RESUMEN
Megalin (low-density lipoprotein receptor-related protein 2) is a giant glycoprotein of about 600 kDa, mediating the endocytosis of more than 60 ligands, including those of proteins, peptides, and drug compounds [S. Goto, M. Hosojima, H. Kabasawa, A. Saito, Int. J. Biochem. Cell Biol. 157, 106393 (2023)]. It is expressed predominantly in renal proximal tubule epithelial cells, as well as in the brain, lungs, eyes, inner ear, thyroid gland, and placenta. Megalin is also known to mediate the endocytosis of toxic compounds, particularly those that cause renal and hearing disorders [Y. Hori et al., J. Am. Soc. Nephrol. 28, 1783-1791 (2017)]. Genetic megalin deficiency causes Donnai-Barrow syndrome/facio-oculo-acoustico-renal syndrome in humans. However, it is not known how megalin interacts with such a wide variety of ligands and plays pathological roles in various organs. In this study, we elucidated the dimeric architecture of megalin, purified from rat kidneys, using cryoelectron microscopy. The maps revealed the densities of endogenous ligands bound to various regions throughout the dimer, elucidating the multiligand receptor nature of megalin. We also determined the structure of megalin in complex with receptor-associated protein, a molecular chaperone for megalin. The results will facilitate further studies on the pathophysiology of megalin-dependent multiligand endocytic pathways in multiple organs and will also be useful for the development of megalin-targeted drugs for renal and hearing disorders, Alzheimer's disease [B. V. Zlokovic et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4229-4234 (1996)], and other illnesses.
Asunto(s)
Microscopía por Crioelectrón , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Animales , Humanos , Ratas , Ligandos , Endocitosis , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/genética , Defectos Congénitos del Transporte Tubular Renal , Miopía , Hernias Diafragmáticas Congénitas , Proteinuria , Pérdida Auditiva SensorineuralRESUMEN
Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome.
Asunto(s)
Agenesia del Cuerpo Calloso/genética , Autofagosomas/metabolismo , Catarata/genética , Endosomas/metabolismo , Lisosomas/metabolismo , Proteínas/genética , Proteínas de Unión al GTP rab/genética , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Secuencia de Aminoácidos , Animales , Autofagosomas/ultraestructura , Autofagia/genética , Proteínas Relacionadas con la Autofagia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Catarata/metabolismo , Catarata/patología , Endosomas/ultraestructura , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas de Membrana de los Lisosomas , Lisosomas/ultraestructura , Fusión de Membrana , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Proteínas/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
The multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (Dab2) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.NEW & NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Túbulos Renales Proximales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Receptores de Superficie Celular/metabolismo , Transcripción Genética , Proteínas Adaptadoras Transductoras de Señales/genética , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Células Cultivadas , Bases de Datos Genéticas , Redes Reguladoras de Genes , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/patología , Hernias Diafragmáticas Congénitas/genética , Hernias Diafragmáticas Congénitas/metabolismo , Hernias Diafragmáticas Congénitas/patología , Humanos , Túbulos Renales Proximales/patología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Ratones Noqueados , Monodelphis , Miopía/genética , Miopía/metabolismo , Miopía/patología , Proteinuria/genética , Proteinuria/metabolismo , Proteinuria/patología , Receptores de Superficie Celular/genética , Defectos Congénitos del Transporte Tubular Renal/genética , Defectos Congénitos del Transporte Tubular Renal/metabolismo , Defectos Congénitos del Transporte Tubular Renal/patologíaRESUMEN
Hereditary motor sensory neuropathy (HMSN/ACC) with agenesis of the corpus callosum (ACC) has been documented in the French-derived populations of Charlevoix and Saguenay/Lac St. Jean in Quebec, Canada, as well as a few sporadic families throughout the world. HMSN/ACC occurs because of loss-of-function mutations in the potassium-chloride cotransporter 3 (KCC3). In HMSN/ACC, motor deficits occur early in infancy with rapid and continual deterioration of motor and sensory fibers into juvenile and adulthood. Genetic work in mice has demonstrated that the disease is caused by loss of KCC3 function in neurons and particularly parvalbumin (PV)-expressing neurons. Currently, there are no treatments or cures for HMSN/ACC other than pain management. As genetic counseling in Quebec has increased as a preventative strategy, most individuals with HSMN/ACC are now adults. The onset of the disease is unknown. In particular, it is unknown if the disease starts early during development and whether it can be reversed by restoring KCC3 function. In this study, we used two separate mouse models that when combined to the PV-CreERT2 tamoxifen-inducible system allowed us to 1) disrupt KCC3 expression in adulthood or juvenile periods; and 2) reintroduce KCC3 expression in mice that first develop with a nonfunctional cotransporter. We show that disrupting or reintroducing KCC3 in the adult mouse has no effect on locomotor behavior, indicating that expression of KCC3 is critical during embryonic development and/or the perinatal period and that once the disease has started, reexpressing a functional cotransporter fails to change the course of HMSN/ACC.
