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1.
Nat Commun ; 10(1): 3506, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31383864

RESUMO

Sensory circuits are typically established during early development, yet how circuit specificity and function are maintained during organismal growth has not been elucidated. To gain insight we quantitatively investigated synaptic growth and connectivity in the Drosophila nociceptive network during larval development. We show that connectivity between primary nociceptors and their downstream neurons scales with animal size. We further identified the conserved Ste20-like kinase Tao as a negative regulator of synaptic growth required for maintenance of circuit specificity and connectivity. Loss of Tao kinase resulted in exuberant postsynaptic specializations and aberrant connectivity during larval growth. Using functional imaging and behavioral analysis we show that loss of Tao-induced ectopic synapses with inappropriate partner neurons are functional and alter behavioral responses in a connection-specific manner. Our data show that fine-tuning of synaptic growth by Tao kinase is required for maintaining specificity and behavioral output of the neuronal network during animal growth.

2.
Cell Rep ; 28(1): 11-20.e9, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269433

RESUMO

Myosin VI is an actin-based cytoskeletal motor implicated in various steps of membrane trafficking. Here, we investigated whether this myosin is crucial for synaptic function and plasticity in neurons. We find that myosin VI localizes at cerebellar parallel fiber to Purkinje cell synapses and that the myosin is indispensable for long-term depression of AMPA-receptor-mediated synaptic signal transmission at this synapse. Moreover, direct visualization of GluA2-containing AMPA receptors in Purkinje cells reveals that the myosin drives removal of AMPA receptors from the surface of dendritic spines in an activity-dependent manner. Co-immunoprecipitation and super-resolution microscopy indicate that specifically the interaction of myosin VI with the clathrin adaptor component α-adaptin is important during long-term depression. Together, these data suggest that myosin VI directly promotes clathrin-mediated endocytosis of AMPA receptors in Purkinje cells to mediate cerebellar long-term depression. Our results provide insights into myosin VI function and the molecular mechanisms underlying synaptic plasticity.

3.
J Biol Chem ; 294(24): 9592-9604, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31040178

RESUMO

Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids. The CLN3 gene encodes a lysosomal membrane protein of unknown function, and CLN3 mutations cause the fatal neurodegenerative lysosomal storage disorder CLN3 (Batten disease) by mechanisms that are poorly understood. To define components critical for lysosomal homeostasis that are affected by this disease, here we quantified the lysosomal proteome in cerebellar cell lines derived from a CLN3 knock-in mouse model of human Batten disease and control cells. We purified lysosomes from SILAC-labeled, and magnetite-loaded cerebellar cells by magnetic separation and analyzed them by MS. This analysis identified 70 proteins assigned to the lysosomal compartment and 3 lysosomal cargo receptors, of which most exhibited a significant differential abundance between control and CLN3-defective cells. Among these, 28 soluble lysosomal proteins catalyzing the degradation of various macromolecules had reduced levels in CLN3-defective cells. We confirmed these results by immunoblotting and selected protease and glycosidase activities. The reduction of 11 lipid-degrading lysosomal enzymes correlated with reduced capacity for lipid droplet degradation and several alterations in the distribution and composition of membrane lipids. In particular, levels of lactosylceramides and glycosphingolipids were decreased in CLN3-defective cells, which were also impaired in the recycling pathway of the exocytic transferrin receptor. Our findings suggest that CLN3 has a crucial role in regulating lysosome composition and their function, particularly in degrading of sphingolipids, and, as a consequence, in membrane transport along the recycling endosome pathway.

4.
Proc Natl Acad Sci U S A ; 116(16): 7963-7972, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30923110

RESUMO

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1-deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1-deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1-deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1-deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1-dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.

