RESUMO
BACKGROUND: Loss-of-function mutations in the PRKN gene, encoding Parkin, are the most common cause of autosomal recessive Parkinson's disease (PD). We have previously identified mitoch ondrial Stomatin-like protein 2 (SLP-2), which functions in the assembly of respiratory chain proteins, as a Parkin-binding protein. Selective knockdown of either Parkin or SLP-2 led to reduced mitochondrial and neuronal function in neuronal cells and Drosophila, where a double knockdown led to a further worsening of Parkin-deficiency phenotypes. Here, we investigated the minimal Parkin region involved in the Parkin-SLP-2 interaction and explored the ability of Parkin-fragments and peptides from this minimal region to restore mitochondrial function. METHODS: In fibroblasts, human induced pluripotent stem cell (hiPSC)-derived neurons, and neuroblastoma cells the interaction between Parkin and SLP-2 was investigated, and the Parkin domain responsible for the binding to SLP-2 was mapped. High resolution respirometry, immunofluorescence analysis and live imaging were used to analyze mitochondrial function. RESULTS: Using a proximity ligation assay, we quantitatively assessed the Parkin-SLP-2 interaction in skin fibroblasts and hiPSC-derived neurons. When PD-associated PRKN mutations were present, we detected a significantly reduced interaction between the two proteins. We found a preferential binding of SLP-2 to the N-terminal part of Parkin, with a highest affinity for the RING0 domain. Computational modeling based on the crystal structure of Parkin protein predicted several potential binding sites for SLP-2 within the Parkin RING0 domain. Amongst these, three binding sites were observed to overlap with natural PD-causing missense mutations, which we demonstrated interfere substantially with the binding of Parkin to SLP-2. Finally, delivery of the isolated Parkin RING0 domain and a Parkin mini-peptide, conjugated to cell-permeant and mitochondrial transporters, rescued compromised mitochondrial function in Parkin-deficient neuroblastoma cells and hiPSC-derived neurons with endogenous, disease causing PRKN mutations. CONCLUSIONS: These findings place further emphasis on the importance of the protein-protein interaction between Parkin and SLP-2 for the maintenance of optimal mitochondrial function. The possibility of restoring an abolished binding to SLP-2 by delivering the Parkin RING0 domain or the Parkin mini-peptide involved in this specific protein-protein interaction into cells might represent a novel organelle-specific therapeutic approach for correcting mitochondrial dysfunction in Parkin-linked PD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Mitocondriais , Neuroblastoma , Doença de Parkinson , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Doença de Parkinson/genética , PeptídeosRESUMO
Autosomal dominant mutations in the gene encoding α-synuclein (SNCA) were the first to be linked with hereditary Parkinson's disease (PD). Duplication and triplication of SNCA has been observed in PD patients, together with mutations at the N-terminal of the protein, among which A30P and A53T influence the formation of fibrils. By overexpressing human α-synuclein in the neuronal system of Drosophila, we functionally validated the ability of IP3K2, an ortholog of the GWAS identified risk gene, Inositol-trisphosphate 3-kinase B (ITPKB), to modulate α-synuclein toxicity in vivo. ITPKB mRNA and protein levels were also increased in SK-N-SH cells overexpressing wild-type α-synuclein, A53T or A30P mutants. Kinase overexpression was detected in the cytoplasmatic and in the nuclear compartments in all α-synuclein cell types. By quantifying mRNAs in the cortex of PD patients, we observed higher levels of ITPKB mRNA when SNCA was expressed more (p < 0.05), compared to controls. A positive correlation was also observed between SNCA and ITPKB expression in the cortex of patients, which was not seen in the controls. We replicated this observation in a public dataset. Our data, generated in SK-N-SH cells and in cortex from PD patients, show that the expression of α-synuclein and ITPKB is correlated in pathological situations.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Mutação , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismoRESUMO
Mutations in the SZT2 gene have been associated with developmental and epileptic encephalopathy-18, a rare severe autosomal recessive neurologic disorder, characterized by psychomotor impairment/intellectual disability, dysmorphic facial features and early onset of refractory seizures. Here we report the generation of the first induced pluripotent stem cell (iPSC) lines from a patient with treatment-resistant epilepsy, carrying compound heterozygous mutations in SZT2 (Mut1: c.498G>T and Mut2: c.6553C>T), and his healthy heterozygous parents. Peripheral blood mononuclear cells were reprogrammed by a non-integrating Sendai virus-based reprogramming system. The generated human iPSC lines exhibited expression of the main pluripotency markers, the potential to differentiate into all three germ layers and presented a normal karyotype. These lines represent a valuable resource to study neurodevelopmental alterations, and to obtain mature, pathology-relevant neuronal populations as an in vitro model to perform functional assays and test the patient's responsiveness to novel antiepileptic treatments.