Asunto(s)
Agenesia del Cuerpo Calloso/terapia , Conducta Animal , Ganglios Espinales/metabolismo , Terapia Genética , Actividad Motora , Enfermedades del Sistema Nervioso Periférico/terapia , Simportadores/metabolismo , Factores de Edad , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/fisiopatología , Animales , Modelos Animales de Enfermedad , Femenino , Ganglios Espinales/fisiopatología , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Parvalbúminas/metabolismo , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Fenotipo , Equilibrio Postural , Prueba de Desempeño de Rotación con Aceleración Constante , Simportadores/genéticaRESUMEN
Mitochondrial intracrines are extracellular signaling proteins, targeted to the mitochondria. The pathway for mitochondrial targeting of mitochondrial intracrines and actions in the mitochondria remains unknown. Megalin/LRP2 mediates the uptake of vitamins and proteins, and is critical for clearance of amyloid-ß protein from the brain. Megalin mutations underlie the pathogenesis of Donnai-Barrow and Lowe syndromes, characterized by brain defects and kidney dysfunction; megalin was not previously known to reside in the mitochondria. Here, we show megalin is present in the mitochondria and associates with mitochondrial anti-oxidant proteins SIRT3 and stanniocalcin-1 (STC1). Megalin shuttles extracellularly-applied STC1, angiotensin II and TGF-ß to the mitochondria through the retrograde early endosome-to-Golgi transport pathway and Rab32. Megalin knockout in cultured cells impairs glycolytic and respiratory capacities. Thus, megalin is critical for mitochondrial biology; mitochondrial intracrine signaling is a continuum of the retrograde early endosome-to-Golgi-Rab32 pathway and defects in this pathway may underlie disease processes in many systems.
Asunto(s)
Péptidos beta-Amiloides/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Mitocondrias/genética , Proteínas de Unión al GTP rab/genética , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Membrana Celular/genética , Glicoproteínas/genética , Células HEK293 , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/patología , Hernias Diafragmáticas Congénitas/genética , Hernias Diafragmáticas Congénitas/metabolismo , Hernias Diafragmáticas Congénitas/patología , Humanos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Mitocondrias/metabolismo , Miopía/genética , Miopía/metabolismo , Miopía/patología , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Síndrome Oculocerebrorrenal/patología , Proteinuria/genética , Proteinuria/metabolismo , Proteinuria/patología , Células RAW 264.7 , Defectos Congénitos del Transporte Tubular Renal/genética , Defectos Congénitos del Transporte Tubular Renal/metabolismo , Defectos Congénitos del Transporte Tubular Renal/patología , Transducción de Señal , Sirtuina 3/genética , Factor de Crecimiento Transformador beta/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na+/K+-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na+/K+-ATPase α1 localization and reduces NMJ denervation in Slc12a6-/- mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.
