RESUMO
Chronic ankle instability (CAI) is associated with altered energy dissipation patterns, but comparisons to lateral ankle sprain (LAS) copers have not been explored. The purpose of this study was to examine differences in relative sagittal plane energy dissipation during a single-leg landing between female CAI and LAS coper participants. We separated 33 females (23.6 ± 4.6 years, 164.3 ± 6.2 cm, 69.4 ± 13.7 kg) into CAI (n = 17) and LAS coper (n = 16) groups. Participants completed 5 single-leg landings followed by a 5-second stabilization. We collected sagittal plane kinematics and joint moments at the ankle, knee, hip, and proximal joints (knee and hip) combined then calculated each joint's energy dissipation at 50, 100, 150, and 200 ms post-landing. We compared the percentage of total energy dissipated by the ankle, knee, hip, and proximal joints during each interval utilizing independent t tests and Cohen's d effect sizes. Statistical significance was set a priori at P < .05. The CAI group had lower relative energy dissipation from the ankle at 50 (24.7 ± 11.5% vs 39.2 ± 11.8%, P < .01, d = 1.25 [0.47, 1.95]), 100 (66.9 ± 19.4% vs 77.7 ± 6.5%, P = .04, d = 0.74 [0.01, 1.42]), and 150 ms (70.7 ± 17.8% vs 81.0 ± 5.7%, P = .03, d = 0.77 [0.04, 1.46]) compared to LAS copers. The CAI group had a greater hip contribution through 150 ms (17.9 ± 10.7% vs 11.7 ± 4.4%, P = .04, d =-0.75 [-1.44, -0.03]) and the proximal joints at 50 (75.3 ± 11.5% vs 60.8 ± 11.8%, P < .01, d = -1.25 [-1.96, -0.47]), 100 (33.1 ± 19.4% vs 22.3 ± 6.5%, P = .04, d = -0.74 [-1.42, -0.01]), and 150 ms (29.3 ± 17.8 vs 19.0 ± 5.7%, P = .03, d =-0.77 [-1.46, -0.04]) compared to LAS copers. Females with CAI may benefit from therapeutic exercises designed to correct a single-leg energy dissipation strategy that relies less on the ankle joint.
Assuntos
Traumatismos do Tornozelo/fisiopatologia , Articulação do Tornozelo/fisiopatologia , Instabilidade Articular/fisiopatologia , Adulto , Fenômenos Biomecânicos , Estudos Transversais , Teste de Esforço , Feminino , Humanos , Adulto JovemRESUMO
We have sequenced the complete genomes of 72 individuals affected with early-onset familial Alzheimer's disease caused by an autosomal dominant, highly penetrant mutation in the presenilin-1 (PSEN1) gene, and performed genome-wide association testing to identify variants that modify age at onset (AAO) of Alzheimer's disease. Our analysis identified a haplotype of single-nucleotide polymorphisms (SNPs) on chromosome 17 within a chemokine gene cluster associated with delayed onset of mild-cognitive impairment and dementia. Individuals carrying this haplotype had a mean AAO of mild-cognitive impairment at 51.0 ± 5.2 years compared with 41.1 ± 7.4 years for those without these SNPs. This haplotype thus appears to modify Alzheimer's AAO, conferring a large (~10 years) protective effect. The associated locus harbors several chemokines including eotaxin-1 encoded by CCL11, and the haplotype includes a missense polymorphism in this gene. Validating this association, we found plasma eotaxin-1 levels were correlated with disease AAO in an independent cohort from the University of California San Francisco Memory and Aging Center. In this second cohort, the associated haplotype disrupted the typical age-associated increase of eotaxin-1 levels, suggesting a complex regulatory role for this haplotype in the general population. Altogether, these results suggest eotaxin-1 as a novel modifier of Alzheimer's disease AAO and open potential avenues for therapy.