Assuntos
Epilepsia Generalizada , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares , Mutação , Heterozigoto , Proteínas do Tecido Nervoso/metabolismoRESUMO
SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.
Assuntos
Ácidos Hidroxâmicos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Acetilação , Animais , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Wistar , VorinostatRESUMO
The Ca2+-dependent activator protein for secretion 2 (CADPS2) is a member of the CAPS/CADPS protein family that plays crucial roles in synaptic vesicle dynamics. Genomic variability in the CADPS2 gene has been associated to autism spectrum disorders and Alzheimer's disease, both characterized by altered neurotransmission. Biological evidence also linked CADPS2 to Parkinson's disease (PD), as a disease-causing mutation in leucine-rich repeat kinase 2 (LRRK2) was reported to increase CADPS2 gene and protein expression. Furthermore, restoration of CADPS2 physiologic levels was able to provide neuroprotection in patient-derived neurons, consistent with the synaptic dysfunction postulated to underlie PD. However, little is known about the influence of PD-related proteins on transcriptional regulation of critical synaptic genes such as CADPS2. Here we aimed at investigating the transcriptional effects of LRRK2 and alpha-synuclein (aSyn) on CADPS2 gene expression, using a combination of in silico analyses and cell biology techniques. First, we identified a predicted promoter in the human CADPS2 genomic sequence, which we then utilized in a luciferase-based gene reporter assay. This approach enabled us to disclose a differential effect of high levels of LRRK2 and aSyn on CADPS2 promoter activity. Specifically, CADPS2 transcriptional activity was enhanced by high cellular levels of LRRK2 and reduced by overexpression of aSyn. Consistently, CADPS2 mRNA levels were diminished in aSyn overexpressing cells. Our results indicate that LRRK2 and aSyn participate in the dysregulation of CADPS2 by altering transcription and support the hypothesis that synaptic dysfunctions, through different mechanisms, might contribute to the neuronal defects of diseases such as PD.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Proteínas de Transporte Vesicular/genética , alfa-Sinucleína/genética , Sequência de Bases , Linhagem Celular , Expressão Gênica , Humanos , Neurônios/metabolismo , Doença de Parkinson/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Ativação Transcricional , Regulação para CimaRESUMO
Group II metabotropic glutamate receptors (mGlu-IIs) modulate hippocampal information processing through several presynaptic actions. We describe a novel postsynaptic inhibitory mechanism mediated by the mGlu2 subtype that activates an inwardly rectifying potassium conductance in the dendrites of DG granule cells of rats and mice. Data from glutamate-uncaging experiments and simulations indicate that mGlu2-activated potassium conductance uniformly reduces the peak amplitude of synaptic inputs arriving in the distal two-thirds of dendrites, with only minor effects on proximal inputs. This unique shunting profile is consistent with a peak expression of the mGlu2-activated conductance at the transition between the proximal and middle third of the dendrites. Further simulations under various physiologically relevant conditions showed that when a shunting conductance was activated in the proximal third of a single dendrite, it effectively modulated input to this specific branch while leaving inputs in neighboring dendrites relatively unaffected. Therefore, the restricted expression of the mGlu2-activated potassium conductance in the proximal third of DG granule cell dendrites represents an optimal localization for achieving the opposing biophysical requirements for uniform yet selective modulation of individual dendritic branches.