Asunto(s)
Agenesia del Cuerpo Calloso/metabolismo , Neuronas Motoras/metabolismo , Unión Neuromuscular/metabolismo , Enfermedades del Sistema Nervioso Periférico/metabolismo , Terminales Presinápticos/metabolismo , Simportadores/deficiencia , Transmisión Sináptica/fisiología , Agenesia del Cuerpo Calloso/tratamiento farmacológico , Agenesia del Cuerpo Calloso/patología , Animales , Carbamazepina/farmacología , Células Cultivadas , Cloruros/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/patología , Neurotransmisores/farmacología , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/patología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/patología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Simportadores/genética , Transmisión Sináptica/efectos de los fármacosRESUMEN
Agenesis of the corpus callosum (AgCC) is a frequent brain disorder found in over 80 human congenital syndromes including ciliopathies. Here, we report a severe AgCC in Ftm/Rpgrip1l knockout mouse, which provides a valuable model for Meckel-Grüber syndrome. Rpgrip1l encodes a protein of the ciliary transition zone, which is essential for ciliogenesis in several cell types in mouse including neuroepithelial cells in the developing forebrain. We show that AgCC in Rpgrip1l(-/-) mouse is associated with a disturbed location of guidepost cells in the dorsomedial telencephalon. This mislocalization results from early patterning defects and abnormal cortico-septal boundary (CSB) formation in the medial telencephalon. We demonstrate that all these defects primarily result from altered GLI3 processing. Indeed, AgCC, together with patterning defects and mispositioning of guidepost cells, is rescued by overexpressing in Rpgrip1l(-/-) embryos, the short repressor form of the GLI3 transcription factor (GLI3R), provided by the Gli3(Δ699) allele. Furthermore, Gli3(Δ699) also rescues AgCC in Rfx3(-/-) embryos deficient for the ciliogenic RFX3 transcription factor that regulates the expression of several ciliary genes. These data demonstrate that GLI3 processing is a major outcome of primary cilia function in dorsal telencephalon morphogenesis. Rescuing CC formation in two independent ciliary mutants by GLI3(Δ699) highlights the crucial role of primary cilia in maintaining the proper level of GLI3R required for morphogenesis of the CC.
Asunto(s)
Cilios/metabolismo , Cuerpo Calloso/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Agenesia del Cuerpo Calloso/embriología , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/metabolismo , Animales , Tipificación del Cuerpo/genética , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/metabolismo , Cuerpo Calloso/enzimología , Cuerpo Calloso/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Encefalocele/genética , Encefalocele/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Noqueados , Mutación , Neocórtex/embriología , Neocórtex/metabolismo , Neocórtex/patología , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Factores de Transcripción del Factor Regulador X , Retinitis Pigmentosa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Gli3 con Dedos de ZincRESUMEN
Single gene disorders of the autophagy pathway are an emerging, novel and diverse group of multisystem diseases in children. Clinically, these disorders prominently affect the central nervous system at various stages of development, leading to brain malformations, developmental delay, intellectual disability, epilepsy, movement disorders, and neurodegeneration, among others. Frequent early and severe involvement of the central nervous system puts the paediatric neurologist, neurogeneticist, and neurometabolic specialist at the forefront of recognizing and treating these rare conditions. On a molecular level, mutations in key autophagy genes map to different stages of this highly conserved pathway and thus lead to impairment in isolation membrane (or phagophore) and autophagosome formation, maturation, or autophagosome-lysosome fusion. Here we discuss 'congenital disorders of autophagy' as an emerging subclass of inborn errors of metabolism by using the examples of six recently identified monogenic diseases: EPG5-related Vici syndrome, beta-propeller protein-associated neurodegeneration due to mutations in WDR45, SNX14-associated autosomal-recessive cerebellar ataxia and intellectual disability syndrome, and three forms of hereditary spastic paraplegia, SPG11, SPG15 and SPG49 caused by SPG11, ZFYVE26 and TECPR2 mutations, respectively. We also highlight associations between defective autophagy and other inborn errors of metabolism such as lysosomal storage diseases and neurodevelopmental diseases associated with the mTOR pathway, which may be included in the wider spectrum of autophagy-related diseases from a pathobiological point of view. By exploring these emerging themes in disease pathogenesis and underlying pathophysiological mechanisms, we discuss how congenital disorders of autophagy inform our understanding of the importance of this fascinating cellular pathway for central nervous system biology and disease. Finally, we review the concept of modulating autophagy as a therapeutic target and argue that congenital disorders of autophagy provide a unique genetic perspective on the possibilities and challenges of pathway-specific drug development.