Assuntos
Doença de Alzheimer/genética , Quimiocina CCL11/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idade de Início , Idoso , Doença de Alzheimer/sangue , Doença de Alzheimer/complicações , Quimiocina CCL11/sangue , Cromossomos Humanos Par 17/genética , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/genética , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The literature on GWAS (genome-wide association studies) data suggests that very large sample sizes (for example, 50,000 cases and 50,000 controls) may be required to detect significant associations of genomic regions for complex disorders such as Alzheimer's disease (AD). Because of the challenges of obtaining such large cohorts, we describe here a novel sequential strategy that combines pooling of DNA and bootstrapping (pbGWAS) in order to significantly increase the statistical power and exponentially reduce expenses. We applied this method to a very homogeneous sample of patients belonging to a unique and clinically well-characterized multigenerational pedigree with one of the most severe forms of early onset AD, carrying the PSEN1 p.Glu280Ala mutation (often referred to as E280A mutation), which originated as a consequence of a founder effect. In this cohort, we identified novel loci genome-wide significantly associated as modifiers of the age of onset of AD (CD44, rs187116, P=1.29 × 10⻹²; NPHP1, rs10173717, P=1.74 × 10⻹²; CADPS2, rs3757536, P=1.54 × 10⻹°; GREM2, rs12129547, P=1.69 × 10⻹³, among others) as well as other loci known to be associated with AD. Regions identified by pbGWAS were confirmed by subsequent individual genotyping. The pbGWAS methodology and the genes it targeted could provide important insights in determining the genetic causes of AD and other complex conditions.
Assuntos
Alanina/genética , Doença de Alzheimer/genética , Predisposição Genética para Doença , Ácido Glutâmico/genética , Presenilina-1/genética , Idade de Início , Doença de Alzheimer/epidemiologia , Estudos de Coortes , Bases de Dados Factuais/estatística & dados numéricos , Feminino , Efeito Fundador , Frequência do Gene , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Mutação/genética , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
PDZ domains are protein-protein interaction modules widely used to assemble membranous signaling complexes including those found in the neuronal synapse. PDZ-containing genes encoded in metazoan genomes vastly outnumber those in prokaryotes, plants, and fungi. By comparing 40 proteomes to track the evolutionary history of the PDZ domain, we observed that the variety of associations between PDZ and other domains expands greatly along the stem leading to metazoans and choanoflagellates. We asked whether the expansion of PDZ domains was due to random or specific sequence changes. Studying the sequence signatures of 58 PDZ lineages that are common to bilaterian animals, we showed that six common amino acid residues are able to classify 96% of PDZ domains to their correct evolutionary lineage. In PDZ domain-ligand cocrystals, four of these "classifying positions" lie in direct contact with the -1 and -3 residues of the ligand. This suggests coevolution of the more flexible regions of the binding interaction as a central mechanism of specialization inherent within the PDZ domain. To identify these positions, we devised two independent algorithms--a metric termed within-clade entropy (WCE) and an average mutual information (AvgMI) score--that both reached similar results. Extending these tools to the choanoflagellate, Monosiga brevicollis, we compared its PDZ domains with their putative metazoan orthologs. Interestingly, the M. brevicollis genes lack conservation at the classifying positions suggesting dissociation between domain organization in multidomain proteins and specific changes within the PDZ domain.
