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1.
Proc Natl Acad Sci U S A ; 119(7)2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35145028

RESUMEN

The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ-knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcgfl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcgfl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca2+-activated large-conductance K+ (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination.


Asunto(s)
Terapia Genética , Actividad Motora/genética , Canales de Potasio Calcio-Activados/metabolismo , Proteína Quinasa C/metabolismo , Células de Purkinje/enzimología , Animales , Señalización del Calcio , Eliminación de Gen , Ratones , Ratones Noqueados , Actividad Motora/fisiología , Canales de Potasio Calcio-Activados/genética , Proteína Quinasa C/genética , Potenciales Sinápticos
2.
Cell Rep ; 37(2): 109831, 2021 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-34644575

RESUMEN

Spinocerebellar ataxias (SCAs) are a group of genetic diseases characterized by progressive ataxia and neurodegeneration, often in cerebellar Purkinje neurons. A SCA1 mouse model, Pcp2-ATXN1[30Q]D776, has severe ataxia in absence of progressive Purkinje neuron degeneration and death. Previous RNA-seq analyses identify cerebellar upregulation of the peptide hormone cholecystokinin (Cck) in Pcp2-ATXN1[30Q]D776 mice. Importantly, absence of Cck1 receptor (Cck1R) in Pcp2-ATXN1[30Q]D776 mice confers a progressive disease with Purkinje neuron death. Administration of a Cck1R agonist, A71623, to Pcp2-ATXN1[30Q]D776;Cck-/- and Pcp2-AXTN1[82Q] mice dampens Purkinje neuron pathology and associated deficits in motor performance. In addition, A71623 administration improves motor performance of Pcp2-ATXN2[127Q] SCA2 mice. Moreover, the Cck1R agonist A71623 corrects mTORC1 signaling and improves expression of calbindin in cerebella of AXTN1[82Q] and ATXN2[127Q] mice. These results indicate that manipulation of the Cck-Cck1R pathway is a potential therapeutic target for treatment of diseases involving Purkinje neuron degeneration.


Asunto(s)
Quimiocinas CC/agonistas , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Células de Purkinje/efectos de los fármacos , Ataxias Espinocerebelosas/tratamiento farmacológico , Tetragastrina/análogos & derivados , Animales , Ataxina-1/genética , Ataxina-1/metabolismo , Atrofia , Conducta Animal/efectos de los fármacos , Calbindinas/metabolismo , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Colecistoquinina/genética , Colecistoquinina/metabolismo , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Degeneración Nerviosa , Neuropéptidos/genética , Neuropéptidos/metabolismo , Células de Purkinje/enzimología , Células de Purkinje/patología , Transducción de Señal , Ataxias Espinocerebelosas/enzimología , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/patología , Tetragastrina/farmacología
3.
FASEB J ; 35(1): e21225, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33337568

RESUMEN

Studies of neuroglial interaction largely depend on cell-specific gene knockout (KO) experiments using Cre recombinase. However, genes known as glial-specific genes have recently been reported to be expressed in neuroglial stem cells, leading to the possibility that a glia-specific Cre driver results in unwanted gene deletion in neurons, which may affect sound interpretation. 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is generally considered to be an oligodendrocyte (OL) marker. Accordingly, Cnp promoter-controlled Cre recombinase has been used to create OL-specific gene targeting mice. However, in this study, using Rosa26-tdTomato-reporter/Cnp-Cre mice, we found that many forebrain neurons and cerebellar Purkinje neurons belong to the lineages of Cnp-expressing neuroglial stem cells. To answer whether gene targeting by Cnp-Cre can induce neuron-autonomous defects, we conditionally deleted an essential autophagy gene, Atg7, in Cnp-Cre mice. The Cnp-Cre-mediated Atg7 KO mice showed extensive p62 inclusion in neurons, including cerebellar Purkinje neurons with extensive neurodegeneration. Furthermore, neuronal areas showing p62 inclusion in Cnp-Cre-mediated Atg7 KO mice overlapped with the neuronal lineage of Cnp-expressing neuroglial stem cells. Moreover, Cnp-Cre-mediated Atg7-KO mice did not develop critical defects in myelination. Our results demonstrate that a large population of central neurons are derived from Cnp-expressing neuroglial stem cells; thus, conditional gene targeting using the Cnp promoter, which is known to be OL-specific, can induce neuron-autonomous phenotypes.


