ABSTRACT
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.
Subject(s)
Cryoelectron Microscopy , Low Density Lipoprotein Receptor-Related Protein-2 , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Low Density Lipoprotein Receptor-Related Protein-2/genetics , Animals , Humans , Rats , Ligands , Endocytosis , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/genetics , Renal Tubular Transport, Inborn Errors , Myopia , Hernias, Diaphragmatic, Congenital , Proteinuria , Hearing Loss, SensorineuralABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Gene Knockout Techniques , Kidney Tubules, Proximal/metabolism , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Receptors, Cell Surface/metabolism , Transcription, Genetic , Adaptor Proteins, Signal Transducing/genetics , Agenesis of Corpus Callosum/genetics , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/pathology , Animals , Apoptosis Regulatory Proteins/genetics , Cells, Cultured , Databases, Genetic , Gene Regulatory Networks , Hearing Loss, Sensorineural/genetics , Hearing Loss, Sensorineural/metabolism , Hearing Loss, Sensorineural/pathology , Hernias, Diaphragmatic, Congenital/genetics , Hernias, Diaphragmatic, Congenital/metabolism , Hernias, Diaphragmatic, Congenital/pathology , Humans , Kidney Tubules, Proximal/pathology , Low Density Lipoprotein Receptor-Related Protein-2/genetics , Male , Mice, Knockout , Monodelphis , Myopia/genetics , Myopia/metabolism , Myopia/pathology , Proteinuria/genetics , Proteinuria/metabolism , Proteinuria/pathology , Receptors, Cell Surface/genetics , Renal Tubular Transport, Inborn Errors/genetics , Renal Tubular Transport, Inborn Errors/metabolism , Renal Tubular Transport, Inborn Errors/pathologyABSTRACT
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.
Subject(s)
Astrocytes/metabolism , Corpus Callosum/metabolism , DCC Receptor/metabolism , Telencephalon/metabolism , Agenesis of Corpus Callosum/genetics , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/pathology , Animals , COS Cells , Cell Line, Tumor , Cell Movement , Cell Shape , Chlorocebus aethiops , Corpus Callosum/embryology , DCC Receptor/genetics , Gene Expression Regulation, Developmental , Genotype , Gestational Age , HEK293 Cells , Humans , Mice, Inbred C57BL , Mice, Knockout , Morphogenesis , Mutation , Netrin-1/genetics , Netrin-1/metabolism , Phenotype , Signal Transduction , Telencephalon/embryologyABSTRACT
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.
Subject(s)
Autophagosomes/metabolism , Autophagy-Related Proteins/metabolism , Lysosomes/metabolism , Mitochondria/metabolism , Vesicular Transport Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Agenesis of Corpus Callosum/genetics , Agenesis of Corpus Callosum/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Proteins/genetics , Cataract/genetics , Cataract/metabolism , Genetic Predisposition to Disease , HeLa Cells , Humans , Microtubule-Associated Proteins/metabolism , Mitophagy , Mutation , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Stability , Protein Transport , Proteolysis , Sf9 Cells , Structure-Activity Relationship , Vesicular Transport Proteins/geneticsABSTRACT
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.
Subject(s)
Agenesis of Corpus Callosum/therapy , Behavior, Animal , Ganglia, Spinal/metabolism , Genetic Therapy , Motor Activity , Peripheral Nervous System Diseases/therapy , Symporters/metabolism , Age Factors , Agenesis of Corpus Callosum/genetics , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/physiopathology , Animals , Disease Models, Animal , Female , Ganglia, Spinal/physiopathology , Gene Expression Regulation, Developmental , Genetic Predisposition to Disease , Male , Mice, Inbred C57BL , Mice, Knockout , Parvalbumins/metabolism , Peripheral Nervous System Diseases/genetics , Peripheral Nervous System Diseases/metabolism , Peripheral Nervous System Diseases/physiopathology , Phenotype , Postural Balance , Rotarod Performance Test , Symporters/geneticsABSTRACT
BACKGROUND: Agenesis of the corpus callosum (ACC) is a relatively common brain malformation in children with developmental disabilities, caused by mutations in many genes. These genetic causes are characterized by their extreme heterogeneity with more than 300 causative genes identified to date. CASE REPORT: We describe two new cases from a three-generation family with ACC and a de novo mutation of the sonic hedgehog (SHH) gene. The affected family members had mild intellectual disability, broad forehead, and widely spaced eyes. A next-generation sequencing (NGS) approach revealed a stop-gain mutation (NM_000193.2:c.1300_1301insA p.Trp434Ter) of the SHH gene; it is the first family to report ACC associated with a single SHH gene mutation. CONCLUSION: ACC with mild intellectual disability and facial dysmorphism may be caused by a mutation in SHH, but further research investigating the genotype-phenotype correlation of SHH mutations is required.
