RESUMO
Polyglutamylation is a dynamic posttranslational modification where glutamate residues are added to substrate proteins by 8 tubulin tyrosine ligase-like (TTLL) family members (writers) and removed by the 6 member Nna1/CCP family of carboxypeptidases (erasers). Genetic disruption of polyglutamylation leading to hyperglutamylation causes neurodegenerative phenotypes in humans and animal models; the best characterized being the Purkinje cell degeneration (pcd) mouse, a mutant of the gene encoding Nna1/CCP1, the prototypic eraser. Emphasizing the functional importance of the balance between glutamate addition and elimination, loss of TTLL1 prevents Purkinje cell degeneration in pcd. However, whether Ttll1 loss protects other vulnerable neurons in pcd, or if elimination of other TTLLs provides protection is largely unknown. Here using a mouse genetic rescue strategy, we characterized the contribution of Ttll1, 4, 5, 7, or 11 to the degenerative phenotypes in cerebellum, olfactory bulb and retinae of pcd mutants. Ttll1 deficiency attenuates Purkinje cell loss and function and reduces olfactory bulb mitral cell death and retinal photoreceptor degeneration. Moreover, degeneration of photoreceptors in pcd is preceded by impaired rhodopsin trafficking to the rod outer segment and likely represents the causal defect leading to degeneration as this too is rescued by elimination of TTLL1. Although TTLLs have similar catalytic properties on model substrates and several are highly expressed in Purkinje cells (e.g. TTLL5 and 7), besides TTLL1 only TTLL4 deficiency attenuated degeneration of Purkinje and mitral cells in pcd. Additionally, TTLL4 loss partially rescued photoreceptor degeneration and impaired rhodopsin trafficking. Despite their common properties, the polyglutamylation profile changes promoted by TTLL1 and TTLL4 deficiencies in pcd mice are very different. We also report that loss of anabolic TTLL5 synergizes with loss of catabolic Nna1/CCP1 to promote photoreceptor degeneration. Finally, male infertility in pcd is not rescued by loss of any Ttll. These data provide insight into the complexity of polyglutamate homeostasis and function in vivo and potential routes to ameliorate disorders caused by disrupted polyglutamylation.
Assuntos
Células de Purkinje , Degeneração Retiniana , Animais , Proteínas de Ligação ao GTP/genética , Ácido Glutâmico/metabolismo , Masculino , Fenótipo , Células de Purkinje/metabolismo , Degeneração Retiniana/metabolismo , Rodopsina/genéticaRESUMO
Nna1 (CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1-6) and is mutated in pcd (Purkinje cell degeneration) mice. Nna1, CCP4, and CCP6 are involved in the post-translational process of polyglutamylation, where they catalyze the removal of polyglutamate side chains. However, it is unknown whether these three cytosolic carboxypeptidases share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescues Purkinje cell degeneration in pcd mice, indicating that they do not have identical functions. Using biotin-based synthetic substrates, we established that the three enzymes are distinguishable based upon individual preferences for glutamate chain length, the amino acid immediately adjacent to the glutamate chain, and whether their activity is enhanced by nearby acidic amino acids. Nna1 and CCP4 remove the C-terminal glutamate from substrates with two or more glutamates, whereas CCP6 requires four or more glutamates. CCP4 behaves as a promiscuous glutamase, with little preference for chain length or neighboring amino acid composition. Besides glutamate chain length dependence, Nna1 and CCP6 exhibit higher k(cat)/K(m) when substrates contain nearby acidic amino acids. All cytosolic carboxypeptidases exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which, together with their other individual preferences for flanking amino acids, greatly increases the potential substrates for these enzymes and the biological processes in which they act. Additionally, Nna1 metabolized substrates mimicking the C terminus of tubulin in a way suggesting that the tyrosinated form of tubulin will accumulate in pcd mice.