Assuntos
Evolução Molecular , Domínios PDZ/genética , Sequência de Aminoácidos , Aminoácidos/genética , Animais , Coanoflagelados/metabolismo , Simulação por Computador , Sequência Conservada , Entropia , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Alinhamento de SequênciaRESUMO
Ordered assembly of the amyloid-beta protein (A beta) into amyloid fibrils is a critical step in Alzheimer's disease (AD). To release the amyloidogenic peptide A beta from the Alzheimer amyloid precursor protein (APP), two secretases act sequentially: first, beta-secretase cleaves close to the membrane within the ectodomain and then gamma-secretase cuts within the transmembrane domain. The sites of gamma-secretase cleavage are after residues 40 or 42 of A beta. Except in those rare cases of AD caused by a mutation, levels of secreted A beta are not elevated; thus, the secretory pathway may be unaffected, and factors other than the extracellular concentration of A beta may contribute to the aggregation properties of the peptide. A beta is also present in intracellular compartments. The two gamma-secretase cleavage products, A beta42 and A beta40, were found in different compartments: A beta42 in the endoplasmic reticulum (ER)/intermediate compartment, and A beta40 in the trans-Golgi network (TGN). The cellular compartments that harbor A beta are target sites for therapeutic intervention. Here we report that in the brain, the principal compartment in which A beta resides is a detergent-insoluble glycolipid-enriched membrane domain (DIG). Also present in the DIG fractions are the endoproteolytic fragments of presenilin-1 (PS1) and APP. The presence of these proteins, which all contribute to the generation of A beta, indicates that the DIG fraction is probably where the intramembranous cleavage of APP occurs.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Caveolinas , Compartimento Celular , Membrana Celular/química , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Química Encefálica , Células CHO , Caveolina 1 , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Colesterol/metabolismo , Cricetinae , Detergentes , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Glicolipídeos/metabolismo , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Proteínas de Membrana/análise , Ratos , Ratos Sprague-Dawley , Solubilidade , Frações Subcelulares/química , Frações Subcelulares/metabolismoRESUMO
Missense mutations in the presenilin 1 (PS1) gene cause the most common form of dominant early-onset familial Alzheimer's disease (FAD) and are associated with increased levels of amyloid beta-peptides (A beta) ending at residue 42 (A beta 42) in plasma and skin fibroblast media of gene carriers. A beta 42 aggregates readily and appears to provide a nidus for the subsequent aggregation of A beta 40 (ref. 4), resulting in the formation of innumerable neuritic plaques. To obtain in vivo information about how PS1 mutations cause AD pathology at such early ages, we characterized the neuropathological phenotype of four PS1-FAD patients from a large Colombian kindred bearing the codon 280 Glu to Ala substitution (Glu280Ala) PS1 mutation. Using antibodies specific to the alternative carboxy-termini of A beta, we detected massive deposition of A beta 42, the earliest and predominant form of plaque A beta to occur in AD (ref. 6-8), in many brain regions. Computer-assisted quantification revealed a significant increase in A beta 42, but not A beta 40, burden in the brains from 4 PS1-FAD patients compared with those from 12 sporadic AD patients. Severe cerebellar pathology included numerous A beta 42-reactive plaques, many bearing dystrophic neurites and reactive glia. Our results in brain tissue are consistent with recent biochemical evidence of increased A beta 42 levels in PS1-FAD patients and strongly suggest that mutant PS1 proteins alter the proteolytic processing of the beta-amyloid precursor protein at the C-terminus of A beta to favor deposition of A beta 42.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Cerebelo/metabolismo , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Mutação Puntual , Idade de Início , Idoso , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Química Encefálica , Cerebelo/patologia , Códon/genética , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Proteínas de Membrana/fisiologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/fisiologia , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/patologia , Presenilina-1RESUMO
The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)-positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi-associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin-membrane interactions may be of key importance for determining the intracellular distribution of selected organelles.
Assuntos
Hipocampo/fisiologia , Cinesinas/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Organelas/fisiologia , Células Piramidais/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Sequência de Bases , Brefeldina A , Carbocianinas , Células Cultivadas , Ciclopentanos/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Corantes Fluorescentes , Complexo de Golgi/fisiologia , Complexo de Golgi/ultraestrutura , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neuritos/ultraestrutura , Organelas/efeitos dos fármacos , Organelas/ultraestrutura , Inibidores da Síntese de Proteínas/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/ultraestrutura , Rodamina 123 , RodaminasRESUMO