5.
Transl Psychiatry ; 9(1): 7, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30664629

RESUMO

In humans, genetic variants of DLGAP1-4 have been linked with neuropsychiatric conditions, including autism spectrum disorder (ASD). While these findings implicate the encoded postsynaptic proteins, SAPAP1-4, in the etiology of neuropsychiatric conditions, underlying neurobiological mechanisms are unknown. To assess the contribution of SAPAP4 to these disorders, we characterized SAPAP4-deficient mice. Our study reveals that the loss of SAPAP4 triggers profound behavioural abnormalities, including cognitive deficits combined with impaired vocal communication and social interaction, phenotypes reminiscent of ASD in humans. These behavioural alterations of SAPAP4-deficient mice are associated with dramatic changes in synapse morphology, function and plasticity, indicating that SAPAP4 is critical for the development of functional neuronal networks and that mutations in the corresponding human gene, DLGAP4, may cause deficits in social and cognitive functioning relevant to ASD-like neurodevelopmental disorders.


Assuntos
Transtorno do Espectro Autista/genética , Disfunção Cognitiva/genética , Proteínas do Tecido Nervoso/genética , Proteínas Associadas SAP90-PSD95/genética , Animais , Comportamento Animal , Modelos Animais de Doenças , Feminino , Relações Interpessoais , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Comportamento Social , Sinapses/metabolismo
6.
PLoS Pathog ; 14(12): e1007527, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30586431

RESUMO

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery-also named injectisome-assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.


Assuntos
Sistemas de Secreção Tipo III , Yersiniose/transmissão , Yersinia enterocolitica/patogenicidade , Humanos , Microscopia de Fluorescência
7.
Sci Transl Med ; 10(466)2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30404864

RESUMO

WNT1 mutations in humans are associated with a new form of osteogenesis imperfecta and with early-onset osteoporosis, suggesting a key role of WNT1 in bone mass regulation. However, the general mode of action and the therapeutic potential of Wnt1 in clinically relevant situations such as aging remain to be established. Here, we report the high prevalence of heterozygous WNT1 mutations in patients with early-onset osteoporosis. We show that inactivation of Wnt1 in osteoblasts causes severe osteoporosis and spontaneous bone fractures in mice. In contrast, conditional Wnt1 expression in osteoblasts promoted rapid bone mass increase in developing young, adult, and aged mice by rapidly increasing osteoblast numbers and function. Contrary to current mechanistic models, loss of Lrp5, the co-receptor thought to transmit extracellular WNT signals during bone mass regulation, did not reduce the bone-anabolic effect of Wnt1, providing direct evidence that Wnt1 function does not require the LRP5 co-receptor. The identification of Wnt1 as a regulator of bone formation and remodeling provides the basis for development of Wnt1-targeting drugs for the treatment of osteoporosis.

8.
Cell Rep ; 24(11): 2946-2956, 2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-30208319

RESUMO

Lysine glutarylation (Kglu) of mitochondrial proteins is associated with glutaryl-CoA dehydrogenase (GCDH) deficiency, which impairs lysine/tryptophan degradation and causes destruction of striatal neurons during catabolic crisis with subsequent movement disability. By investigating the role of Kglu modifications in this disease, we compared the brain and liver glutarylomes of Gcdh-deficient mice. In the brain, we identified 73 Kglu sites on 37 mitochondrial proteins involved in various metabolic degradation pathways. Ultrastructural immunogold studies indicated that glutarylated proteins are heterogeneously distributed in mitochondria, which are exclusively localized in glial cells. In liver cells, all mitochondria contain Kglu-modified proteins. Glutarylation reduces the catalytic activities of the most abundant glutamate dehydrogenase (GDH) and the brain-specific carbonic anhydrase 5b and interferes with GDH-protein interactions. We propose that Kglu contributes to the functional heterogeneity of mitochondria and may metabolically adapt glial cells to the activity and metabolic demands of neighboring GCDH-deficient neurons.

9.
Neuron ; 99(6): 1155-1169.e9, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30174115

RESUMO

Cellular prion protein (PrPC) modulates cell adhesion and signaling in the brain. Conversion to its infectious isoform causes neurodegeneration, including Creutzfeldt-Jakob disease in humans. PrPC undergoes rapid plasma membrane turnover and extracellular release via exosomes. However, the intracellular transport of PrPC and its potential impact on prion disease progression is barely understood. Here we identify critical components of PrPC trafficking that also link intracellular and extracellular PrPC turnover. PrPC associates with muskelin, dynein, and KIF5C at transport vesicles. Notably, muskelin coordinates bidirectional PrPC transport and facilitates lysosomal degradation over exosomal PrPC release. Muskelin gene knockout consequently causes PrPC accumulation at the neuronal surface and on secreted exosomes. Moreover, prion disease onset is accelerated following injection of pathogenic prions into muskelin knockout mice. Our data identify an essential checkpoint in PrPC turnover. They propose a novel connection between neuronal intracellular lysosome targeting and extracellular exosome trafficking, relevant to the pathogenesis of neurodegenerative conditions.