Asunto(s)
2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/deficiencia , Enfermedades Neurodegenerativas/enzimología , Neuroglía/enzimología , Células de Purkinje/enzimología , Células Madre/enzimología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia/genética , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Noqueados , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Neuroglía/patología , Células de Purkinje/patología , Células Madre/patología
4.
Neurochem Int ; 142: 104920, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33238153

RESUMEN

The immunohistochemical pattern of kynurenine aminotransferase-2 (KAT-2) - the key role enzyme in the production of neuroactive and neuroprotective kynurenic acid (KYNA) - was studied in the cerebellum of mice. It is known from literature that KAT-2 is localized mainly in astrocytes in different parts of the cerebrum. Kynurenine aminotransferase (KAT) activity in the cerebellum is relatively low and alternative production routes for KYNA have been described there. Therefore we examined the immunohistochemical pattern of KAT-2 in this part of the brain. Surprisingly, the cellular localization of KAT-2 in mice was proven to be unique; it localized characteristically in Purkinje cells and in some other types of neurons (not identified) but was not found in astrocytes nor microglia. The exclusive neuronal, but not glial localization of KAT-2 in the cerebellum is novel and may be related to its low activity and to the alternative pathways for KYNA production that have been described.


Asunto(s)
Cerebelo/citología , Cerebelo/enzimología , Neuronas/enzimología , Transaminasas/metabolismo , Animales , Cerebelo/química , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/química , Células de Purkinje/química , Células de Purkinje/enzimología , Especificidad de la Especie , Transaminasas/análisis
5.
J Neuroinflammation ; 17(1): 301, 2020 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-33054763

RESUMEN

BACKGROUND: Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity. METHODS: Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N1-acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox-/-) and Sat1-KO (Sat1-/-) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses. RESULTS: Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the α-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice. CONCLUSIONS: These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of α-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.


Asunto(s)
Acetiltransferasas/deficiencia , Ataxia/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/deficiencia , Células de Purkinje/enzimología , Acetiltransferasas/genética , Animales , Apoptosis/fisiología , Ataxia/genética , Ataxia/patología , Cerebelo/enzimología , Cerebelo/patología , Inflamación/enzimología , Inflamación/genética , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Células de Purkinje/patología , Poliamino Oxidasa
6.
Brain Behav Evol ; 95(1): 45-55, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32155640

RESUMEN

While in birds and mammals the cerebellum is a highly convoluted structure that consists of numerous transverse lobules, in most amphibians and reptiles it consists of only a single unfolded sheet. Orthogonal to the lobules, the cerebellum is comprised of sagittal zones that are revealed in the pattern of afferent inputs, the projection patterns of Purkinje cells, and Purkinje cell response properties, among other features. The expression of several molecular markers, such as aldolase C, is also parasagittally organized. Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme expressed in the cerebellar Purkinje cells of the vertebrate cerebellum. In birds, mammals, and some lizards (Ctenophoresspp.), ZII is expressed in a heterogenous fashion of alternating sagittal bands of high (ZII+) and low (ZII-) expression Purkinje cells. In contrast, turtles and snakes express ZII homogenously (ZII+) in their cerebella, but the pattern in crocodilians is unknown. Here, we examined the expression of ZII in two crocodilian species (Crocodylus niloticus and Alligator mississippiensis) to help determine the evolutionary origin of striped ZII expression in vertebrates. We expected crocodilians to express ZII in a striped (ZII+/ZII-) manner because of their close phylogenetic relationship to birds and their larger and more folded cerebellum compared to that of snakes and turtles. Contrary to our prediction, all Purkinje cells in the crocodilian cerebellum had a generally homogenous expression of ZII (ZII+) rather than clear ZII+/- stripes. Our results suggest that either ZII stripes were lost in three groups (snakes, turtles, and crocodilians) or ZII stripes evolved independently three times (lizards, birds, and mammals).