Asunto(s)
Autofagia/fisiología , Encefalopatías Metabólicas Innatas/genética , Encefalopatías Metabólicas Innatas/metabolismo , Enfermedades por Almacenamiento Lisosomal del Sistema Nervioso/genética , Enfermedades por Almacenamiento Lisosomal del Sistema Nervioso/metabolismo , Agenesia del Cuerpo Calloso/diagnóstico , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/metabolismo , Encefalopatías Metabólicas Innatas/diagnóstico , Catarata/diagnóstico , Catarata/genética , Catarata/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal del Sistema Nervioso/diagnóstico , Lisosomas/genética , Lisosomas/metabolismo , Paraplejía Espástica Hereditaria/diagnóstico , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismoRESUMEN
Epigenetic mechanisms are important in different neurological disorders, and one such mechanism is histone acetylation. The multivalent chromatin regulator BRPF1 (bromodomain- and plant homeodomain-linked (PHD) zinc finger-containing protein 1) recognizes different epigenetic marks and activates three histone acetyltransferases, so it is both a reader and a co-writer of the epigenetic language. The three histone acetyltransferases are MOZ, MORF, and HBO1, which are also known as lysine acetyltransferase 6A (KAT6A), KAT6B, and KAT7, respectively. The MORF gene is mutated in four neurodevelopmental disorders sharing the characteristic of intellectual disability and frequently displaying callosal agenesis. Here, we report that forebrain-specific inactivation of the mouse Brpf1 gene caused early postnatal lethality, neocortical abnormalities, and partial callosal agenesis. With respect to the control, the mutant forebrain contained fewer Tbr2-positive intermediate neuronal progenitors and displayed aberrant neurogenesis. Molecularly, Brpf1 loss led to decreased transcription of multiple genes, such as Robo3 and Otx1, important for neocortical development. Surprisingly, elevated expression of different Hox genes and various other transcription factors, such as Lhx4, Foxa1, Tbx5, and Twist1, was also observed. These results thus identify an important role of Brpf1 in regulating forebrain development and suggest that it acts as both an activator and a silencer of gene expression in vivo.
Asunto(s)
Agenesia del Cuerpo Calloso/genética , Encéfalo/anomalías , Encéfalo/crecimiento & desarrollo , Proteínas Portadoras/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Adaptadoras Transductoras de Señales , Agenesia del Cuerpo Calloso/metabolismo , Animales , Conducta Animal , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Cuerpo Calloso/crecimiento & desarrollo , Cuerpo Calloso/metabolismo , Proteínas de Unión al ADN , Eliminación de Gen , Silenciador del Gen , Ratones , Ratones Noqueados , Neurogénesis , Activación TranscripcionalRESUMEN
Brain development requires a precise balance between expansion of the neural progenitor pool and the production of postmitotic neurons and glia. Disruption of this equilibrium results in a myriad of structural abnormalities and disorders of the nervous system. The molecular mechanism that restricts neural progenitor expansion is poorly understood. Here we show that the tumor suppressor neurofibromatosis 2 (Nf2; merlin) limits the expansion of neural progenitor cells (NPCs) in the mammalian dorsal telencephalon. Nf2 is localized at the apical region of NPCs. In the absence of Nf2, NPCs of the cortical hem, hippocampal primordium and neocortical primordium overexpand, while production of Cajal-Retzius cells and hippocampal neurons decreases, resulting in severe malformation of the hippocampus in adult mice. We further show that Nf2 functions by inhibiting the Yap/Taz transcriptional coactivators, probably through a mechanism that is distinct from the canonical Hippo pathway. Overexpressing human YAP in NPCs causes a hippocampal malformation phenotype that closely resembles that of Nf2 mutants and, importantly, deleting Yap in the Nf2 mutant background largely restores hippocampal development. Our studies uncover Nf2 as an important inhibitor of neural progenitor expansion and establish Yap/Taz as key downstream effectors of Nf2 during brain development.