10.
J Bone Miner Res ; 33(12): 2186-2201, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30075049

RESUMO

Skeletal pathologies are frequently observed in lysosomal storage disorders, yet the relevance of specific lysosomal enzymes in bone remodeling cell types is poorly defined. Two lysosomal enzymes, ie, cathepsin K (Ctsk) and Acp5 (also known as tartrate-resistant acid phosphatase), have long been known as molecular marker proteins of differentiated osteoclasts. However, whereas the cysteine protease Ctsk is directly involved in the degradation of bone matrix proteins, the molecular function of Acp5 in osteoclasts is still unknown. Here we show that Acp5, in concert with Acp2 (lysosomal acid phosphatase), is required for dephosphorylation of the lysosomal mannose 6-phosphate targeting signal to promote the activity of specific lysosomal enzymes. Using an unbiased approach we identified the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), mutated in mucopolysaccharidosis type VI (MPS-VI), as an osteoclast marker, whose activity depends on dephosphorylation by Acp2 and Acp5. Similar to Acp2/Acp5-/- mice, Arsb-deficient mice display lysosomal storage accumulation in osteoclasts, impaired osteoclast activity, and high trabecular bone mass. Of note, the most prominent lysosomal storage accumulation was observed in osteocytes from Arsb-deficient mice, yet this pathology did not impair production of sclerostin (Sost) and Fgf23. Because the influence of enzyme replacement therapy (ERT) on bone remodeling in MPS-VI is still unknown, we additionally treated Arsb-deficient mice by weekly injection of recombinant human ARSB from 12 to 24 weeks of age. We found that the high bone mass phenotype of Arsb-deficient mice and the underlying bone cell deficits were fully corrected by ERT in the trabecular compartment. Taken together, our results do not only show that the function of Acp5 in osteoclasts is linked to dephosphorylation and activation of lysosomal enzymes, they also provide an important proof-of-principle for the feasibility of ERT to correct bone cell pathologies in lysosomal storage disorders. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

11.
Mol Cell Proteomics ; 17(8): 1612-1626, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29773673

RESUMO

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

12.
Cell Mol Life Sci ; 75(17): 3251-3267, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29520422

RESUMO

A disintegrin and metalloproteinase 10 (ADAM10) plays a major role in the ectodomain shedding of important surface molecules with physiological and pathological relevance including the amyloid precursor protein (APP), the cellular prion protein, and different cadherins. Despite its therapeutic potential, there is still a considerable lack of knowledge how this protease is regulated. We have previously identified tetraspanin15 (Tspan15) as a member of the TspanC8 family to specifically associate with ADAM10. Cell-based overexpression experiments revealed that this binding affected the maturation process and surface expression of the protease. Our current study shows that Tspan15 is abundantly expressed in mouse brain, where it specifically interacts with endogenous ADAM10. Tspan15 knockout mice did not reveal an overt phenotype but showed a pronounced decrease of the active and mature form of ADAM10, an effect which augmented with aging. The decreased expression of active ADAM10 correlated with an age-dependent reduced shedding of neuronal (N)-cadherin and the cellular prion protein. APP α-secretase cleavage and the expression of Notch-dependent genes were not affected by the loss of Tspan15, which is consistent with the hypothesis that different TspanC8s cause ADAM10 to preferentially cleave particular substrates. Analyzing spine morphology revealed no obvious differences between Tspan15 knockout and wild-type mice. However, Tspan15 expression was elevated in brains of an Alzheimer's disease mouse model and of patients, suggesting that upregulation of Tspan15 expression reflects a cellular response in a disease state. In conclusion, our data show that Tspan15 and most likely also other members of the TspanC8 family are central modulators of ADAM10-mediated ectodomain shedding in vivo.