Asunto(s)
Caimanes y Cocodrilos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células de Purkinje/enzimología , Animales
7.
Cell Cycle ; 19(2): 153-159, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31876231

RESUMEN

Spinocerebellar ataxias (SCA) are a genetically heterogeneous family of cerebellar neurodegenerative diseases characterized by abnormal firing of Purkinje neurons and degeneration. We recently demonstrated the slowed firing rates seen in several SCAs share a common etiology of hyper-activation of the Src family of non-receptor tyrosine kinases (SFKs). However, the lack of clinically available neuroactive SFK inhibitors lead us to investigate alternative mechanisms to modulate SFK activity. Previous studies demonstrate that SFK activity can be enhanced by the removal of inhibitory phospho-marks by receptor-protein-tyrosine phosphatases (RPTPs). In this Extra View we show that MTSS1 inhibits SFK activity through the binding and inhibition of a subset of the RPTP family members, and lowering RPTP activity in cerebellar slices with peptide inhibitors increases the suppressed Purkinje neuron basal firing rates seen in two different SCA models. Together these results identify RPTPs as novel effectors of Purkinje neuron basal firing, extending the MTSS1/SFK regulatory circuit we previously described and expanding the therapeutic targets for SCA patients.


Asunto(s)
Potenciales de Acción/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Células de Purkinje/enzimología , Potenciales de Acción/efectos de los fármacos , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Ratones , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Células de Purkinje/efectos de los fármacos , Ataxias Espinocerebelosas/enzimología , Ataxias Espinocerebelosas/fisiopatología
8.
Glia ; 67(8): 1526-1541, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30989755

RESUMEN

Mitochondrial dysfunction causes neurodegeneration but whether impairment of mitochondrial homeostasis in astrocytes contributes to this pathological process remains largely unknown. The m-AAA protease exerts quality control and regulatory functions crucial for mitochondrial homeostasis. AFG3L2, which encodes one of the subunits of the m-AAA protease, is mutated in spinocerebellar ataxia SCA28 and in infantile syndromes characterized by spastic-ataxia, epilepsy and premature death. Here, we investigate the role of Afg3l2 and its redundant homologue Afg3l1 in the Bergmann glia (BG), radial astrocytes of the cerebellum that have functional connections with Purkinje cells (PC) and regulate glutamate homeostasis. We show that astrocyte-specific deletion of Afg3l2 in the mouse leads to late-onset motor impairment and to degeneration of BG, which display aberrant morphology, altered expression of the glutamate transporter EAAT2, and a reactive inflammatory signature. The neurological and glial phenotypes are drastically exacerbated when astrocytes lack both Afg31l and Afg3l2, and therefore, are totally depleted of the m-AAA protease. Moreover, mitochondrial stress responses and necroptotic markers are induced in the cerebellum. In both mouse models, targeted BG show a fragmented mitochondrial network and loss of mitochondrial cristae, but no signs of respiratory dysfunction. Importantly, astrocyte-specific deficiency of Afg3l1 and Afg3l2 triggers secondary morphological degeneration and electrophysiological changes in PCs, thus demonstrating a non-cell-autonomous role of glia in neurodegeneration. We propose that astrocyte dysfunction amplifies both neuroinflammation and glutamate excitotoxicity in patients carrying mutations in AFG3L2, leading to a vicious circle that contributes to neuronal death.


Asunto(s)
Proteasas ATP-Dependientes/deficiencia , ATPasas Asociadas con Actividades Celulares Diversas/deficiencia , Astrocitos/enzimología , Cerebelo/enzimología , Metaloendopeptidasas/deficiencia , Mitocondrias/enzimología , Enfermedades Neurodegenerativas/enzimología , Proteasas ATP-Dependientes/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , Animales , Astrocitos/patología , Cerebelo/patología , Modelos Animales de Enfermedad , Femenino , Inflamación/enzimología , Inflamación/patología , Masculino , Metaloendopeptidasas/genética , Ratones Transgénicos , Mitocondrias/patología , Enfermedades Neurodegenerativas/patología , Células de Purkinje/enzimología , Células de Purkinje/patología
9.
Neurol Res ; 41(3): 234-241, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30462592