Assuntos
Carboxipeptidases/genética , Proteínas de Ligação ao GTP/genética , Degeneração Neural/metabolismo , Ácido Poliglutâmico/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Sequência de Aminoácidos , Animais , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Camundongos , Camundongos Transgênicos , Degeneração Neural/patologia , Células de Purkinje/metabolismo , Células de Purkinje/patologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Especificidade por Substrato , Tubulina (Proteína)/metabolismoRESUMO
Cerebellin precursor protein 1 (Cbln1) is the prototype of a family of secreted neuronal glycoproteins (Cbln1-4) and its genetic elimination results in synaptic alterations in cerebellum (CB) and striatum. In CB, Cbln1 acts as a bi-functional ligand bridging pre-synaptic ß-neurexins on granule cells to post-synaptic Grid2 on Purkinje neurons. Although much is known concerning the action of Cbln1, little is known of the function of its other family members. Here, we show that Cbln1 and Cbln2 have similar binding activities to ß-neurexins and Grid2 and the targeted ectopic expression of Cbln2 to Purkinje cells in transgenic mice rescues the cerebellar deficits in Cbln1-null animals: suggesting that the two proteins have redundant function mediated by their common receptor binding properties. Cbln1 and Cbln2 are also co-expressed in the endolysosomal compartment of the thalamic neurons responsible for the synaptic alterations in striatum of Cbln1-null mice. Therefore, to determine whether the two family members have similar functions, we generated Cbln2-null mice. Cbln2-null mice do not show the synaptic alterations evident in striatum of Cbln1-null mice. Thus, Cbln2 can exhibit functional redundancy with Cbln1 in CB but it does not have the same properties as Cbln1 in thalamic neurons, implying one or both utilize different receptors/mechanisms in this brain region.
Assuntos
Proteínas do Tecido Nervoso/fisiologia , Precursores de Proteínas/fisiologia , Animais , Feminino , Células HEK293 , Humanos , Lisossomos/enzimologia , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Ligação Proteica/genética , Precursores de Proteínas/biossíntese , Precursores de Proteínas/deficiência , Precursores de Proteínas/genética , Células de Purkinje/enzimologia , Células de Purkinje/metabolismoRESUMO
Cerebellin precursor protein (Cbln1) is essential for synapse integrity in cerebellum through assembly into complexes that bridge pre-synaptic ß-neurexins (Nrxn) to post-synaptic GluRδ2. However, GluRδ2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluRδ2 and Nrxns1-3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, (deleted in colorectal cancer (DCC) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln null mice. Thus, Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less well-characterized role of netrin/DCC in adult brain.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Precursores de Proteínas/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Camundongos , Camundongos Knockout , Neurônios , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cbln1, a glycoprotein secreted from granule cells and GluRdelta2 in the postsynaptic densities of Purkinje cells are components of an incompletely understood pathway essential for integrity and plasticity of parallel fiber-Purkinje cell synapses. We show that Cbln1 undergoes anterograde transport from granule cells to Purkinje cells and Bergmann glia, and enters the endolysosomal trafficking system, raising the possibility that Cbln1 exerts its activity on or within Purkinje cells and Bergmann glia. Cbln1 is absent in Purkinje cells and Bergmann glia of GluRdelta2-null mice, suggesting a mechanistic convergence on Cbln1 trafficking. Ectopic expression of Cbln1 in Purkinje cells of L7-cbln1 transgenic mice reveals Cbln1 undergoes anterograde and retrograde trans-neuronal trafficking even across synapses that lack GluRDelta2, indicating that it is not universally essential for Cbln1 transport. The L7-cbln1 transgene also ameliorates the locomotor deficits of cbln1-null mice, indicating that the presence and/or release of Cbln1 from the postsynaptic neuron has functional consequences.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Precursores de Proteínas/metabolismo , Transporte Proteico/fisiologia , Células de Purkinje/metabolismo , Animais , Cerebelo/citologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Neuroglia/metabolismo , Precursores de Proteínas/genética , Células de Purkinje/citologia , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismoRESUMO
Cbln1 and the orphan glutamate receptor GluRdelta2 are pre- and postsynaptic components, respectively, of a novel transneuronal signaling pathway regulating synapse structure and function. We show here that Cbln1 is secreted from cerebellar granule cells in complex with a related protein, Cbln3. However, cbln1- and cbln3-null mice have different phenotypes and cbln1 cbln3 double-null mice have deficits identical to those of cbln1 knockout mice. The basis for these discordant phenotypes is that Cbln1 and Cbln3 reciprocally regulate each other's degradation and secretion such that cbln1-null mice lack both Cbln1 and Cbln3, whereas cbln3-null mice lack Cbln3 but have an approximately sixfold increase in Cbln1. Unlike Cbln1, Cbln3 cannot form homomeric complexes and is secreted only when bound to Cbln1. Structural modeling and mutation analysis reveal that, by constituting a steric clash that is masked upon binding Cbln1 in a "hide-and-run" mechanism of endoplasmic reticulum retention, a single arginine confers the unique properties of Cbln3.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Precursores de Proteínas/metabolismo , Precursores de Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Retículo Endoplasmático/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Fenótipo , Ligação Proteica , Precursores de Proteínas/deficiência , Precursores de Proteínas/genéticaRESUMO