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células-Madre Neurales/metabolismo , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/genética , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Animales , Tipificación del Cuerpo , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Polaridad Celular , Femenino , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Hipocampo/embriología , Hipocampo/metabolismo , Hipocampo/patología , Ratones , Ratones Noqueados , Células-Madre Neurales/patología , Neurofibromina 2/genética , Fenotipo , Fosfoproteínas/genética , Embarazo , Factores de Transcripción/genética , Activación Transcripcional , Proteínas Señalizadoras YAPRESUMEN
Hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC) is an autosomal recessive disease of the central and peripheral nervous system that presents as early-onset polyneuropathy. Patients are hypotonic and areflexic from birth, with abnormal facial features and atrophic muscles. Progressive peripheral neuropathy eventually confines them to a wheelchair in the second decade of life, and death occurs by the fourth decade. We here define the neuropathologic features of the disease in autopsy tissues from eight cases. Both developmental and neurodegenerative features were found. Hypoplasia or absence of the major telencephalic commissures and a hypoplasia of corticospinal tracts to half the normal size, were the major neurodevelopmental defects we observed. Despite being a neurodegenerative disease, preservation of brain weight and a conspicuous absence of neuronal or glial cell death were signal features of this disease. Small tumor-like overgrowths of axons, termed axonomas, were found in the central and peripheral nervous system, indicating attempted axonal regeneration. We conclude that the neurodegenerative deficits in HMSN/ACC are primarily caused by an axonopathy superimposed upon abnormal development, affecting peripheral but also central nervous system axons, all ultimately because of a genetic defect in the axonal cotransporter KCC3.
Asunto(s)
Agenesia del Cuerpo Calloso/patología , Axones/patología , Encéfalo/patología , Enfermedades del Sistema Nervioso Periférico/patología , Sistema Nervioso Periférico/patología , Simportadores/genética , Adulto , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/fisiopatología , Autopsia , Axones/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Degeneración Nerviosa , Sistema Nervioso Periférico/metabolismo , Sistema Nervioso Periférico/fisiopatología , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Fenotipo , Pronóstico , Simportadores/metabolismo , Adulto JovenRESUMEN
Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are known to be involved in corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear. Here, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis to determine the genomic insertion site. Transgenes were inserted into chromosome 18 â¼12.0 Mb from the centromere. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1(TAS). Interestingly, Cables1(TAS) mRNA consisted of exons 1-3 of Cables1 and part of the transgene that encoded a novel truncated Cables1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1(TAS) in the fetal cerebrum, but not that of wild-type Cables1. To investigate whether a dominant negative effect of Cables1(TAS) or complete loss of function of Cables1 gives rise to ACC, we produced Cables1-null mutant mice. ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of Cables1(TAS) impairs callosal formation. Moreover, ACC frequency in Cables1(+/TAS) mice was significantly lower than that in Cables1(-/TAS) mice, indicating that wild-type Cables1 interfered with the dominant negative effect of Cables1(TAS). This study indicated that truncated Cables1 causes ACC and wild-type Cables1 contributes to callosal formation.