RESUMEN

OBJECTIVES: Mechanisms underlying Purkinje cell (PC) death, which leads to many diseases in humans, are still poorly elucidated. Progressive PC degeneration occurs in shaker mutant rat due to an X-linked recessive mutation leading to gait ataxia and total-body tremors. Chemoablation of the inferior olive (IO) and olivocerebellar deafferentation temporally accelerated PC death from the natural 6-8 week time-course to 1-2 weeks in the shaker mutant rat. The present study posits that IO chemoablation leads to the accelerated and augmented upregulation of the executioner active caspase-3 that triggers apoptosis of at-risk PCs throughout the ordinary phenotypic manifestation of the shaker mutation. METHODS: Immunofluorescence and double labeling for calbindin and active caspase-3 were used in vermal cerebellar sections from IO-chemoablated rats to demonstrate the effect of IO chemoablation on active caspase-3 expression in at-risk PCs. RESULTS: Active caspase-3 expression was enhanced in the anterior degeneration (ADC) and posterior degeneration (PDC) compartments to reach a peak in both degeneration compartments at 24 h following the injections for IO chemoablation. DISCUSSION: Consequently, it can be deduced that active caspase-3 expression in shaker mutant rats is modifiable suggesting the possibility of targeting it therapeutically in an attempt to rescue PCs from death. Abbreviation PC: Purkinje cell; IO: inferior olive; ADC: Anterior degeneration compartment; PDC: Posterior degeneration compartment; ISC: Intermediate survival compartment; FNSC: Flocculonodular survival compartment.


Asunto(s)
Caspasa 3/metabolismo , Vermis Cerebeloso/enzimología , Enfermedades Neurodegenerativas/metabolismo , Núcleo Olivar/fisiopatología , Células de Purkinje/enzimología , Animales , Vermis Cerebeloso/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Enfermedades Neurodegenerativas/patología , Células de Purkinje/patología , Distribución Aleatoria , Ratas Mutantes
10.
EMBO J ; 37(23)2018 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-30420557

RESUMEN

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration (pcd) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders.


Asunto(s)
Carboxipeptidasas/deficiencia , Cerebelo/enzimología , Neuronas Motoras/enzimología , Nervios Periféricos/enzimología , Células de Purkinje/enzimología , Columna Vertebral/enzimología , Degeneraciones Espinocerebelosas/enzimología , Cerebelo/patología , Femenino , Proteínas de Unión al GTP , Humanos , Masculino , Neuronas Motoras/patología , Péptidos/genética , Péptidos/metabolismo , Nervios Periféricos/patología , Procesamiento Proteico-Postraduccional , Células de Purkinje/patología , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina , Columna Vertebral/patología , Degeneraciones Espinocerebelosas/genética , Degeneraciones Espinocerebelosas/patología
11.
J Neurosci ; 38(34): 7409-7419, 2018 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-30006360

RESUMEN

Inositol hexakisphosphate kinases (IP6Ks) regulate various biological processes. Among pyrophosphates generated by IP6Ks, diphosphoinositol pentakisphosphate (IP7), and bis-diphosphoinositol tetrakisphosphate have been extensively characterized. IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, IP6K1, IP6K2, and IP6K3, which have distinct biological functions. We report that IP6K2 binds protein 4.1.N with high affinity and specificity. Nuclear translocation of 4.1N, which is required for its principal functions, is dependent on IP6K2. Both of these proteins are highly expressed in granule cells of the cerebellum where their interaction regulates Purkinje cell morphology and cerebellar synapses. The deletion of IP6K2 in male/female mice elicits substantial defects in synaptic influences of granule cells upon Purkinje cells as well as notable impairment of locomotor function. Moreover, the disruption of IP6K2-4.1N interactions impairs cell viability. Thus, IP6K2 and its interaction with 4.1N appear to be major determinants of cerebellar disposition and psychomotor behavior.SIGNIFICANCE STATEMENT Inositol phosphates are produced by a family of inositol hexakisphosphate kinases (IP6Ks)-IP6K1, IP6K2, and IP6K3. Of these, the physiological roles of IP6K2 in the brain have been least characterized. In the present study, we report that IP6K2 binds selectively to the neuronal protein 4.1N. Both of these proteins are highly expressed in granule cells of the cerebellum. Using IP6K2 knock-out (KO) mice, we establish that IP6K2-4.1N interactions in granule cells regulate Purkinje cell morphology, the viability of cerebellar neurons, and psychomotor behavior.