Assuntos
Dendritos/metabolismo , Giro Denteado/metabolismo , Inibição Neural/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Giro Denteado/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/deficiência , Receptores de Glutamato Metabotrópico/genéticaRESUMO
Low-threshold (T-type) Ca(2+) channels encoded by the Ca(V)3 genes endow neurons with oscillatory properties that underlie slow waves characteristic of the non-rapid eye movement (NREM) sleep EEG. Three Ca(V)3 channel subtypes are expressed in the thalamocortical (TC) system, but their respective roles for the sleep EEG are unclear. Ca(V)3.3 protein is expressed abundantly in the nucleus reticularis thalami (nRt), an essential oscillatory burst generator. We report the characterization of a transgenic Ca(V)3.3(-/-) mouse line and demonstrate that Ca(V)3.3 channels are indispensable for nRt function and for sleep spindles, a hallmark of natural sleep. The absence of Ca(V)3.3 channels prevented oscillatory bursting in the low-frequency (4-10 Hz) range in nRt cells but spared tonic discharge. In contrast, adjacent TC neurons expressing Ca(V)3.1 channels retained low-threshold bursts. Nevertheless, the generation of synchronized thalamic network oscillations underlying sleep-spindle waves was weakened markedly because of the reduced inhibition of TC neurons via nRt cells. T currents in Ca(V)3.3(-/-) mice were <30% compared with those in WT mice, and the remaining current, carried by Ca(V)3.2 channels, generated dendritic [Ca(2+)](i) signals insufficient to provoke oscillatory bursting that arises from interplay with Ca(2+)-dependent small conductance-type 2 K(+) channels. Finally, naturally sleeping Ca(V)3.3(-/-) mice showed a selective reduction in the power density of the σ frequency band (10-12 Hz) at transitions from NREM to REM sleep, with other EEG waves remaining unaltered. Together, these data identify a central role for Ca(V)3.3 channels in the rhythmogenic properties of the sleep-spindle generator and provide a molecular target to elucidate the roles of sleep spindles for brain function and development.
Assuntos
Canais de Cálcio Tipo T/fisiologia , Sono/fisiologia , Tálamo/fisiologia , Animais , Ondas Encefálicas , Sinalização do Cálcio , Eletroencefalografia , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Sono REMRESUMO
Impaired function or expression of group II metabotropic glutamate receptors (mGluRIIs) is observed in brain disorders such as schizophrenia. This class of receptor is thought to modulate activity of neuronal circuits primarily by inhibiting neurotransmitter release. Here, we characterize a postsynaptic excitatory response mediated by somato-dendritic mGluRIIs in hippocampal CA3 pyramidal cells and in stratum oriens interneurons. The specific mGluRII agonists DCG-IV or LCCG-1 induced an inward current blocked by the mGluRII antagonist LY341495. Experiments with transgenic mice revealed a significant reduction of the inward current in mGluR3(-/-) but not in mGluR2(-/-) mice. The excitatory response was associated with periods of synchronized activity at theta frequency. Furthermore, cholinergically induced network oscillations exhibited decreased frequency when mGluRIIs were blocked. Thus, our data indicate that hippocampal responses are modulated not only by presynaptic mGluRIIs that reduce glutamate release but also by postsynaptic mGluRIIs that depolarize neurons and enhance CA3 network activity.