Asunto(s)
Agenesia del Cuerpo Calloso/genética , Proteínas Portadoras/genética , Ciclinas/deficiencia , Ciclinas/genética , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Animales , Exones , Estudios de Asociación Genética , Homocigoto , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Ratones Transgénicos , Mutagénesis Insercional , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Spock3/Testican-3 is a nervous system-expressed heparan sulfate proteoglycan belonging to a subgroup of the BM-40/SPARC/osteonectin family, the role of which in brain development is unclear. Because Spock1, a member of the Spock family, inhibits their attachment to substrates and the neurite outgrowth of cultured neuronal cells, Spock3 is also thought to be similarly involved in the neuronal development. In the present study, we established a Spock3-mutant mouse harboring a deletion extending from the presumptive upstream regulatory region to exon 4 of the Spock3 locus and performed histological and behavioral studies on these mutant mice. In wild-type (WT) mice, all Spock members were clearly expressed during brain development. In adults, intense Spock1 and Spock2 expressions were observed throughout the entire brain; whereas, Spock3 expression was no longer visible except in the thalamic nuclei. Thus, Spock3 expression is mostly confined to the developmental stage of the brain. In adult mutant mice, the cells of all cortical layers were swollen. The corpus callosum was narrowed around the central region along the rostral-caudal axis and many small spaces were observed without myelin sheaths throughout the entire corpus callosum. In addition, the cortical input and output fibers did not form into thick bundled fibers as well as the WT counterparts did. Moreover, a subpopulation of corticospinal axonal fibers penetrated into the dorsal striatum with moderately altered orientations. Consistent with these modifications of brain structures, the mutant mice exhibited decreased anxiety-like behavior and lowered sociability. Together, these results demonstrate that Spock3 plays an important role in the formation or maintenance of major neuronal structures in the brain.
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Agenesia del Cuerpo Calloso/genética , Ansiedad/genética , Axones/metabolismo , Conducta Animal/fisiología , Cuerpo Calloso/metabolismo , Proteoglicanos/genética , Conducta Social , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Animales , Ansiedad/metabolismo , Ansiedad/patología , Axones/patología , Cuerpo Calloso/patología , Masculino , Ratones , Neuronas/metabolismo , Neuronas/patología , Proteoglicanos/metabolismoRESUMEN
In man, mutations of the megalin-encoding gene causes the rare Donnai-Barrow/Facio-Oculo-Acoustico-Renal Syndrome, which is partially characterized by high-grade myopia. Previous studies of renal megalin function have established that megalin is crucial for conservation of renal filtered nutrients including vitamin A; however, the role of megalin in ocular physiology and development is presently unknown. Therefore, we investigate ocular megalin expression and the ocular phenotype of megalin-deficient mice. Topographical and subcellular localization of megalin as well as the ocular phenotype of megalin-deficient mice were examined with immunological techniques using light, confocal and electron microscopy. We identified megalin in the retinal pigment epithelium (RPE) and non-pigmented ciliary body epithelium (NPCBE) in normal mouse eyes. Immunocytochemical investigations furthermore showed that megalin localizes to vesicular structures in the RPE and NPCBE cells. Histological investigations of ocular mouse tissue also identified a severe myopia phenotype as well as enlarged RPE melanosomes and abnormal ciliary body development in the megalin-deficient mice. In conclusion, the complex ocular phenotype observed in the megalin-deficient mice suggests that megalin-mediated developmental abnormalities may contribute to the high myopia phenotype observed in the Donnai-Barrow Syndrome patients and, thus, that megalin harbors important roles in ocular development and physiology. Finally, our data show that megalin-deficient mice may provide a valuable model for future studies of megalin in ocular physiology and pathology.
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Cuerpo Ciliar/metabolismo , Regulación de la Expresión Génica , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/biosíntesis , Melanosomas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Animales , Cuerpo Ciliar/patología , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/patología , Hernias Diafragmáticas Congénitas/genética , Hernias Diafragmáticas Congénitas/metabolismo , Hernias Diafragmáticas Congénitas/patología , Humanos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Melanosomas/genética , Melanosomas/patología , Ratones , Ratones Mutantes , Miopía/genética , Miopía/metabolismo , Miopía/patología , Proteinuria/genética , Proteinuria/metabolismo , Proteinuria/patología , Defectos Congénitos del Transporte Tubular Renal/genética , Defectos Congénitos del Transporte Tubular Renal/metabolismo , Defectos Congénitos del Transporte Tubular Renal/patología , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Pediatric neurodegenerative diseases are a heterogeneous group of diseases that result from specific genetic and biochemical defects. In recent years, studies have revealed a wide spectrum of abnormal cellular functions that include impaired proteolysis, abnormal lipid trafficking, accumulation of lysosomal content, and mitochondrial dysfunction. Within neurons, elaborated degradation pathways such as the ubiquitin-proteasome system and the autophagy-lysosomal pathway are critical for maintaining homeostasis and normal cell function. Recent evidence suggests a pivotal role for autophagy in major adult and pediatric neurodegenerative diseases. We herein review genetic, pathological, and molecular evidence for the emerging link between autophagy dysfunction and lysosomal storage disorders such as Niemann-Pick type C, progressive myoclonic epilepsies such as Lafora disease, and leukodystrophies such as Alexander disease. We also discuss the recent discovery of genetically deranged autophagy in Vici syndrome, a multisystem disorder, and the implications for the role of autophagy in development and disease. Deciphering the exact mechanism by which autophagy contributes to disease pathology may open novel therapeutic avenues to treat neurodegeneration. To this end, an outlook on novel therapeutic approaches targeting autophagy concludes this review.
