT is a physiological inhibitor of bone remodeling. INTRODUCTION: The absence of significant changes in bone mineral density caused by decline or overproduction of CT in humans has raised the question, whether the pharmacological action of CT as an inhibitor of bone resorption is also of physiological relevance. To study the physiological role of mammalian CT, we have analyzed the age-dependent bone phenotype of two mouse models, one lacking CT and alphaCGRP (Calca-/-), the other one lacking only alphaCGRP (alphaCGRP-/-). MATERIALS AND METHODS: Bones from wildtype, Calca-/- -mice and alphaCGRP-/- -mice were analyzed at the ages of 6, 12 and 18 months using undecalcified histology. Differences of bone remodeling were quantified by static and dynamic histomorphometry as well as by measuring the urinary collagen degradation products. To rule out secondary mechanisms underlying the observed phenotype, we determined serum concentrations of relevant hormones using commercially available antibody-based detection kits. RESULTS: Whereas alphaCGRP-/- -mice display an osteopenia at all ages analyzed, the Calca-/- -mice develop a phenotype of high bone turnover with age. Histomorphometric analysis performed at the age of 12 months revealed significant increases of bone formation and bone resorption specifically in the Calca-/- -mice. This severe phenotype that can result in hyperostotic lesions, can not be explained by obvious endocrine abnormalities other than the absence of CT. CONCLUSIONS: In addition to the previously described increase of bone formation in the Calca-deficient mice, we have observed that there is also an increase of bone resorption with age. This suggests that CT has a dual action as an inhibitor of bone remodeling, which may explain why alterations of CT serum levels in humans do not result in major changes of bone mineral density.


Assuntos
Densidade Óssea , Remodelação Óssea , Peptídeo Relacionado com Gene de Calcitonina/sangue , Calcitonina/sangue , Envelhecimento/sangue , Envelhecimento/genética , Animais , Densidade Óssea/genética , Doenças Ósseas Metabólicas/sangue , Doenças Ósseas Metabólicas/genética , Remodelação Óssea/genética , Calcitonina/deficiência , Peptídeo Relacionado com Gene de Calcitonina/deficiência , Humanos , Camundongos , Camundongos Knockout
17.
J Clin Invest ; 124(2): 675-86, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24401273

RESUMO

High blood pressure is the leading risk factor for death worldwide. One of the hallmarks is a rise of peripheral vascular resistance, which largely depends on arteriole tone. Ca2+-activated chloride currents (CaCCs) in vascular smooth muscle cells (VSMCs) are candidates for increasing vascular contractility. We analyzed the vascular tree and identified substantial CaCCs in VSMCs of the aorta and carotid arteries. CaCCs were small or absent in VSMCs of medium-sized vessels such as mesenteric arteries and larger retinal arterioles. In small vessels of the retina, brain, and skeletal muscle, where contractile intermediate cells or pericytes gradually replace VSMCs, CaCCs were particularly large. Targeted disruption of the calcium-activated chloride channel TMEM16A, also known as ANO1, in VSMCs, intermediate cells, and pericytes eliminated CaCCs in all vessels studied. Mice lacking vascular TMEM16A had lower systemic blood pressure and a decreased hypertensive response following vasoconstrictor treatment. There was no difference in contractility of medium-sized mesenteric arteries; however, responsiveness of the aorta and small retinal arterioles to the vasoconstriction-inducing drug U46619 was reduced. TMEM16A also was required for peripheral blood vessel contractility, as the response to U46619 was attenuated in isolated perfused hind limbs from mutant mice. Out data suggest that TMEM16A plays a general role in arteriolar and capillary blood flow and is a promising target for the treatment of hypertension.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Canais de Cloreto/metabolismo , Hipertensão/fisiopatologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Anoctamina-1 , Arteríolas/patologia , Pressão Sanguínea/fisiologia , Encéfalo/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Eletrofisiologia , Antagonistas de Estrogênios/farmacologia , Células HEK293 , Humanos , Hipertensão/tratamento farmacológico , Potenciais da Membrana/efeitos dos fármacos , Artérias Mesentéricas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/citologia , Proteínas de Neoplasias/metabolismo , Pericitos/metabolismo , Retina/metabolismo , Tamoxifeno/farmacologia , Fatores de Tempo , Resistência Vascular , Vasoconstritores/farmacologia
18.
Nat Commun ; 5: 5215, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25333900

RESUMO

The hormone calcitonin (CT) is primarily known for its pharmacologic action as an inhibitor of bone resorption, yet CT-deficient mice display increased bone formation. These findings raised the question about the underlying cellular and molecular mechanism of CT action. Here we show that either ubiquitous or osteoclast-specific inactivation of the murine CT receptor (CTR) causes increased bone formation. CT negatively regulates the osteoclast expression of Spns2 gene, which encodes a transporter for the signalling lipid sphingosine 1-phosphate (S1P). CTR-deficient mice show increased S1P levels, and their skeletal phenotype is normalized by deletion of the S1P receptor S1P3. Finally, pharmacologic treatment with the nonselective S1P receptor agonist FTY720 causes increased bone formation in wild-type, but not in S1P3-deficient mice. This study redefines the role of CT in skeletal biology, confirms that S1P acts as an osteoanabolic molecule in vivo and provides evidence for a pharmacologically exploitable crosstalk between osteoclasts and osteoblasts.