Asunto(s)
Cerebelo/fisiología , Proteínas del Citoesqueleto/fisiología , Proteínas de la Membrana/fisiología , Actividad Motora/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Neuropéptidos/fisiología , Fosfotransferasas (Aceptor del Grupo Fosfato)/fisiología , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Supervivencia Celular , Cerebelo/citología , Cerebelo/enzimología , Conducta Exploratoria , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Neuronas/enzimología , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Unión Proteica , Desempeño Psicomotor/fisiología , Células de Purkinje/enzimología , Células de Purkinje/fisiología , Prueba de Desempeño de Rotación con Aceleración Constante , Sinapsis/fisiología
12.
Nature ; 557(7706): 510-515, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29769718

RESUMEN

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars sti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.


Asunto(s)
Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/metabolismo , Mutación , Biosíntesis de Proteínas , Células de Purkinje/enzimología , Células de Purkinje/patología , Alanina/metabolismo , Alanina-ARNt Ligasa/química , Animales , Dominio Catalítico , Muerte Celular , Femenino , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Células de Purkinje/metabolismo , Serina/metabolismo
13.
J Clin Invest ; 128(5): 2076-2088, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29494346

RESUMEN

Increasing evidence suggests that synapse dysfunctions are a major determinant of several neurodevelopmental and neurodegenerative diseases. Here we identify protein kinase N1 (PKN1) as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber-forming (PF-forming) cerebellar granule cells (Cgcs). Postnatal Pkn1-/- animals showed a defective PF-Purkinje cell (PF-PC) synapse formation. In vitro, Pkn1-/- Cgcs exhibited deregulated axonal outgrowth, elevated AKT phosphorylation, and higher levels of neuronal differentiation-2 (NeuroD2), a transcription factor preventing presynaptic maturation. Concomitantly, Pkn1-/- Cgcs had a reduced density of presynaptic sites. By inhibiting AKT with MK-2206 and siRNA-mediated knockdown, we found that AKT hyperactivation is responsible for the elongated axons, higher NeuroD2 levels, and reduced density of presynaptic specifications in Pkn1-/- Cgcs. In line with our in vitro data, Pkn1-/- mice showed AKT hyperactivation, elevated NeuroD2 levels, and reduced expression of PF-PC synaptic markers during stages of PF maturation in vivo. The long-term effect of Pkn1 knockout was further seen in cerebellar atrophy and mild ataxia. In summary, our results demonstrate that PKN1 functions as a developmentally active gatekeeper of AKT activity, thereby fine-tuning axonal outgrowth and presynaptic differentiation of Cgcs and subsequently the correct PF-PC synapse formation.


Asunto(s)
Axones/enzimología , Proyección Neuronal , Proteína Quinasa C/metabolismo , Células de Purkinje/enzimología , Sinapsis/metabolismo , Animales , Compuestos Heterocíclicos con 3 Anillos/farmacología , Ratones , Ratones Noqueados , Proteína Quinasa C/genética , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células de Purkinje/citología , Sinapsis/genética
14.
Cell Res ; 28(3): 296-306, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29451229

RESUMEN

The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.