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Autofagia , Encefalopatías Metabólicas/patología , Encéfalo/patología , Metabolismo Energético , Enfermedades Neurodegenerativas/patología , Neuronas/patología , Factores de Edad , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Enfermedad de Alexander/metabolismo , Enfermedad de Alexander/patología , Animales , Autofagia/genética , Encéfalo/metabolismo , Encefalopatías Metabólicas/genética , Encefalopatías Metabólicas/metabolismo , Encefalopatías Metabólicas/terapia , Catarata/metabolismo , Catarata/patología , Niño , Metabolismo Energético/genética , Predisposición Genética a la Enfermedad , Humanos , Enfermedad de Lafora/metabolismo , Enfermedad de Lafora/patología , Degeneración Nerviosa , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/terapia , Neuronas/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Fenotipo , Pronóstico , Factores de RiesgoRESUMEN
Disruption of the potassium/chloride cotransporter 3 (KCC3), encoded by the SLC12A6 gene, causes hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC), a neurodevelopmental and neurodegenerative disorder affecting both the peripheral nervous system and CNS. However, the precise role of KCC3 in the maintenance of ion homeostasis in the nervous system and the pathogenic mechanisms leading to HMSN/ACC remain unclear. We established two Slc12a6 Cre/LoxP transgenic mouse lines expressing C-terminal truncated KCC3 in either a neuron-specific or ubiquitous fashion. Our results suggest that neuronal KCC3 expression is crucial for axon volume control. We also demonstrate that the neuropathic features of HMSN/ACC are predominantly due to a neuronal KCC3 deficit, while the auditory impairment is due to loss of non-neuronal KCC3 expression. Furthermore, we demonstrate that KCC3 plays an essential role in inflammatory pain pathways. Finally, we observed hypoplasia of the corpus callosum in both mouse mutants and a marked decrease in axonal tracts serving the auditory cortex in only the general deletion mutant. Together, these results establish KCC3 as an important player in both central and peripheral nervous system maintenance.
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Agenesia del Cuerpo Calloso/genética , Modelos Animales de Enfermedad , Neuropatía Hereditaria Motora y Sensorial/genética , Fenotipo , Simportadores/deficiencia , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Animales , Femenino , Neuropatía Hereditaria Motora y Sensorial/metabolismo , Neuropatía Hereditaria Motora y Sensorial/patología , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Trastornos Heredodegenerativos del Sistema Nervioso/patología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Simportadores/biosíntesis , Simportadores/genéticaRESUMEN
BACKGROUND: The reabsorption of filtered plasma proteins, hormones and vitamins by the renal proximal tubules is vital for body homeostasis. Studies of megalin-deficient mice suggest that the large multi-ligand endocytic receptor megalin plays an essential role in this process. In humans, dysfunctional megalin causes the extremely rare Donnai-Barrow/Facio-Oculo-Acustico-Renal (DB/FOAR) syndrome characterized by a characteristic and multifaceted phenotype including low-molecular-weight proteinuria. In this study, we examined the role of megalin for tubular protein reabsorption in humans through analysis of proximal tubular function in megalin-deficient patients. METHODS: Direct sequencing of the megalin-encoding gene (LRP2) was performed in a family in which three children presented with classical DB/FOAR manifestations. Renal consequences of megalin deficiency were investigated through immunohistochemical analyses of renal biopsy material and immunoblotting of urine samples. RESULTS: In the patients, a characteristic urinary protein profile with increased urinary excretion of vitamin D-binding protein, retinol-binding protein and albumin was associated with absence of, or reduced, proximal tubular endocytic uptake as shown by renal immunohistochemistry. In the absence of tubular uptake, urinary albumin excretion was in the micro-albuminuric range suggesting that limited amounts of albumin are filtered in human glomeruli. CONCLUSIONS: This study demonstrated that megalin plays an essential role for human proximal tubular protein reabsorption and suggests that only limited amounts of albumin is normally filtered in the human glomeruli. Finally, we propose that the characteristic urinary protein profile of DB/FOAR patients may be utilized as a diagnostic marker of megalin dysfunction.