Assuntos
Região CA3 Hipocampal/fisiologia , Rede Nervosa/fisiologia , Células Piramidais/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Aminoácidos/farmacologia , Animais , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/metabolismo , Ciclopropanos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Glicina/análogos & derivados , Glicina/farmacologia , Interneurônios/metabolismo , Interneurônios/fisiologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Rede Nervosa/metabolismo , Técnicas de Patch-Clamp , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/genética , Ritmo Teta/efeitos dos fármacos , Ritmo Teta/fisiologia , Xantenos/farmacologiaRESUMO
In Parkinson's disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear, but they are hypothesized to involve the autophagy-lysosome pathway (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, and LRRK2 kinase activity has been shown to be involved in pS129-aSyn inclusion modulation. We observed selective downregulation of the novel PD risk factor RIT2 in vitro and in vivo. Rit2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of Rit2 operated neuroprotection against AAV-A53T-aSyn. Furthermore, Rit2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. On the other hand, reduction of Rit2 levels leads to defects in the ALP, similar to those induced by the G2019S-LRRK2 mutation. Our data indicate that Rit2 is required for correct lysosome function, inhibits overactive LRRK2 to ameliorate ALP impairment, and counteracts aSyn aggregation and related deficits. Targeting Rit2 could represent an effective strategy to combat neuropathology in familial and idiopathic PD.
RESUMO
Group II metabotropic glutamate receptors (mGluR2, encoded by Grm2, and mGluR3, encoded by Grm3) are inhibitory autoreceptors that negatively modulate the adenylate cyclase signaling cascade. Within the hippocampus, mGluR2 is believed to play a key role in the induction of long-term depression (LTD) at mossy fiber-CA3 synapses. Here, we used Grm2/3 double knockout (dko) mice to investigate to what extent group II mGluRs are necessary for mossy fiber LTD. Surprisingly, we found that these mice displayed prominent mossy fiber LTD. However, the induction of this form of LTD was sensitive to the external Ca(2+) concentration. Mossy fiber LTD in Grm2/3 dko mice was indistinguishable from that in wild-type mice at 4 mM Ca(2+) , but largely absent at 2 mM external Ca(2+) . Mossy fiber LTD in Grm2/3 dko mice was not blocked by the N-methyl-D-aspartic acid (NMDA) receptor antagonist D-AP5, confirming that the observed response did not reflect NMDA receptor-dependent LTD in contaminating associational-commissural fibers, and enabling us to use the NMDA receptor-mediated EPSC to monitor mossy fiber LTD. Using whole-cell recordings, we demonstrated that LTD of the NMDA receptor-mediated EPSC in Grm2/3 dko mice was not affected by intracellular application of BAPTA and CsF to block postsynaptic Ca(2+) and G-protein-mediated effects. This presynaptic LTD was, however, blocked by the AMPA/kainate receptor antagonist, NBQX. Thus, an activity-dependent, external Ca(2+) concentration-sensitive form of mossy fiber LTD can be induced in Grm2/3 dko mice. Two mGluR antagonists also failed to block mossy fiber LTD under 4 mM conditions in wild-type mice, strengthening the conclusion that group II mGluRs are not obligatory for mossy fiber LTD.
Assuntos
Hipocampo/anatomia & histologia , Depressão Sináptica de Longo Prazo/genética , Fibras Musgosas Hipocampais/fisiologia , Receptores de Glutamato Metabotrópico/deficiência , Animais , Biofísica , Cálcio/metabolismo , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estimulação Elétrica/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Técnicas In Vitro , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Magnésio/metabolismo , Camundongos , Camundongos Knockout , Fibras Musgosas Hipocampais/efeitos dos fármacos , Técnicas de Patch-Clamp , Receptores de Glutamato Metabotrópico/antagonistas & inibidoresRESUMO
Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.
Assuntos
Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mapas de Interação de Proteínas , Aminoácidos/deficiência , Animais , Proteínas Sanguíneas/farmacologia , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Organogênese/efeitos dos fármacos , Análise de Componente Principal , Mapas de Interação de Proteínas/efeitos dos fármacos , Sirolimo/farmacologiaRESUMO
A novel imidazobenzazepine template (5a) with potent dual H(1)/5-HT(2A) antagonist activity was identified. Application of a zwitterionic approach to this poorly selective and poorly developable starting point successfully delivered a class of high quality leads, 3-[4-(3-R(1)-2-R-5H-imidazo[1,2-b][2]benzazepin-11-yl)-1-piperazinyl]-2,2-dimethylpropanoic acids (e.g., 9, 19, 20, and 21), characterized by potent and balanced H(1)/5-HT(2A) receptor antagonist activities and good developability profiles.