Assuntos
Calcitonina/metabolismo , Lisofosfolipídeos/metabolismo , Osteoclastos/citologia , Osteogênese , Esfingosina/análogos & derivados , Alelos , Animais , Osso e Ossos/metabolismo , Colagenases/metabolismo , Cruzamentos Genéticos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/citologia , Osteoporose/fisiopatologia , Fenótipo , Porosidade , Receptores da Calcitonina/metabolismo , Transdução de Sinais , Esfingosina/metabolismo
19.
PLoS One ; 7(10): e46155, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23056253

RESUMO

Regulation of ion and pH homeostasis is essential for normal neuronal function. The sodium-driven chloride bicarbonate exchanger NCBE (Slc4a10), a member of the SLC4 family of bicarbonate transporters, uses the transmembrane gradient of sodium to drive cellular net uptake of bicarbonate and to extrude chloride, thereby modulating both intracellular pH (pH(i)) and chloride concentration ([Cl(-)](i)) in neurons. Here we show that NCBE is strongly expressed in the retina. As GABA(A) receptors conduct both chloride and bicarbonate, we hypothesized that NCBE may be relevant for GABAergic transmission in the retina. Importantly, we found a differential expression of NCBE in bipolar cells: whereas NCBE was expressed on ON and OFF bipolar cell axon terminals, it only localized to dendrites of OFF bipolar cells. On these compartments, NCBE colocalized with the main neuronal chloride extruder KCC2, which renders GABA hyperpolarizing. NCBE was also expressed in starburst amacrine cells, but was absent from neurons known to depolarize in response to GABA, like horizontal cells. Mice lacking NCBE showed decreased visual acuity and contrast sensitivity in behavioral experiments and smaller b-wave amplitudes and longer latencies in electroretinograms. Ganglion cells from NCBE-deficient mice also showed altered temporal response properties. In summary, our data suggest that NCBE may serve to maintain intracellular chloride and bicarbonate concentration in retinal neurons. Consequently, lack of NCBE in the retina may result in changes in pH(i) regulation and chloride-dependent inhibition, leading to altered signal transmission and impaired visual function.


Assuntos
Antiportadores de Cloreto-Bicarbonato/fisiologia , Retina/fisiologia , Simportadores de Sódio-Bicarbonato/fisiologia , Acuidade Visual/fisiologia , Células Amácrinas/metabolismo , Animais , Antiportadores de Cloreto-Bicarbonato/deficiência , Antiportadores de Cloreto-Bicarbonato/genética , Sensibilidades de Contraste/genética , Sensibilidades de Contraste/fisiologia , Eletrorretinografia , Gânglios/citologia , Gânglios/metabolismo , Gânglios/fisiologia , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microscopia Confocal , Estimulação Luminosa , Retina/citologia , Retina/metabolismo , Células Bipolares da Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/fisiologia , Simportadores de Sódio-Bicarbonato/deficiência , Simportadores de Sódio-Bicarbonato/genética , Simportadores/metabolismo , Acuidade Visual/genética , Cotransportadores de K e Cl-
20.
EMBO Mol Med ; 4(10): 1057-71, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22933323

RESUMO

The V-ATPase is a multisubunit complex that transports protons across membranes. Mutations of its B1 or a4 subunit are associated with distal renal tubular acidosis and deafness. In the kidney, the a4 subunit is expressed in intercalated cells of the distal nephron, where the V-ATPase controls acid/base secretion, and in proximal tubule cells, where its role is less clear. Here, we report that a4 KO mice suffer not only from severe acidosis but also from proximal tubule dysfunction with defective endocytic trafficking, proteinuria, phosphaturia and accumulation of lysosomal material and we provide evidence that these findings may be also relevant in patients. In the inner ear, the a4 subunit co-localized with pendrin at the apical side of epithelial cells lining the endolymphatic sac. As a4 KO mice were profoundly deaf and displayed enlarged endolymphatic fluid compartments mirroring the alterations in pendrin KO mice, we propose that pendrin and the proton pump co-operate in endolymph homeostasis. Thus, our mouse model gives new insights into the divergent functions of the V-ATPase and the pathophysiology of a4-related symptoms.


Assuntos
Acidose Tubular Renal/fisiopatologia , Túbulos Renais Proximais/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Humanos , Lactente , Camundongos , Camundongos Knockout , Subunidades Proteicas/deficiência , Subunidades Proteicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/deficiência
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