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Agenesia del Cuerpo Calloso/patología , Albúminas/metabolismo , Pérdida Auditiva Sensorineural/patología , Túbulos Renales Proximales/patología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Mutación/genética , Miopía/patología , Proteinuria/patología , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/metabolismo , Preescolar , Femenino , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/metabolismo , Hernias Diafragmáticas Congénitas , Humanos , Túbulos Renales Proximales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Miopía/genética , Miopía/metabolismo , Fenotipo , Proteinuria/genética , Proteinuria/metabolismo , Defectos Congénitos del Transporte Tubular RenalRESUMEN
Missense and protein-truncating mutations of the human potassium-chloride co-transporter 3 gene (KCC3) cause hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), which is a severe neurodegenerative disease characterized by axonal dysfunction and neurodevelopmental defects. We previously reported that KCC3-truncating mutations disrupt brain-type creatine kinase-dependent activation of the co-transporter through the loss of its last 140 amino acids. Here, we report a novel and more distal HMSN/ACC-truncating mutation (3402C â T; R1134X) that eliminates only the last 17 residues of the protein. This small truncation disrupts the interaction with brain-type creatine kinase in mammalian cells but also affects plasma membrane localization of the mutant transporter. Although it is not truncated, the previously reported HMSN/ACC-causing 619C â T (R207C) missense mutation also leads to KCC3 loss of function in Xenopus oocyte flux assay. Immunodetection in Xenopus oocytes and in mammalian cultured cells revealed a decreased amount of R207C at the plasma membrane, with significant retention of the mutant proteins in the endoplasmic reticulum. In mammalian cells, curcumin partially corrected these mutant protein mislocalizations, with more protein reaching the plasma membrane. These findings suggest that mis-trafficking of mutant protein is an important pathophysiological feature of HMSN/ACC causative KCC3 mutations.
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Agenesia del Cuerpo Calloso/metabolismo , Sustitución de Aminoácidos , Neuropatía Hereditaria Motora y Sensorial/metabolismo , Mutación Missense , Proteínas del Tejido Nervioso/metabolismo , Simportadores/metabolismo , Agenesia del Cuerpo Calloso/genética , Secuencia de Aminoácidos , Animales , Células HeLa , Neuropatía Hereditaria Motora y Sensorial/genética , Humanos , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas , Eliminación de Secuencia , Simportadores/genética , Xenopus laevisRESUMEN
The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.
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Astrocitos/metabolismo , Cuerpo Calloso/metabolismo , Receptor DCC/metabolismo , Telencéfalo/metabolismo , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/patología , Animales , Células COS , Línea Celular Tumoral , Movimiento Celular , Forma de la Célula , Chlorocebus aethiops , Cuerpo Calloso/embriología , Receptor DCC/genética , Regulación del Desarrollo de la Expresión Génica , Genotipo , Edad Gestacional , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis , Mutación , Netrina-1/genética , Netrina-1/metabolismo , Fenotipo , Transducción de Señal , Telencéfalo/embriologíaRESUMEN
Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome-lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended "shepherd's staff" architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome-lysosome fusion. The hEPG5-GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5's overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.