Assuntos
Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/uso terapêutico , Transtornos do Sono-Vigília/tratamento farmacológico , HumanosRESUMO
The optimisation of an HTS hit series (1) leading to the identification of structurally novel, selective, orally bioavailable mGluR2 positive modulators GSK1331258 and GSK1331268 is described. Structure-activity relationships, attenuation of dopaminergic activity, and potentiation of mGluR2 responses in rat hippocampal MPP-DG synapses are also reported.
Assuntos
Benzimidazóis/química , Piperazinas/química , Receptores de Glutamato Metabotrópico/metabolismo , Administração Oral , Regulação Alostérica , Animais , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Dopamina/metabolismo , Ensaios de Triagem em Larga Escala , Piperazinas/síntese química , Piperazinas/farmacologia , Ratos , Relação Estrutura-Atividade , Potenciais Sinápticos/efeitos dos fármacosRESUMO
The Parkinson's disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a crucial modulator of the autophagy-lysosome pathway, but unclarity exists on the precise mechanics of its role and the direction of this modulation. In particular, LRRK2 is involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and a significant proportion of LRRK2 patients presenting Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to obtain a comprehensive knowledge on LRRK2-associated pathology. Here, we investigated a G2019S-LRRK2 recombinant cell line exhibiting accumulation of endogenous, phosphorylated alpha-synuclein. We found that G2019S-LRRK2 leads to accumulation of LC3 and abnormalities in lysosome morphology and proteolytic activity in a kinase-dependent fashion, but independent from constitutively active Rab10. Notably, LRRK2 inhibition was ineffective upon upstream blockade of autophagosome-lysosome fusion events, highlighting this step as critical for alpha-synuclein clearance.
RESUMO
The finding that the mGlu2/3 metabotropic glutamate receptor agonist, LY404039, improves clinical symptoms in schizophrenia warrants a search for a possible interaction between mGlu2/3 receptors and dopamine D2 receptors. Here, this topic is examined in striatal tissue of mice lacking either mGlu2 or mGlu3 receptor. Such mice are known to be behaviorally supersensitive to dopamine receptor agonists. Therefore, to determine the basis of this dopamine supersensitivity, the proportion of dopamine D2(High) receptors was measured in the striata of mGlu2 and mGlu3 receptor knockout mice. The proportion of D2(High) receptors was found to be elevated by 220% in the striata of both knockouts. To measure the functional dopamine supersensitivity, the D2 agonist (+)PHNO was used to stimulate the incorporation of GTP-gamma-S in the striatal homogenates in the presence of drugs that blocked the dopamine D1, D3, and D5 receptors. Compared with control striata, the mGlu2 receptor knockout tissues were 67-fold more sensitive to (+)PHNO, while the mGlu3 receptor knockout tissues were 17-fold more sensitive. These data suggest that group II mGlu receptors-mGlu2 receptors in particular-may normally regulate D2 receptors by reducing the proportion of high-affinity D2 receptors in membranes. Such regulation may contribute to the antipsychotic action of mGlu2/3 receptor agonists.
Assuntos
Corpo Estriado/efeitos dos fármacos , Dopamina/farmacologia , Receptores de AMPA/deficiência , Receptores de Dopamina D2/metabolismo , Receptores de Glutamato Metabotrópico/deficiência , Vitamina K 1/análogos & derivados , Animais , Ligação Competitiva/efeitos dos fármacos , Domperidona/farmacocinética , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacocinética , Relação Dose-Resposta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Camundongos , Camundongos Knockout , Oxazinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Vitamina K 1/farmacologiaRESUMO
Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.