Cbln1 is a cerebellum-specific protein of previously unknown function that is structurally related to the C1q and tumor necrosis factor families of proteins. We show that Cbln1 is a glycoprotein secreted from cerebellar granule cells that is essential for three processes in cerebellar Purkinje cells: the matching and maintenance of pre- and postsynaptic elements at parallel fiber-Purkinje cell synapses, the establishment of the proper pattern of climbing fiber-Purkinje cell innervation, and induction of long-term depression at parallel fiber-Purkinje cell synapses. Notably, the phenotype of cbln1-null mice mimics loss-of-function mutations in the orphan glutamate receptor, GluR delta2, a gene selectively expressed in Purkinje neurons. Therefore, Cbln1 secreted from presynaptic granule cells may be a component of a transneuronal signaling pathway that controls synaptic structure and plasticity.
Assuntos
Cerebelo/citologia , Proteínas do Tecido Nervoso/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Precursores de Proteínas/fisiologia , Sinapses/metabolismo , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Ataxia/genética , Comportamento Animal , Northern Blotting/métodos , Western Blotting/métodos , Células Cultivadas , Clonagem Molecular/métodos , Espinhas Dendríticas , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Regulação da Expressão Gênica , Humanos , Hibridização In Situ/métodos , Técnicas In Vitro , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão/métodos , Atividade Motora/genética , Mutagênese/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/ultraestrutura , Técnicas de Patch-Clamp/métodos , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Radioimunoensaio/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sinapses/ultraestrutura , Transfecção/métodos , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo.
Assuntos
Cerebelo/metabolismo , Glicosilação , Proteínas do Tecido Nervoso/metabolismo , Precursores de Proteínas/metabolismo , Sinapses/fisiologia , Animais , Células Cultivadas , Camundongos Endogâmicos C57BL , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Precursores de Proteínas/genéticaRESUMO
The identification of mRNAs that have restricted expression patterns in the brain represents powerful tools with which to characterize and manipulate the nervous system. Here, we describe a strategy using microarray technology (Affymetrix Mouse Genome 430 2.0 Arrays) to identify mRNA transcripts that are candidate markers of cerebellar Purkinje neurons. Initially, gene expression profiles were compared between cerebella of 4-month-old Purkinje cell degeneration (pcd(3J)) mice, in which most Purkinje cells had already degenerated and wild-type littermates with a normal complement of Purkinje neurons. Of 14,563 probe sets expressed in wild-type cerebellum, 797 showed a significant (p<0.0001) reduction in pcd(3J) mice. These probes could represent transcripts with varying levels of specificity for Purkinje cells as well as transcripts in other cell types that decline as a secondary consequence of Purkinje cell loss. Ranking of the probe signals revealed that well-known Purkinje cell-specific transcripts such as calbindin and L7/pcp2 clustered in a group that was <33% of wild-type levels. Therefore, to identify potentially new Purkinje cell-specific transcripts that cluster with the known markers, more stringent selection criteria were applied (<33% of wild-type signal and p<0.0001). With these criteria, 55 independent transcripts were identified of which 33 were annotated genes and 22 were ESTs and RIKEN cDNAs. A literature search revealed that 25 of the 33 annotated genes were expressed in Purkinje cells, with no data being available on the other 8. Thus, the additional 8 annotated and 22 un-annotated genes are clustered with many genes expressed in Purkinje cells making them candidate markers. To confirm the microarray data, eight representative annotated genes were selected including five reported to be in Purkinje neurons and three for which no data was available. Semi-quantitative RT-PCR demonstrated reduced expression of all eight transcripts in cerebella from pcd(3J) mice. The promoters of genes expressed selectively in subsets of neurons can be used to direct heterologous gene expression in transgenic mice and the more restricted the expression pattern the greater their utility. Therefore, microarray analysis was used to assess expression levels of all 55 transcripts in cerebral cortex, striatum, substantia nigra and ventral tegmental area. This permitted the identification of a set of genes whose promoters might have utility for selectively targeting gene expression to cerebellar Purkinje cells.