Assuntos
Proteína ADAM10/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Tetraspaninas/genética , Proteína ADAM10/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Células Cultivadas , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Ligação Proteica , Ratos , Sinapses/metabolismo , Tetraspaninas/metabolismo
13.
Mol Psychiatry ; 2018 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-29467497

RESUMO

Atypical brain connectivity is a major contributor to the pathophysiology of neurodevelopmental disorders (NDDs) including autism spectrum disorders (ASDs). TAOK2 is one of several genes in the 16p11.2 microdeletion region, but whether it contributes to NDDs is unknown. We performed behavioral analysis on Taok2 heterozygous (Het) and knockout (KO) mice and found gene dosage-dependent impairments in cognition, anxiety, and social interaction. Taok2 Het and KO mice also have dosage-dependent abnormalities in brain size and neural connectivity in multiple regions, deficits in cortical layering, dendrite and synapse formation, and reduced excitatory neurotransmission. Whole-genome and -exome sequencing of ASD families identified three de novo mutations in TAOK2 and functional analysis in mice and human cells revealed that all the mutations impair protein stability, but they differentially impact kinase activity, dendrite growth, and spine/synapse development. Mechanistically, loss of Taok2 activity causes a reduction in RhoA activation, and pharmacological enhancement of RhoA activity rescues synaptic phenotypes. Together, these data provide evidence that TAOK2 is a neurodevelopmental disorder risk gene and identify RhoA signaling as a mediator of TAOK2-dependent synaptic development.

14.
Cell Rep ; 22(4): 1040-1053, 2018 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-29386126

RESUMO

Variants in the phospholipase D3 (PLD3) gene have genetically been linked to late-onset Alzheimer's disease. We present a detailed biochemical analysis of PLD3 and reveal its endogenous localization in endosomes and lysosomes. PLD3 reaches lysosomes as a type II transmembrane protein via a (for mammalian cells) uncommon intracellular biosynthetic route that depends on the ESCRT (endosomal sorting complex required for transport) machinery. PLD3 is sorted into intraluminal vesicles of multivesicular endosomes, and ESCRT-dependent sorting correlates with ubiquitination. In multivesicular endosomes, PLD3 is subjected to proteolytic cleavage, yielding a stable glycosylated luminal polypeptide and a rapidly degraded N-terminal membrane-bound fragment. This pathway closely resembles the delivery route of carboxypeptidase S to the yeast vacuole. Our experiments reveal a biosynthetic route of PLD3 involving proteolytic processing and ESCRT-dependent sorting for its delivery to lysosomes in mammalian cells.

15.
Biochim Biophys Acta Mol Cell Res ; 1864(11 Pt B): 2162-2168, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28693924

RESUMO

The Golgi-resident site-1 protease (S1P) is a key regulator of cholesterol homeostasis and ER stress responses by converting latent transcription factors sterol regulatory element binding proteins (SREPBs) and activating transcription factor 6 (ATF6), as well as viral glycoproteins to their active forms. S1P is also essential for lysosome biogenesis via proteolytic activation of the hexameric GlcNAc-1-phosphotransferase complex required for modification of newly synthesized lysosomal enzymes with the lysosomal targeting signal, mannose 6-phosphate. In the absence of S1P, the catalytically inactive α/ß-subunit precursor of GlcNAc-1-phosphotransferase fails to be activated and results in missorting of newly synthesized lysosomal enzymes, and lysosomal accumulation of non-degraded material, which are biochemical features of defective GlcNAc-1-phosphotransferase subunits and the associated pediatric lysosomal diseases mucolipidosis type II and III. The early embryonic death of S1P-deficient mice and the importance of various S1P-regulated biological processes, including lysosomal homeostasis, cautioned for clinical inhibition of S1P. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.