Assuntos
Sistemas CRISPR-Cas , Neurônios Dopaminérgicos/metabolismo , Edição de Genes , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/biossíntese , Células-Tronco Pluripotentes Induzidas/metabolismo , Reação em Cadeia da Polimerase , Transgenes , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Microscopia de FluorescênciaRESUMO
Dual metabotropic glutamate 2/3 (mGlu2/3) receptor agonists have been examined with success in the clinic with positive proof of efficacy in several tests of anxiety and schizophrenia. Moreover, a large body of evidence has accumulated that these drugs have significant neuroprotective potential. An important discussion in the field deals with dissecting effects on mGlu2 versus effects on mGlu3 receptors, which is relevant for the potential use of subtype-selective agonists or allosteric activators. We addressed this issue using mGlu2 and mGlu3 receptor knock-out mice. We used mixed cultures of cortical cells in which astrocytes and neurons were plated at different times and could therefore originate from different mice. Cultures were challenged with NMDA for the induction of excitotoxic neuronal death. The mGlu2/3 receptor agonist, (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), was equally neuroprotective in cultures containing neurons from wild-type, mGlu2-/-, or mGlu3-/- mice. Neuroprotection was instead abolished when astrocytes lacked mGlu3 receptors, unless neuronal mGlu2 receptors were also absent. The latter condition partially restored the protective activity of LY379268. Cultures in which neurons originated from mGlu2-/- mice were also intrinsically resistant to NMDA toxicity. In in vivo experiments, systemic administration of LY379268 protected striatal neurons against NMDA toxicity in wild-type and mGlu2-/- mice but not in mGlu3-/- mice. In addition, LY379268 was protective against nigrostriatal degeneration induced by low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine only in mice lacking mGlu2 receptors. We conclude that neuroprotection by mGlu2/3 receptor agonists requires the activation of astrocytic mGlu3 receptors, whereas, unexpectedly, activation of mGlu2 receptors might be harmful to neurons exposed to toxic insults.
Assuntos
Degeneração Neural/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Receptores de Glutamato Metabotrópico/deficiência , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/genética , Degeneração Neural/patologia , Receptores de Glutamato Metabotrópico/agonistasRESUMO
UNLABELLED: OBJECTIVES AND MATERIALS AND METHODS: The aims of the present study were (1) to determine the neuronal activation pattern elicited by the group II mGlu antagonist LY341495 and (2) to evaluate the contribution of each group II mGlu subtype by using wild-type (WT) and knockout (KO) mice lacking either mGlu2 or mGlu3. c-Fos expression was used as a marker of neuronal activation. RESULTS AND DISCUSSION: In WT mice, LY341495 induced widespread c-Fos expression in 68 out of 92 brain areas, including limbic areas such as the amygdala, septum, prefrontal cortex, and hippocampus. LY341495-induced c-Fos response was markedly decreased in the medial part of the central amygdala (CeM) and lateral septum (LS) in mGlu3-KO mice, as well as in the lateral parabrachial nucleus (LPB) in both KO strains. In the majority of investigated areas, LY341495-induced c-Fos expression was similar in KO and WT mice. Analysis of the cellular and subcellular distribution of mGlu2 and mGlu3 revealed a prevailing presence of mGlu3-immunoreactivity in the CeM in glial processes and in postsynapstic neuronal elements, whereas only rare presynaptic axon terminals were found immunoreactive for mGlu2. CONCLUSION: In conclusion, our data indicate that group II mGlu blockade increases neuronal activation in a variety of brain areas, including many stress- and anxiety-related areas. The activation of two key brain areas, the CeM and LS, is mediated via mGlu3, while activation in the LPB involves both subtypes. Moreover, in the majority of investigated areas, LY341495-mediated neuronal activation appears to require a complex cross talk between group II mGlu subtypes or the action of LY341495 on additional receptors.
Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Genes fos/efeitos dos fármacos , Genes fos/genética , Receptores de Glutamato Metabotrópico/deficiência , Aminoácidos/farmacologia , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/ultraestrutura , Animais , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Genes fos/imunologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/ultraestrutura , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/imunologia , Septo do Cérebro/efeitos dos fármacos , Septo do Cérebro/metabolismo , Septo do Cérebro/ultraestrutura , Xantenos/farmacologiaRESUMO
Group II metabotropic glutamate receptors (mGluR2 and mGluR3, also called mGlu2 and mGlu3, encoded by GRM2 and GRM3, respectively) are therapeutic targets for several psychiatric disorders. GRM3 may also be a schizophrenia susceptibility gene. mGluR2-/- and mGluR3-/- mice provide the only unequivocal means to differentiate between these receptors, yet interpretation of in vivo findings may be complicated by secondary effects on expression of other genes. To address this issue, we examined the expression of NMDA receptor subunits (NR1, NR2A, NR2B) and glutamate transporters (EAAT1-3), as well as the remaining group II mGluR, in the hippocampus of mGluR2-/- and mGluR3-/- mice, compared with wild-type controls. mGluR2 mRNA was increased in mGluR3-/- mice, and vice versa. NR2A mRNA was increased in both knockout mice. EAAT1 (GLAST) mRNA and protein, and EAAT2 (GLT-1) protein, were reduced in mGluR3-/- mice, whereas EAAT3 (EAAC1) mRNA was decreased in mGluR2-/- mice. Transcripts for NR1 and NR2B were unchanged. The findings show a compensatory upregulation of the remaining group II metabotropic glutamate receptor in the knockout mice. Upregulation of NR2A expression suggests modified NMDA receptor signaling in mGluR2-/- and mGluR3-/- mice, and downregulation of glutamate transporter expression suggests a response to altered synaptic glutamate levels. The results show a mutual interplay between mGluR2 and mGluR3, and also provide a context in which to interpret behavioral and electrophysiological results in these mice.
Assuntos
Regulação da Expressão Gênica/genética , Proteínas de Transporte de Glutamato da Membrana Plasmática/deficiência , Proteínas de Transporte de Glutamato da Membrana Plasmática/genética , Hipocampo/metabolismo , Receptores de Glutamato Metabotrópico/deficiência , Receptores de Glutamato Metabotrópico/genética , Sistema X-AG de Transporte de Aminoácidos/biossíntese , Sistema X-AG de Transporte de Aminoácidos/deficiência , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Regulação para Baixo/genética , Transportador 1 de Aminoácido Excitatório/antagonistas & inibidores , Transportador 1 de Aminoácido Excitatório/biossíntese , Transportador 1 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/antagonistas & inibidores , Transportador 2 de Aminoácido Excitatório/biossíntese , Transportador 2 de Aminoácido Excitatório/genética , Transportador 3 de Aminoácido Excitatório/antagonistas & inibidores , Transportador 3 de Aminoácido Excitatório/biossíntese , Transportador 3 de Aminoácido Excitatório/genética , Proteínas de Transporte de Glutamato da Membrana Plasmática/biossíntese , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Receptores de Glutamato/biossíntese , Receptores de Glutamato/genética , Receptores de Glutamato Metabotrópico/biossíntese , Regulação para Cima/genéticaRESUMO
Primary neuronal culture from rodents is a well-established model to investigate cellular neurobiology in vitro. However, for this purpose cell cultures need to be generated expressly, requiring extensive animal handling. Furthermore, often the preparation of fresh culture generates an excess of cells that are ultimately wasted. Therefore the ability to successfully cryopreserve primary neural cells would represent an important resource for neuroscience research and would allow to significantly reduce the sacrifice of animals. We describe here a novel freezing medium that allows long-term cryopreservation of primary mouse neurons prepared from E15.5 embryos. Combining imaging, biochemical and electrophysiological analyses, we found that cryopreserved cultures are viable and mature regarding morphology and functionality. These findings suggest that cryopreserved neurons are a valuable alternative to acutely dissociated neural cultures.