We have indirectly analyzed the role of tau in generating the highly organized microtubule (MT) array of the axon. Axons contain MT arrays of uniform polarity orientation, plus ends distal to the cell body (Heidemann, S. R., J. M. Landers, and M. A. Hamborg. 1981. J. Cell Biol. 91:661-673). Surprisingly, these MTs do not radiate from a single discrete nucleating structure in the cell body (Sharp, G. A., K. Weber, and M. Osborn. 1982. Eur. J. Cell Biol. 29: 97-103), but rather stop and start at multiple sites along the length of the axon (Bray, D., and M. B. Bunge. 1981. J. Neurocytol. 10:589-605). When Sf9 ovarian cells are induced to express high levels of tau protein, they develop cellular processes which are similar in appearance to axons and which contain dense arrays of MTs (Knops, J., K. S. Kosik, G. Lee, J. D. Pardee, L. Cohen-Gould, and L. McConlogue. 1991. J. Cell Biol. 114:725-734). We have analyzed the organization of MTs within these arrays, and determined it to be similar, but not identical, to the organization of MTs within the axon. The caliber, MT number, and MT density vary significantly from process to process, but on average are manyfold higher in the tau-induced processes than typically found in axons. Greater than 89% of the MTs in the processes are oriented with their plus ends distal to the cell body, and this proportion is even higher in the processes that are most similar to axons with regard to caliber, MT number, and MT density. Similar to the situation in the axon, MTs are discontinuous along the length of the tau-induced processes, and do not emanate from any observable nucleating structure in the cell body. We have also identified bundles of MTs throughout the cell bodies of the Sf9 cells induced to express tau. Similar to the MT arrays in the processes, these MT bundles are not visibly associated with any other cytological structures that might regulate their polarity orientation. Nevertheless, these bundles consist of MTs most (greater than 82%) of which have the same polarity orientation. Collectively, these results suggest that tau may play a fundamental role in generating MT organization in the axon. In particular, a key property of tau may be to bundle MTs preferentially with the same polarity orientation.
Assuntos
Axônios/ultraestrutura , Microtúbulos/metabolismo , Proteínas tau/metabolismo , Linhagem Celular , Expressão Gênica , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Proteínas tau/genéticaRESUMO
Growth cones extend dynamic protrusions called filopodia and lamellipodia as exploratory probes that signal the direction of neurite growth. Gelsolin, as an actin filament-severing protein, may serve an important role in the rapid shape changes associated with growth cone structures. In wild-type (wt) hippocampal neurons, antibodies against gelsolin labeled the neurite shaft and growth cone. The behavior of filopodia in cultured hippocampal neurons from embryonic day 17 wt and gelsolin null (Gsn-) mice (Witke, W., A.H. Sharpe, J.H. Hartwig, T. Azuma, T.P. Stossel, and D.J. Kwiatkowski. 1995. Cell. 81:41-51.) was recorded with time-lapse video microscopy. The number of filopodia along the neurites was significantly greater in Gsn- mice and gave the neurites a studded appearance. Dynamic studies suggested that most of these filopodia were formed from the region of the growth cone and remained as protrusions from the newly consolidated shaft after the growth cone advanced. Histories of individual filopodia in Gsn- mice revealed elongation rates that did not differ from controls but an impaired retraction phase that probably accounted for the increased number of filopodia long the neutrite shaft. Gelsolin appears to function in the initiation of filopodial retraction and in its smooth progression.
Assuntos
Gelsolina/genética , Camundongos Knockout/fisiologia , Neuritos/fisiologia , Animais , Tamanho Celular/fisiologia , Células Cultivadas , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Vídeo , Neuritos/ultraestrutura , Neurônios/química , Neurônios/fisiologia , Neurônios/ultraestrutura , Pseudópodes/fisiologiaRESUMO
In the present study, we present evidence about the cellular functions of KIF2, a kinesin-like superfamily member having a unique structure in that its motor domain is localized at the center of the molecule (Noda Y., Y. Sato-Yoshitake, S. Kondo, M. Nangaku, and N. Hirokawa. 1995. J. Cell Biol. 129:157-167.). Using subcellular fractionation techniques, isopicnic sucrose density centrifugation of microsomal fractions from developing rat cerebral cortex, and immunoisolation with KIF2 antibodies, we have now identified a type of nonsynaptic vesicle that associates with KIF2. This type of organelle lacks synaptic vesicle markers (synapsin, synaptophysin), amyloid precursor protein, GAP-43, or N-cadherin. On the other hand, it contains betagc, which is a novel variant of the beta subunit of the IGF-1 receptor, which is highly enriched in growth cone membranes. Both betagc and KIF2 are upregulated by NGF in PC12 cells and highly concentrated in growth cones of developing neurons. We have also analyzed the consequences of KIF2 suppression by antisense oligonucleotide treatment on nerve cell morphogenesis and the distribution of synaptic and nonsynaptic vesicle markers. KIF2 suppression results in a dramatic accumulation of betagc within the cell body and in its complete disappearance from growth cones; no alterations in the distribution of synapsin, synaptophysin, GAP-43, or amyloid percursor protein are detected in KIF2-suppressed neurons. Instead, all of them remained highly enriched at nerve terminals. KIF2 suppression also produces a dramatic inhibition of neurite outgrowth; this phenomenon occurs after betagc has disappeared from growth cones. Taken collectively, our results suggest an important role for KIF2 in neurite extension, a phenomenon that may be related with the anterograde transport of a type of nonsynaptic vesicle that contains as one of its components a growth cone membrane receptor for IGF-1, a growth factor implicated in nerve cell development.