Assuntos
Colesterol/metabolismo , Mucolipidoses/genética , Pró-Proteína Convertases/genética , Proteólise , Serina Endopeptidases/genética , Animais , Colesterol/genética , Estresse do Retículo Endoplasmático/genética , Complexo de Golgi/metabolismo , Humanos , Lisossomos/genética , Camundongos , Mucolipidoses/patologia , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética
16.
Nat Neurosci ; 20(8): 1085-1095, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28604684

RESUMO

Nociception is an evolutionarily conserved mechanism to encode and process harmful environmental stimuli. Like most animals, Drosophila melanogaster larvae respond to a variety of nociceptive stimuli, including noxious touch and temperature, with stereotyped escape responses through activation of multimodal nociceptors. How behavioral responses to these different modalities are processed and integrated by the downstream network remains poorly understood. By combining trans-synaptic labeling, ultrastructural analysis, calcium imaging, optogenetics and behavioral analyses, we uncovered a circuit specific for mechanonociception but not thermonociception. Notably, integration of mechanosensory input from innocuous and nociceptive sensory neurons is required for robust mechanonociceptive responses. We further show that neurons integrating mechanosensory input facilitate primary nociceptive output by releasing short neuropeptide F, the Drosophila neuropeptide Y homolog. Our findings unveil how integration of somatosensory input and neuropeptide-mediated modulation can produce robust modality-specific escape behavior.


Assuntos
Comportamento Animal/fisiologia , Drosophila melanogaster/metabolismo , Nociceptores/metabolismo , Células Receptoras Sensoriais/metabolismo , Tato/fisiologia , Animais , Larva/metabolismo , Optogenética/métodos
17.
J Immunol ; 199(1): 172-185, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28550201

RESUMO

The invariant chain (CD74) mediates assembly and targeting of MHC class II (MHCII) complexes. In endosomes, CD74 undergoes sequential degradation by different proteases, including cathepsin S (CatS) and the intramembrane protease signal peptide peptidase-like 2a (SPPL2a). In their absence, CD74 N-terminal fragments (NTFs) accumulate. In SPPL2a-/- B cells, such an NTF impairs endosomal trafficking and BCR signal transduction. In mice, this leads to a loss of splenic B cells beyond the transitional stage 1. To gain insight into CD74 determinants and the role of MHCII, we compared B cells from CatS-/- , SPPL2a-/- , and SPPL2a-MHCII double-deficient mice. We assessed differentiation of B cells in bone marrow and spleen and analyzed their endosomal morphology, BCR expression, and signal transduction. We demonstrate that MHCII is dispensable for the B cell phenotype of SPPL2a-/- mice, further supporting a CD74-intrinsic effect. Despite significant vacuolization of endosomal compartments similar to SPPL2a-/- B cells, CatS-/- traditional stage 1 B cells show unimpaired degradation of endocytic cargo, have intact BCR signaling, and do not exhibit any relevant defects in maturation. This could indicate that CD74 NTF-induced structural changes of endosomes are not directly involved in these processes. We further found that the block of CD74 degradation in CatS-/- B cells is incomplete, so that NTF levels are significantly lower than in SPPL2a-/- B cells. This suggests a dose dependency and threshold for the CD74 NTF-associated impairment of B cell signaling and maturation. In addition, different functional properties of the longer, MHCII-bound CD74 NTF could contribute to the milder phenotype of CatS-/- B cells.


Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/imunologia , Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Ácido Aspártico Endopeptidases/deficiência , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Catepsinas/deficiência , Catepsinas/genética , Catepsinas/metabolismo , Diferenciação Celular , Endossomos/imunologia , Endossomos/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Transdução de Sinais
18.
EMBO Rep ; 18(6): 962-981, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28420656