Subject(s)
Agenesis of Corpus Callosum/genetics , Hedgehog Proteins/genetics , Adult , Agenesis of Corpus Callosum/metabolism , Child , China , Codon, Nonsense/genetics , Corpus Callosum/pathology , Craniofacial Abnormalities/genetics , Developmental Disabilities/genetics , Family , Female , Hedgehog Proteins/metabolism , Humans , Intellectual Disability/genetics , Male , Mutation/genetics , Nervous System Malformations/genetics , Pedigree , PhenotypeABSTRACT
Williams-Beuren syndrome (WBS) is caused by microdeletions of 28 genes and is characterized by cognitive disorder and hypotrophic corpus callosum (CC). Nsun5 gene, which encodes cytosine-5 RNA methyltransferase, is located in the deletion loci of WBS. We have reported that single-gene knockout of Nsun5 (Nsun5-KO) in mice impairs spatial cognition. Herein, we report that postnatal day (PND) 60 Nsun5-KO mice showed the volumetric reduction of CC with a decline in the number of myelinated axons and loose myelin sheath. Nsun5 was highly expressed in callosal oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs) from PND7 to PND28. The numbers of OPCs and OLs in CC of PND7-28 Nsun5-KO mice were significantly reduced compared to wild-type littermates. Immunohistochemistry and Western blot analyses of myelin basic protein (MBP) showed the hypomyelination in the CC of PND28 Nsun5-KO mice. The Nsun5 deletion suppressed the proliferation of OPCs but did not affect transition of radial glial cells into OPCs or cell cycle exit of OPCs. The protein levels, rather than transcriptional levels, of CDK1, CDK2 and Cdc42 in the CC of PND7 and PND14 Nsun5-KO mice were reduced. These findings point to the involvement of Nsun5 deletion in agenesis of CC observed in WBS.
Subject(s)
Corpus Callosum/metabolism , Methyltransferases/genetics , Myelin Sheath/metabolism , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/pathology , Animals , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Cell Proliferation , Corpus Callosum/growth & development , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Disease Models, Animal , Methyltransferases/deficiency , Mice , Mice, Knockout , Oligodendroglia/cytology , Oligodendroglia/metabolism , Williams Syndrome/metabolism , Williams Syndrome/pathologyABSTRACT
The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5-/- mutants. By aging, epg5-/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.
Subject(s)
Agenesis of Corpus Callosum/metabolism , Autophagy-Related Proteins/metabolism , Cataract/metabolism , Gene Knockout Techniques , Zebrafish Proteins/metabolism , Zebrafish/genetics , Amino Acid Sequence , Animals , Autophagosomes/metabolism , Autophagy-Related Proteins/chemistry , Autophagy-Related Proteins/genetics , Base Sequence , Gene Expression Regulation, Developmental , Goblet Cells/pathology , Intestines/pathology , Intestines/ultrastructure , Larva/ultrastructure , Lysosomes/metabolism , Membrane Fusion , Models, Biological , Motor Neurons/metabolism , Motor Neurons/pathology , Mutagenesis/genetics , Mutation/genetics , Organ Specificity , Zebrafish/embryology , Zebrafish Proteins/chemistry , Zebrafish Proteins/geneticsABSTRACT
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.
Subject(s)
Amyloid beta-Peptides/genetics , Low Density Lipoprotein Receptor-Related Protein-2/genetics , Mitochondria/genetics , rab GTP-Binding Proteins/genetics , Agenesis of Corpus Callosum/genetics , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/pathology , Amyloid beta-Peptides/metabolism , Animals , Brain/metabolism , Brain/pathology , Cell Membrane/genetics , Glycoproteins/genetics , HEK293 Cells , Hearing Loss, Sensorineural/genetics , Hearing Loss, Sensorineural/metabolism , Hearing Loss, Sensorineural/pathology , Hernias, Diaphragmatic, Congenital/genetics , Hernias, Diaphragmatic, Congenital/metabolism , Hernias, Diaphragmatic, Congenital/pathology , Humans , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Mice , Mitochondria/metabolism , Myopia/genetics , Myopia/metabolism , Myopia/pathology , Oculocerebrorenal Syndrome/genetics , Oculocerebrorenal Syndrome/metabolism , Oculocerebrorenal Syndrome/pathology , Proteinuria/genetics , Proteinuria/metabolism , Proteinuria/pathology , RAW 264.7 Cells , Renal Tubular Transport, Inborn Errors/genetics , Renal Tubular Transport, Inborn Errors/metabolism , Renal Tubular Transport, Inborn Errors/pathology , Signal Transduction , Sirtuin 3/genetics , Transforming Growth Factor beta/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
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.