Asunto(s)
Proteínas AAA/metabolismo , Calcio/metabolismo , Metaloendopeptidasas/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/enzimología , Proteasas ATP-Dependientes/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , Animales , Canales de Calcio/metabolismo , Humanos , Metaloendopeptidasas/genética , Ratones , Mitocondrias/genética , Modelos Animales , Células de Purkinje/enzimología , Paraplejía Espástica Hereditaria/genética , Ataxias Espinocerebelosas/genética
15.
Hum Mol Genet ; 27(8): 1396-1410, 2018 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-29432535

RESUMEN

Among the many types of neurons expressing protein kinase C (PKC) enzymes, cerebellar Purkinje neurons are particularly reliant on appropriate PKC activity for maintaining homeostasis. The importance of PKC enzymes in Purkinje neuron health is apparent as mutations in PRKCG (encoding PKCγ) cause cerebellar ataxia. PRKCG has also been identified as an important node in ataxia gene networks more broadly, but the functional role of PKC in other forms of ataxia remains unexplored, and the mechanisms by which PKC isozymes regulate Purkinje neuron health are not well understood. Here, we investigated how PKC activity influences neurodegeneration in inherited ataxia. Using mouse models of spinocerebellar ataxia type 1 (SCA1) and 2 (SCA2) we identify an increase in PKC-mediated substrate phosphorylation in two different forms of inherited cerebellar ataxia. Normalizing PKC substrate phosphorylation in SCA1 and SCA2 mice accelerates degeneration, suggesting that the increased activity observed in these models is neuroprotective. We also find that increased phosphorylation of PKC targets limits Purkinje neuron membrane excitability, suggesting that PKC activity may support Purkinje neuron health by moderating excitability. These data suggest a functional role for PKC enzymes in ataxia gene networks, and demonstrate that increased PKC activity is a protective modifier of degeneration in inherited cerebellar ataxia.


Asunto(s)
Ataxina-1/genética , Ataxina-2/genética , Proteína Quinasa C/genética , Células de Purkinje/enzimología , Ataxias Espinocerebelosas/genética , Animales , Ataxina-1/metabolismo , Ataxina-2/metabolismo , Cerebelo/enzimología , Cerebelo/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Microtomía , Fosforilación , Cultivo Primario de Células , Proteína Quinasa C/metabolismo , Células de Purkinje/patología , Transducción de Señal , Ataxias Espinocerebelosas/enzimología , Ataxias Espinocerebelosas/patología , Ataxias Espinocerebelosas/prevención & control , Técnicas de Cultivo de Tejidos
16.
Brain Res ; 1675: 8-19, 2017 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-28855102

RESUMEN

Ethanol and age-induced pathologies of the Purkinje neuron (PN) may result from histone deacetylases (HDACs), enzymes which repress transcription through coiling of the DNA. The purposes of this study were to investigate expression patterns of Class 1 and IIa HDACs in PN and the effects of aging and alcohol on the density of HDACs and histone acetylation in PN. Ninety, eight month old rats (30/diet) were fed a liquid ethanol, liquid control, or rat chow diet for 10, 20, or 40weeks (30/treatment duration). Double immunocytochemical labeling on tissue sections from these rats used antibodies against HDAC isoforms or acetylated histones, and calbindin, a marker for PN. Fluorescent intensities were also measured. Results showed a significant age but not an alcohol-related decrease in the densities of HDACs 2, 3, and 7. In contrast, there were age related-increases in the densities of phosphorylated form of HDAC (4, 5, 7) PN and in PN nuclei expressing HDAC 7. There were also a trend towards ethanol-induced inhibition of acetylation as the density of AH2b PN nuclei and AH3 and AH2b fluorescent intensity was significantly lower in the EF compared to the PF rats. This study presents unique data concerning which HDACs are commonly expressed in PN and indicates that aging rather than lengthy alcohol expression alters expression of the HDACs studied here. These results also suggest that lengthy ethanol consumption may inhibit histone deacetylation in PN.