Assuntos
Cinesinas/fisiologia , Neuritos/fisiologia , Neurônios/fisiologia , Organelas/metabolismo , Precursor de Proteína beta-Amiloide/análise , Animais , Anticorpos Monoclonais , Córtex Cerebral/química , Proteína GAP-43 , Cinesinas/análise , Cinesinas/genética , Cinesinas/imunologia , Glicoproteínas de Membrana/análise , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/análise , Neurônios/citologia , Oligonucleotídeos Antissenso/farmacologia , Organelas/química , Células PC12 , Ratos , Receptor IGF Tipo 1/análise , Frações Subcelulares/química , Sinapsinas/análise , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Sinaptofisina/análise , Tionucleotídeos/farmacologia , Regulação para CimaRESUMO
In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin-radixin or ezrin-moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin-moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8-10 times slower than the one of growth cones from control neurons. Radixin-moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phenomenon that appears to be directly linked with the alterations in growth cone structure and motility. Taken collectively, our data suggest that by regulating key aspects of growth cone development and maintenance, radixin and moesin modulate neurite formation and the development of neuronal polarity.
Assuntos
Proteínas Sanguíneas/genética , Proteínas do Citoesqueleto , Cones de Crescimento/fisiologia , Proteínas de Membrana/genética , Proteínas dos Microfilamentos , Proteínas/genética , Células Piramidais/citologia , Actinas/metabolismo , Animais , Elementos Antissenso (Genética) , Proteínas Sanguíneas/metabolismo , Polaridade Celular/fisiologia , Células Cultivadas , Citoesqueleto/fisiologia , Expressão Gênica/fisiologia , Cones de Crescimento/química , Hipocampo/citologia , Proteínas de Membrana/metabolismo , Neuritos/química , Neuritos/fisiologia , Proteínas/metabolismo , Células Piramidais/química , Células Piramidais/ultraestrutura , Ratos , Frações Subcelulares/química , TionucleotídeosRESUMO
Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP-43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.
Assuntos
Hipocampo/metabolismo , Cinesinas/biossíntese , Morfogênese/efeitos dos fármacos , Neurônios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Animais , Sequência de Bases , Transporte Biológico Ativo , Polaridade Celular , Células Cultivadas , Proteína GAP-43 , Hipocampo/efeitos dos fármacos , Imuno-Histoquímica , Cinesinas/efeitos dos fármacos , Cinesinas/isolamento & purificação , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Sinapsinas/metabolismoRESUMO
The ways in which the various microtubule-associated proteins (MAPs) contribute to cellular function are unknown beyond the ability of these proteins to modify microtubule dynamics. One member of the MAP family, tau protein, is restricted in its distribution to the axonal compartment of neurons, and has therefore prompted studies that attempt to relate tau function to the generation or maintenance of this structure. Sf9 cells from a moth ovary, when infected with a baculovirus containing a tau cDNA insert, elaborate very long processes. This single gene product expressed in a foreign host cell grossly alters the normal rounded morphology of these cells. The slender, relatively nonbranched appearance of these processes as well as their uniform caliber resembles the light-microscopic appearance of axons observed in several neuronal culture systems. Immunolabeling of the tau-expressing Sf9 cells demonstrated tau reactivity in the induced processes, and EM that microtubule bundles were present in the processes. Microtubule stabilization alone was insufficient to generate processes, since taxol treatment did not alter the overall cell shape, despite the induction of microtubule bundling within the cell body.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Proteínas do Tecido Nervoso/fisiologia , Alcaloides/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Baculoviridae/genética , Linhagem Celular , Citocalasina D/farmacologia , Vetores Genéticos , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/efeitos dos fármacos , Mariposas , Paclitaxel , Reação em Cadeia da Polimerase , Transfecção , Proteínas tauRESUMO
The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.
Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Melanoma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Humanos , Melanoma/patologia , Proteínas dos Microfilamentos/efeitos dos fármacos , Microinjeções , Microtúbulos/efeitos dos fármacos , Células Tumorais Cultivadas , Proteínas tau/farmacologiaRESUMO
The classical adherens junction that holds epithelial cells together consists of a protein complex in which members of the cadherin family linked to various catenins are the principal components. delta-catenin is a mammalian brain protein in the Armadillo repeat superfamily with sequence similarity to the adherens junction protein p120(ctn). We found that delta-catenin can be immunoprecipitated as a complex with other components of the adherens junction, including cadherin and beta-catenin, from transfected cells and brain. The interaction with cadherin involves direct contact within the highly conserved juxtamembrane region of the COOH terminus, where p120(ctn) also binds. In developing mouse brain, staining with delta-catenin antibodies is prominent towards the apical boundary of the neuroepithelial cells in the ventricular zone. When transfected into Madin-Darby canine kidney (MDCK) epithelial cells delta-catenin colocalized with cadherin, p120(ctn), and beta-catenin. The Arm domain alone was sufficient for achieving localization and coimmunoprecipitation with cadherin. The ectopic expression of delta-catenin in MDCK cells altered their morphology, induced the elaboration of lamellipodia, interfered with monolayer formation, and increased scattering in response to hepatocyte growth factor treatment. We propose that delta-catenin can regulate adhesion molecules to implement the organization of large cellular arrays necessary for tissue morphogenesis.
Assuntos
Movimento Celular/fisiologia , Proteínas de Drosophila , Proteínas do Tecido Nervoso/fisiologia , Transativadores , Animais , Proteínas do Domínio Armadillo , Sequência de Bases , Sítios de Ligação/genética , Encéfalo/metabolismo , Caderinas/genética , Caderinas/isolamento & purificação , Caderinas/fisiologia , Cateninas , Adesão Celular/fisiologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/fisiologia , Linhagem Celular , Tamanho Celular/fisiologia , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/isolamento & purificação , Proteínas do Citoesqueleto/fisiologia , Primers do DNA/genética , Cães , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fosfoproteínas , Transfecção , beta Catenina , delta CateninaRESUMO
An almost bewildering number of findings concerning Alzheimer's disease mask the significant recent progress in understanding the molecular basis of some inherited forms of this disease and the proteolytic processing of proteins related to the disease. Alzheimer's disease is an amyloidosis, a condition in which certain proteins or protein fragments precipitate in various tissues as amyloid, fibrillar aggregates with a beta-pleated sheet conformation. Alzheimer's is also characterized by neuritic lesions and cell death. Some rare forms of the disease are now known to arise from a mutation in an amyloidogenic protein. Another recent insight is the discovery of an endosomal-lysosomal processing pathway capable of generating protein fragments that can deposit extracellularly as amyloid fibrils. Key future directions for cellular-based research in Alzheimer's disease include the study of membrane trafficking and the passage of intracellular material to the extracellular milieu, molecular signaling among intracellular compartments, the interaction between organelles and the neuronal cytoskeleton, and the nature of cytoskeletal reorganization after neuronal injury.
Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Encéfalo/patologia , Glicosilação , Humanos , Dados de Sequência Molecular , Neuritos/ultraestrutura , Emaranhados Neurofibrilares/ultraestrutura , TransfecçãoRESUMO
Synapsin I and synapsin II are neuron-specific phosphoproteins that have a role in the regulation of neurotransmitter release and in the formation of nerve terminals. After depletion of synapsin II by antisense oligonucleotides, rat hippocampal neurons in culture were unable to consolidate their minor processes and did not elongate axons. These aberrant morphological changes were accompanied by an abnormal distribution of intracellular filamentous actin (F-actin). In addition, synapsin II suppression resulted in a selective decrease in the amounts of several synaptic vesicle-associated proteins. These data suggest that synapsin II participates in cytoskeletal organization during the early stages of nerve cell development.