Subject(s)
Agenesis of Corpus Callosum/metabolism , Motor Neurons/metabolism , Neuromuscular Junction/metabolism , Peripheral Nervous System Diseases/metabolism , Presynaptic Terminals/metabolism , Symporters/deficiency , Synaptic Transmission/physiology , Agenesis of Corpus Callosum/drug therapy , Agenesis of Corpus Callosum/pathology , Animals , Carbamazepine/pharmacology , Cells, Cultured , Chlorides/metabolism , Disease Models, Animal , Mice, Inbred C57BL , Mice, Transgenic , Motor Neurons/drug effects , Motor Neurons/pathology , Neuromuscular Junction/drug effects , Neuromuscular Junction/pathology , Neurotransmitter Agents/pharmacology , Peripheral Nervous System Diseases/drug therapy , Peripheral Nervous System Diseases/pathology , Presynaptic Terminals/drug effects , Presynaptic Terminals/pathology , Sodium-Potassium-Exchanging ATPase/metabolism , Spinal Cord/drug effects , Spinal Cord/metabolism , Spinal Cord/pathology , Symporters/genetics , Synaptic Transmission/drug effectsABSTRACT
Mutations in GPSM2 cause Chudley-McCullough syndrome (CMCS), an autosomal recessive neurological disorder characterized by early-onset sensorineural deafness and brain anomalies. Here, we show that mutation of the mouse orthologue of GPSM2 affects actin-rich stereocilia elongation in auditory and vestibular hair cells, causing deafness and balance defects. The G-protein subunit Gαi3, a well-documented partner of Gpsm2, participates in the elongation process, and its absence also causes hearing deficits. We show that Gpsm2 defines an â¼200 nm nanodomain at the tips of stereocilia and this localization requires the presence of Gαi3, myosin 15 and whirlin. Using single-molecule tracking, we report that loss of Gpsm2 leads to decreased outgrowth and a disruption of actin dynamics in neuronal growth cones. Our results elucidate the aetiology of CMCS and highlight a new molecular role for Gpsm2/Gαi3 in the regulation of actin dynamics in epithelial and neuronal tissues.
Subject(s)
Actins/metabolism , Agenesis of Corpus Callosum/genetics , Arachnoid Cysts/genetics , Carrier Proteins/genetics , Growth Cones/metabolism , Hair Cells, Auditory/metabolism , Hair Cells, Vestibular/metabolism , Hearing Loss, Sensorineural/genetics , Neurons/metabolism , Stereocilia/metabolism , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/physiopathology , Animals , Arachnoid Cysts/metabolism , Arachnoid Cysts/physiopathology , Cell Cycle Proteins , Deafness/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Hearing Loss, Sensorineural/metabolism , Hearing Loss, Sensorineural/physiopathology , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/metabolism , Mice , Mutation , Myosins/metabolism , Postural Balance , Sensation Disorders/geneticsABSTRACT
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.
Subject(s)
Agenesis of Corpus Callosum/genetics , Autophagosomes/metabolism , Cataract/genetics , Endosomes/metabolism , Lysosomes/metabolism , Proteins/genetics , rab GTP-Binding Proteins/genetics , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/pathology , Amino Acid Sequence , Animals , Autophagosomes/ultrastructure , Autophagy/genetics , Autophagy-Related Proteins , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cataract/metabolism , Cataract/pathology , Endosomes/ultrastructure , Gene Expression Regulation , HeLa Cells , Humans , Lysosomal Membrane Proteins , Lysosomes/ultrastructure , Membrane Fusion , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Protein Binding , Proteins/metabolism , Qa-SNARE Proteins/genetics , Qa-SNARE Proteins/metabolism , Qb-SNARE Proteins/genetics , Qb-SNARE Proteins/metabolism , Qc-SNARE Proteins/genetics , Qc-SNARE Proteins/metabolism , R-SNARE Proteins/genetics , R-SNARE Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Synaptosomal-Associated Protein 25/genetics , Synaptosomal-Associated Protein 25/metabolism , Vesicular Transport Proteins , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
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.