Asunto(s)
Envejecimiento/metabolismo , Etanol/administración & dosificación , Regulación Enzimológica de la Expresión Génica , Histona Desacetilasas/biosíntesis , Células de Purkinje/enzimología , Envejecimiento/efectos de los fármacos , Consumo de Bebidas Alcohólicas/metabolismo , Consumo de Bebidas Alcohólicas/patología , Animales , Histona Desacetilasas/genética , Masculino , Células de Purkinje/efectos de los fármacos , Células de Purkinje/patología , Ratas , Ratas Endogámicas F344
17.
Dev Neurobiol ; 77(10): 1175-1187, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28589675

RESUMEN

Cyclin-dependent kinase 5 (Cdk5) is recognized as a unique member among other Cdks due to its versatile roles in many biochemical processes in the nervous system. The proper development of neuronal dendrites is required for the formation of complex neural networks providing the physiological basis of various neuronal functions. We previously reported that sparse dendrites were observed on cultured Cdk5-null Purkinje cells and Purkinje cells in Wnt1cre -mediated Cdk5 conditional knockout (KO) mice. In the present study, we generated L7cre -mediated p35; p39 double KO (L7cre -p35f/f ; p39-/- ) mice whose Cdk5 activity was eliminated specifically in Purkinje cells of the developing cerebellum. Consequently, these mice exhibited defective Purkinje cell migration, motor coordination deficiency and a Purkinje dendritic abnormality similar to what we have observed before, suggesting that dendritic growth of Purkinje cells was cell-autonomous in vivo. We found that mixed and overlay cultures of WT cerebellar cells rescued the dendritic deficits in Cdk5-null Purkinje cells, however, indicating that Purkinje cell dendritic development was also supported by non-cell-autonomous factors. We then again rescued these abnormalities in vitro by applying exogenous brain-derived neurotrophic factor (BDNF). Based on the results from culture experiments, we attempted to rescue the developmental defects of Purkinje cells in L7cre -p35f/f ; p39-/- mice by using a TrkB agonist. We observed partial rescue of morphological defects of dendritic structures of Purkinje cells. These results suggest that Cdk5 activity is required for Purkinje cell dendritic growth in cell-autonomous and non-cell-autonomous manners. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1175-1187, 2017.


Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Dendritas/enzimología , Proyección Neuronal/fisiología , Células de Purkinje/enzimología , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Calbindinas/metabolismo , Células Cultivadas , Cerebelo/efectos de los fármacos , Cerebelo/enzimología , Cerebelo/crecimiento & desarrollo , Cerebelo/patología , Dendritas/efectos de los fármacos , Dendritas/patología , Miembro Anterior/efectos de los fármacos , Miembro Anterior/fisiopatología , Inmunohistoquímica , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Fuerza Muscular/efectos de los fármacos , Fuerza Muscular/fisiología , Proyección Neuronal/efectos de los fármacos , Células de Purkinje/efectos de los fármacos , Células de Purkinje/patología , Receptor trkB/agonistas , Receptor trkB/metabolismo , Prueba de Desempeño de Rotación con Aceleración Constante
18.
Cell Tissue Res ; 368(3): 441-458, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28191598

RESUMEN

Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate. Diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid, another second messenger. Of the DGK family, DGKε is the only DGK isoform that exhibits substrate specificity for DG with an arachidonoyl acyl chain at the sn-2 position. Recently, we demonstrated that hydrophobic residues in the N-terminus of DGKε play an important role in targeting the endoplasmic reticulum in transfected cells. However, its cellular expression and subcellular localization in the brain remain elusive. In the present study, we investigate this issue using specific DGKε antibody. DGKε was richly expressed in principal neurons of higher brain regions, including pyramidal cells in the hippocampus and neocortex, medium spiny neurons in the striatum and Purkinje cells in the cerebellum. In Purkinje cells, DGKε was localized to the subsurface cisterns and colocalized with inositol 1,4,5-trisphosphate receptor-1 in dendrites and axons. In dendrites of Purkinje cells, DGKε was also distributed in close apposition to DG lipase-α, which catalyzes arachidonoyl-DG to produce 2-arachidonoyl glycerol, a major endocannabinoid in the brain. Behaviorally, DGKε-knockout mice exhibited hyper-locomotive activities and impaired motor coordination and learning. These findings suggest that DGKε plays an important role in neuronal and brain functions through its distinct neuronal expression and subcellular localization and also through coordinated arrangement with other molecules involving the phosphoinositide signaling pathway.