RESUMO

Oligomeric amyloid-ß (Aß) 1-42 disrupts synaptic function at an early stage of Alzheimer's disease (AD). Multiple posttranslational modifications of Aß have been identified, among which N-terminally truncated forms are the most abundant. It is not clear, however, whether modified species can induce synaptic dysfunction on their own and how altered biochemical properties can contribute to the synaptotoxic mechanisms. Here, we show that a prominent isoform, pyroglutamated Aß3(pE)-42, induces synaptic dysfunction to a similar extent like Aß1-42 but by clearly different mechanisms. In contrast to Aß1-42, Aß3(pE)-42 does not directly associate with synaptic membranes or the prion protein but is instead taken up by astrocytes and potently induces glial release of the proinflammatory cytokine TNFα. Moreover, Aß3(pE)-42-induced synaptic dysfunction is not related to NMDAR signalling and Aß3(pE)-42-induced impairment of synaptic plasticity cannot be rescued by D1-agonists. Collectively, the data point to a scenario where neuroinflammatory processes together with direct synaptotoxic effects are caused by posttranslational modification of soluble oligomeric Aß and contribute synergistically to the onset of synaptic dysfunction in AD.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Sinapses/fisiologia , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Animais , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neuroimunomodulação , Plasticidade Neuronal , Fragmentos de Peptídeos/genética , Isoformas de Proteínas , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Fator de Necrose Tumoral alfa/biossíntese
19.
Hum Mol Genet ; 26(9): 1678, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334871

RESUMO

Mutations in the depalmitoylation enzyme, palmitoyl protein thioesterase (PPT1), result in the early onset neurodegenerative disease known as Infantile Neuronal Ceroid Lipofuscinosis. Here, we provide proteomic evidence suggesting that PPT1 deficiency could be considered as a ciliopathy. Analysis of membrane proteins from brain enriched for acylated proteins from neonate Ppt1 knock out and control mice revealed a list of 88 proteins with differential expression levels. Amongst them, we identified Rab3IP, which regulates ciliogenesis in concert with Rab8 and Rab11. Immunostaining analysis revealed that PPT1 is localized in the cilia. Indeed, an unbiased proteomics analysis on isolated cilia revealed 660 proteins, which differed in their abundance levels between wild type and Ppt1 knock out. We demonstrate here that Rab3IP, Rab8 and Rab11 are palmitoylated, and that palmitoylation of Rab11 is required for correct intracellular localization. Cells and brain preparations from Ppt1-/- mice exhibited fewer cells with cilia and abnormally longer cilia, with both acetylated tubulin and Rab3IP wrongly distributed along the length of cilia. Most importantly, the analysis revealed a difference in the distribution and levels of the modified proteins in cilia in the retina of mutant mice versus the wildtype, which may be important in the early neurodegenerative phenotype. Overall, our results suggest a novel link between palmitoylated proteins, cilial organization and the pathophysiology of Neuronal Ceroid Lipofuscinosis.


Assuntos
Proteínas de Membrana/fisiologia , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Animais , Encéfalo/metabolismo , Cílios/metabolismo , Cílios/patologia , Células HEK293 , Humanos , Lipoilação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutação , Células NIH 3T3 , Neurônios/metabolismo , Proteômica/métodos , Retina/metabolismo , Tioléster Hidrolases/deficiência
20.
Small ; 13(3)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28084694

RESUMO

Osteocytes-the central regulators of bone remodeling-are enclosed in a network of microcavities (lacunae) and nanocanals (canaliculi) pervading the mineralized bone. In a hitherto obscure process related to aging and disease, local plugs in the lacuno-canalicular network disrupt cellular communication and impede bone homeostasis. By utilizing a suite of high-resolution imaging and physics-based techniques, it is shown here that the local plugs develop by accumulation and fusion of calcified nanospherites in lacunae and canaliculi (micropetrosis). Two distinctive nanospherites phenotypes are found to originate from different osteocytic elements. A substantial deviation in the spherites' composition in comparison to mineralized bone further suggests a mineralization process unlike regular bone mineralization. Clearly, mineralization of osteocyte lacunae qualifies as a strong marker for degrading bone material quality in skeletal aging. The understanding of micropetrosis may guide future therapeutics toward preserving osteocyte viability to maintain mechanical competence and fracture resistance of bone in elderly individuals.


Assuntos
Envelhecimento/patologia , Osso e Ossos/patologia , Calcificação Fisiológica , Nanosferas/química , Osteopetrose/patologia , Idoso de 80 Anos ou mais , Matriz Óssea/ultraestrutura , Feminino , Humanos , Nanosferas/ultraestrutura , Osteócitos/ultraestrutura
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