Assuntos
Proteínas de Ligação ao Cálcio , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/fisiologia , Neurônios/citologia , Sinapsinas/metabolismo , Sinaptofisina/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Hipocampo/citologia , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Neurônios/metabolismo , Neurônios/ultraestrutura , Oligonucleotídeos Antissenso/farmacologia , Ratos , Sinapsinas/genética , Sinaptotagminas , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismoRESUMO
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD.
Assuntos
Encéfalo/metabolismo , Ligases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/metabolismo , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Ubiquitinas/metabolismo , Encéfalo/enzimologia , Tronco Encefálico/enzimologia , Tronco Encefálico/metabolismo , Linhagem Celular , Detergentes , Congelamento , Glicosilação , Humanos , Corpos de Lewy/enzimologia , Corpos de Lewy/metabolismo , Ligases/genética , Mutação de Sentido Incorreto , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Transtornos Parkinsonianos/enzimologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismo , Especificidade por Substrato , Sinucleínas , alfa-SinucleínaRESUMO
The study of individuals with autosomal dominant Alzheimer's disease affords one of the best opportunities to characterize the biological and cognitive changes of Alzheimer's disease that occur over the course of the preclinical and symptomatic stages. Unifying the knowledge gained from the past three decades of research in the world's largest single-mutation autosomal dominant Alzheimer's disease kindred - a family in Antioquia, Colombia with the E280A mutation in the Presenilin1 gene - will provide new directions for Alzheimer's research and a framework for generalizing the findings from this cohort to the more common sporadic form of Alzheimer's disease. As this specific mutation is virtually 100% penetrant for the development of the disease by midlife, we use a previously defined median age of onset for mild cognitive impairment for this cohort to examine the trajectory of the biological and cognitive markers of the disease as a function of the carriers' estimated years to clinical onset. Studies from this cohort suggest that structural and functional brain abnormalities - such as cortical thinning and hyperactivation in memory networks - as well as differences in biofluid and in vivo measurements of Alzheimer's-related pathological proteins distinguish Presenilin1 E280A mutation carriers from non-carriers as early as childhood, or approximately three decades before the median age of onset of clinical symptoms. We conclude our review with discussion on future directions for Alzheimer's disease research, with specific emphasis on ways to design studies that compare the generalizability of research in autosomal dominant Alzheimer's disease to the larger sporadic Alzheimer's disease population.
Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/diagnóstico por imagem , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Adolescente , Adulto , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Compostos de Anilina , Doenças Assintomáticas , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Criança , Colômbia , Imagem de Tensor de Difusão , Progressão da Doença , Eletroencefalografia , Etilenoglicóis , Feminino , Neuroimagem Funcional , Humanos , Imageamento por Ressonância Magnética , Masculino , Testes de Estado Mental e Demência , Pessoa de Meia-Idade , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/líquido cefalorraquidiano , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Tomografia Computadorizada de Emissão de Fóton Único , Adulto JovemRESUMO
We show here that antisense MAP2 oligonucleotides inhibit neurite outgrowth in cultured cerebellar macroneurons. Unlike control neurons, which first extend a lamellipodial veil followed by a consolidation phase during which the cells extend minor neurites, MAP2-suppressed cells persist with lamellipodia and later become rounded. The induction of microtubules containing tyrosinated tubulin, which parallels neurite outgrowth in control neurons, was blocked under antisense conditions. The small but significant increase in acetylated microtubules was not affected. In contrast, the suppression of tau, which selectively blocks axonal elongation, completely prevented the increase of acetylated microtubules, but did not modify the induction of labile microtubules. These results suggest that MAP2 and tau have different functions: the initial establishment of neurites depends upon MAP2, whereas further neurite elongation depends upon tau and microtubule stabilization.