Assuntos
Transtornos Heredodegenerativos do Sistema Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Células de Purkinje/fisiologia , Animais , Cerebelo/patologia , Cerebelo/fisiologia , Genes Recessivos , Marcadores Genéticos , Genótipo , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Camundongos , Camundongos Mutantes Neurológicos , Células de Purkinje/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Excessive extracellular zinc may contribute to neuronal cell death following ischemia and seizures, although the mechanisms mediating zinc-induced cell death remain largely unknown. In this study, we examined potential cellular and molecular mechanisms associated with zinc neurotoxicity and determined the neuroprotective effects of the superoxide dismutase (SOD)/catalase mimetic, EUK-134. Cortical neuron cultures exposed to zinc for 24 h exhibited concentration-dependent increases in lactate dehydrogenase (LDH) release and number of apoptotic cell bodies. Both effects were prevented by treatment with EUK-134. Zinc exposure resulted in increased release of cytochrome c from the mitochondria into the cytosol. Treatment with EUK-134 blocked this parameter of mitochondrial dysfunction. Exposure of cultures to zinc for 4 h produced an elevation of reactive oxygen species (ROS) as determined by increased 2,7-dichlorofluorescein (DCF) fluorescence, which was followed by an increase in lipid peroxidation. EUK-134 completely attenuated ROS production and subsequent oxidative damage. Finally, zinc exposure activated NF-kappaB, an effect also prevented by EUK-134. These data indicate that multiple cellular and molecular mechanisms are involved in zinc neurotoxicity. As all these mechanisms appear to be sensitive to treatment with EUK-134, our data suggest that oxidative stress occurs early in the cascade of events triggered by zinc.
Assuntos
Catalase , Córtex Cerebral/efeitos dos fármacos , Líquido Intracelular/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Salicilatos/farmacologia , Superóxido Dismutase , Zinco/farmacologia , Animais , Catalase/química , Catalase/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/química , Superóxido Dismutase/farmacologia , Zinco/toxicidadeRESUMO
PEP-19/PCP4 maps within the Down syndrome critical region and encodes a small, predominantly neuronal, IQ motif protein. Pep-19 binds calmodulin and inhibits calmodulin-dependent signaling, which is critical for synaptic function, and therefore alterations in Pep-19 levels may affect synaptic plasticity and behavior. To investigate its possible role, we generated and characterized pep-19/pcp4-null mice. Synaptic plasticity at excitatory synapses of cerebellar Purkinje cells, which express the highest levels of Pep-19, was dramatically altered in pep-19/pcp4-null mice. Instead of long-term depression, pep-19/pcp4-null mice exhibited long-term potentiation at parallel fiber-Purkinje cell synapses. The mutant mice have a marked deficit in their ability to learn a locomotor task, as measured by improved performance upon repeated testing on an accelerating rotarod. Thus, our data indicate that pep-19/pcp4 is a critical determinant of synaptic plasticity in cerebellum and locomotor learning.