Subject(s)
Agenesis of Corpus Callosum/pathology , Axons/pathology , Brain/pathology , Peripheral Nervous System Diseases/pathology , Peripheral Nervous System/pathology , Symporters/genetics , Adult , Agenesis of Corpus Callosum/genetics , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/physiopathology , Autopsy , Axons/metabolism , Brain/metabolism , Brain/physiopathology , Female , Genetic Predisposition to Disease , Humans , Male , Nerve Degeneration , Peripheral Nervous System/metabolism , Peripheral Nervous System/physiopathology , Peripheral Nervous System Diseases/genetics , Peripheral Nervous System Diseases/metabolism , Peripheral Nervous System Diseases/physiopathology , Phenotype , Prognosis , Symporters/metabolism , Young AdultABSTRACT
We report on seven novel patients with a submicroscopic 22q12 deletion. The common phenotype constitutes a contiguous gene deletion syndrome on chromosome 22q12.1q12.2, featuring NF2-related schwannoma of the vestibular nerve, corpus callosum agenesis and palatal defects. Combining our results with the literature, eight patients are recorded with palatal defects in association with haploinsufficiency of 22q12.1, including the MN1 gene. These observations, together with the mouse expression data and the finding of craniofacial malformations including cleft palate in a Mn1-knockout mouse model, suggest that this gene is a candidate gene for cleft palate in humans.
Subject(s)
Agenesis of Corpus Callosum/genetics , Chromosome Deletion , Chromosomes, Human, Pair 22 , Cleft Palate/genetics , Neuroma, Acoustic/genetics , Tumor Suppressor Proteins/deficiency , Adolescent , Agenesis of Corpus Callosum/diagnosis , Agenesis of Corpus Callosum/metabolism , Animals , Child , Child, Preschool , Chromosome Mapping , Cleft Palate/diagnosis , Cleft Palate/metabolism , Female , Gene Expression , Haploinsufficiency , Humans , Male , Mice , Mice, Knockout , Neuroma, Acoustic/diagnosis , Neuroma, Acoustic/metabolism , Sequence Analysis, DNA , Trans-Activators , Tumor Suppressor Proteins/geneticsABSTRACT
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.
Subject(s)
Autophagy/physiology , Brain Diseases, Metabolic, Inborn/genetics , Brain Diseases, Metabolic, Inborn/metabolism , Lysosomal Storage Diseases, Nervous System/genetics , Lysosomal Storage Diseases, Nervous System/metabolism , Agenesis of Corpus Callosum/diagnosis , Agenesis of Corpus Callosum/genetics , Agenesis of Corpus Callosum/metabolism , Brain Diseases, Metabolic, Inborn/diagnosis , Cataract/diagnosis , Cataract/genetics , Cataract/metabolism , Humans , Lysosomal Storage Diseases, Nervous System/diagnosis , Lysosomes/genetics , Lysosomes/metabolism , Spastic Paraplegia, Hereditary/diagnosis , Spastic Paraplegia, Hereditary/genetics , Spastic Paraplegia, Hereditary/metabolismABSTRACT
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.