Asunto(s)
Cerebelo/enzimología , Diacilglicerol Quinasa/metabolismo , Células de Purkinje/enzimología , Animales , Encéfalo/enzimología , Cerebelo/citología , Cerebelo/ultraestructura , Diacilglicerol Quinasa/genética , Células HeLa , Humanos , Immunoblotting , Inmunohistoquímica , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Aprendizaje , Locomoción , Ratones , Ratones Noqueados , Células PC12 , Fosfatidilinositoles/metabolismo , Desempeño Psicomotor , Células de Purkinje/ultraestructura , Ratas , Ratas Wistar , Sistemas de Mensajero Secundario , Distribución Tisular
19.
Mol Brain ; 9: 7, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26772978

RESUMEN

BACKGROUND: The cerebellum is responsible for coordinating motor functions and has a unique laminated architecture. Purkinje cells are inhibitory neurons and represent the only output from the cerebellar cortex. Tyrosine hydroxylase (TH) is the key enzyme for the synthesis of catecholamines, including dopamine and noradrenaline, and it is normally not expressed in cerebellar neurons. RESULTS: We report here that the low-density lipoprotein receptors (Lrp) 5 and 6, Wnt co-receptors, are required for the development of the cerebellum and for suppressing ectopic TH expression in Purkinje cells. Simultaneous inactivation of Lrp 5 and 6 by Nestin-Cre results in defective lamination and foliation of the cerebellum during postnatal development. Surprisingly, TH is ectopically expressed by Purkinje cells, although they still keep its other neurochemical characteristics. These phenotypes are also observed in the cerebellum of GFAP-Cre;ß-catenin(flox/flox) mice, and AAV2-Cre-mediated gene deletion leads to ectopic TH expression in Purkinje cells of ß-catenin(flox/flox) mice as well. CONCLUSIONS: Our results revealed a new role of the canonical Lrp5/6-ß-catenin pathway in regulating the morphogenesis of the cerebellum during postnatal development.


Asunto(s)
Cerebelo/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Células de Purkinje/enzimología , Tirosina 3-Monooxigenasa/metabolismo , beta Catenina/metabolismo , Animales , Western Blotting , Catecolaminas/metabolismo , Diferenciación Celular , Cerebelo/crecimiento & desarrollo , Cerebelo/patología , Dependovirus/metabolismo , Integrasas/metabolismo , Ratones Noqueados , Neuronas/metabolismo
20.
Mol Neurobiol ; 53(8): 5149-60, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-26399641

RESUMEN

Purkinje cell dendritic development is severely compromised after chronic activation of protein kinase C (PKC). In a recent transgenic mouse model of spinocerebellar ataxia 14, the ser361-to-gly (S361G) mutation of the protein kinase C gamma (PKCγ) gene was expressed in Purkinje cells. Purkinje cells from these mutant mice in organotypic slice cultures have the same stunted dendritic tree as Purkinje cells after pharmacological activation of PKC. Because the transgene is exclusively present in Purkinje cells, cerebellar tissue from these mice is an attractive starting material for searching genes which might be interacting with PKCγ in Purkinje cells for inducing the stunted dendritic growth. We have performed a microarray analysis and identified several candidate genes with an increased messenger RNA (mRNA) expression in the PKCγ-S361G transgenic Purkinje cells. Out of these candidates, we have further studied carbonic anhydrase 8 (CA8). We show here that CA8 mRNA and protein expression is strongly induced in PKCγ-S361G transgenic Purkinje cells. Overexpression of CA8 in Purkinje cells in dissociated cultures strongly inhibited Purkinje cell dendritic development and produced a dendritic phenotype similar to PKCγ-S361G. There was no evidence for a direct binding of CA8 to either PKCγ or the type 1 IP3 receptor. Knockdown of CA8 with miRNA did not alter Purkinje cell dendritic development and did not protect Purkinje cells in dissociated cultures from the stunted dendritic growth induced by PKCγ-S361G or by PKC activation. Our results indicate that CA8 is a novel important regulator of Purkinje cell dendritic development and that its expression is controlled by PKCγ activity.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Dendritas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Quinasa C/metabolismo , Células de Purkinje/enzimología , Animales , Forma de la Célula , Cerebelo/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones Transgénicos , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Regulación hacia Arriba/genética
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