Subject(s)
Cilia/metabolism , Corpus Callosum/metabolism , Kruppel-Like Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Adaptor Proteins, Signal Transducing/deficiency , Agenesis of Corpus Callosum/embryology , Agenesis of Corpus Callosum/genetics , Agenesis of Corpus Callosum/metabolism , Animals , Body Patterning/genetics , Ciliary Motility Disorders/genetics , Ciliary Motility Disorders/metabolism , Corpus Callosum/enzymology , Corpus Callosum/pathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Models, Animal , Encephalocele/genetics , Encephalocele/metabolism , Gene Expression Regulation, Developmental , Humans , Kruppel-Like Transcription Factors/genetics , Mice , Mice, Knockout , Mutation , Neocortex/embryology , Neocortex/metabolism , Neocortex/pathology , Nerve Tissue Proteins/genetics , Neurons/metabolism , Polycystic Kidney Diseases/genetics , Polycystic Kidney Diseases/metabolism , Regulatory Factor X Transcription Factors , Retinitis Pigmentosa , Transcription Factors/genetics , Transcription Factors/metabolism , Zinc Finger Protein Gli3ABSTRACT
Descrito por primera vez en 1987, el Síndrome de Pai se considera una variante rara de la displasia fronto-nasal. Consiste en el fallo del cierre de la línea media y sus signos son encéfalo-cráneo-faciales. Los hallazgos que conforman el síndrome son: la presencia de una variedad de pólipos (intranasal, cutáneos y alveolar del maxilar superior), fisura ósea y labiopalatina en la línea media, lipoma intracraneal y agenesia parcial o total del cuerpo calloso. Su causa es desconocida y su presentación esporádica. La incidencia se estima en 1 de cada 20.000 a 40.000 recién nacidos, siendo el sexo femenino el más afectado. El objetivo de este artículo es presentar el primer caso clínico documentado en México con estas características, y de acuerdo al último caso publicado en 2014 por Mee Hong, es el número 38 de la literatura mundial (AU)
Described by the first time in 1987, Pai's Syndrome is considered a rare variant of the displasia fronto-nasal. It consists of the fault of the closing of the middle line and his signs are encephalo-craneo-facial. The findings of the syndrome are the presence of a variety of polyps (intranasal, cutaneous and alveolar of the upper jaw), bony and lip-palate cleft in the middle line, intracranial lipoma and partial or total agenesia of the corpus callosum. The etiology of this syndrome is not known, and its presentation is sporadic. The incidence is estimated in 1 of every 20.000-40.000 newborn children, being the most affected feminine sex. The aim of this article is to present the first clinical case reported in Mexico with these characteristics and that in agreement to the last case published in 2014 for Mee Hong, it is number 38 of the world literature (AU)
Subject(s)
Child , Infant, Newborn , Humans , Agenesis of Corpus Callosum/genetics , Agenesis of Corpus Callosum/pathology , Nasal Polyps/congenital , Nasal Polyps/complications , Lipoma/chemically induced , Lipoma/metabolism , Agenesis of Corpus Callosum/metabolism , Agenesis of Corpus Callosum/surgery , Nasal Polyps/rehabilitation , Nasal Polyps/surgery , Lipoma/complications , Lipoma/surgeryABSTRACT
Donnai-Barrow syndrome (DBS; MIM 222448) is characterized by typical craniofacial anomalies (major hypertelorism with bulging eyes), high grade myopia, deafness and low molecular weight proteinuria. The disorder results from mutations in the low density lipoprotein receptor-related protein 2 gene LRP2 that maps to chromosome 2q31.1. LRP2 encodes megalin, a multi-ligand endocytic receptor. Herein, we describe the clinical presentation of 4 patients from 2 unrelated Saudi families. Two novel LRP2 mutations, a homozygous nonsense mutation (c.4968C>G; p.Tyr1656*) and a missense mutation (c.12062G>A; p.Cys4021Tyr), were detected in the first and second family respectively. Interestingly, intrafamilial phenotypic variability was observed in one family, while DBS features were atypical in the second family. Differential diagnosis of DBS includes several syndromes associating hypertelorism with high grade myopia, and several syndromal forms of CDH, which are briefly summarized in this study.
Subject(s)
Agenesis of Corpus Callosum/genetics , Hearing Loss, Sensorineural/genetics , Hernias, Diaphragmatic, Congenital/genetics , Low Density Lipoprotein Receptor-Related Protein-2/genetics , Myopia/genetics , Proteinuria/genetics , Renal Tubular Transport, Inborn Errors/genetics , Adolescent , Agenesis of Corpus Callosum/metabolism , Child , Child, Preschool , Codon, Nonsense , DNA Mutational Analysis , Female , Gene Expression , Hearing Loss, Sensorineural/metabolism , Hernias, Diaphragmatic, Congenital/metabolism , Humans , Infant , Infant, Newborn , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Male , Mutation, Missense , Myopia/metabolism , Proteinuria/metabolism , Renal Tubular Transport, Inborn Errors/metabolism , Young AdultABSTRACT
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.
Subject(s)
Agenesis of Corpus Callosum/genetics , Brain/abnormalities , Brain/growth & development , Carrier Proteins/genetics , Gene Expression Regulation, Developmental , Adaptor Proteins, Signal Transducing , Agenesis of Corpus Callosum/metabolism , Animals , Behavior, Animal , Brain/metabolism , Carrier Proteins/metabolism , Corpus Callosum/growth & development , Corpus Callosum/metabolism , DNA-Binding Proteins , Gene Deletion , Gene Silencing , Mice , Mice, Knockout , Neurogenesis , Transcriptional ActivationABSTRACT
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.