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1.
Int J Mol Sci ; 23(16)2022 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-36012727

RESUMO

A popular approach to spatiotemporally target genes using the loxP/Cre recombination system is stereotaxic microinjection of adeno-associated virus (AAV) expressing Cre recombinase (AAV_Cre) in specific neuronal structures. Here, we report that AAV_Cre microinjection in the ventral tegmental area (VTA) of ErbB4 Cyt-1-floxed (ErbB4 Cyt-1fl/fl) mice at titers commonly used in the literature (~1012-1013 GC/mL) can have neurotoxic effects on dopaminergic neurons and elicit behavioral abnormalities. However, these effects of AAV_Cre microinjection are independent of ErbB4 Cyt-1 recombination because they are also observed in microinjected wild-type (WT) controls. Mice microinjected with AAV_Cre (1012-1013 GC/mL) exhibit reductions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) expression, loss of dopaminergic neurons, and they behaviorally become hyperactive, fail to habituate in the open field and exhibit sensorimotor gating deficits compared to controls microinjected with AAV_GFP. Importantly, these AAV_Cre non-specific effects are: (1) independent of serotype, (2) occur with vectors expressing either Cre or Cre-GFP fusion protein and (3) preventable by reducing viral titers by 1000-fold (1010 GC/mL), which retains sufficient recombination activity to target floxed genes. Our studies emphasize the importance of including AAV_Cre-injected WT controls in experiments because recombination-independent effects on gene expression, neurotoxicity and behaviors could be erroneously attributed to consequences of gene ablation.


Assuntos
Dependovirus , Neurônios Dopaminérgicos , Transdução Genética , Animais , Dependovirus/genética , Dependovirus/metabolismo , Neurônios Dopaminérgicos/metabolismo , Integrases/genética , Integrases/metabolismo , Camundongos , Receptor ErbB-4/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
2.
Med Biol Eng Comput ; 60(10): 2995-3007, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36018532

RESUMO

Computerized techniques for image analysis are critical for progress in cell biology. The complexity of the data in current methods eliminates the need for manual image analysis and usually requires the application of multiple algorithms sequentially to the images. Our aim was to develop a software for immunohistochemical analysis of brain dopaminergic neurons combining several computational approaches to automatically analyze and quantify their number in the substantia nigra after a neurotoxic injury. For this purpose, we used a Parkinson's disease animal model to test our application. The dopaminergic neurotoxin, 6-hydroxydopamine, was administered in adult male rats to damage dopaminergic neurons in substantia nigra and to induce hemiparkinsonism. The lesion was corroborated by behavioral evaluation in response to apomorphine and amphetamine. The animals were euthanized and their brains processed for tyrosine hydroxylase immunohistochemistry for dopamine neuron identification. Neurons positive for tyrosine hydroxylase were evaluated in substantia nigra by light microscopy. The images were used to show quantification applicability. To test our software counting accuracy and validity, automatic dopamine neuron number was correlated with the data obtained by three independent observers. Several parameters were used to depict neuronal function in dataset images from control and lesioned brains. In conclusion, we could perform an automated quantification of dopaminergic neurons and corroborate the validity and accuracy of a freely available software.


Assuntos
Neurônios Dopaminérgicos , Tirosina 3-Mono-Oxigenase , Animais , Neurônios Dopaminérgicos/metabolismo , Masculino , Oxidopamina/toxicidade , Ratos , Software , Substância Negra/metabolismo , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/metabolismo
3.
Eur J Pharmacol ; 932: 175204, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-35964655

RESUMO

Current treatments for Parkinson's Disease (PD) only provide symptomatic relief; however, they don't delay the disease progression, hence new treatment options should be considered. Carvedilol is a nonselective ß & α1 blocker with additional effects as an antioxidant, anti-inflammatory and neuro protective properties. In this research, an insilico study was conducted to primarily evaluate carvedilol as an anti-parkinsonian and anti-tau protein target. PASS prediction was performed followed by a docking study of carvedilol. Carvedilol yielded promising results and forward guided this study onto its in vivo evaluation. The in vivo study aimed to assess the neuro-protective effects of carvedilol in rotenone-induced rat model of PD and investigate the potential underlying mechanisms. The effects of carvedilol (2.5, 5, and 10 mg/kg) on the measured parameters of open field, catalepsy, Y-maze tests as well as brain histology, and tyrosine hydroxylase (TH) were evaluated. The effective doses (5 and 10 mg/kg) were further tested for their potential anti-tau protein effects. Carvedilol (5 and 10 mg/kg) prevented rotenone-induced motor deficits, spatial memory dysfunction, and histological damage. Additionally, carvedilol significantly inhibited rotenone-induced decrease in TH expression in the striata of the rats. These effects were associated with reduction of rotenone-induced neuro-inflammation, microglial activation and release of glial fibrillary acidic protein (GFAP), along with reduction in N-methyl-D-aspartate receptors activation, alpha-synculein and phospho-Tau (P-Tau) protein expression. Carvedilol also reduced tau protein hyper-phosphosrylation by Glycogen synthase 3ß (GSK 3ß) inhibition and Phosphoinositide 3-kinase (PI3K) stimulation. Collectively, these results suggest that carvedilol might be a possible candidate for management of PD.


Assuntos
Fármacos Neuroprotetores , Síndromes Neurotóxicas , Doença de Parkinson , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Carvedilol/farmacologia , Carvedilol/uso terapêutico , Proteína Glial Fibrilar Ácida/metabolismo , Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato , Rotenona/toxicidade , Tirosina 3-Mono-Oxigenase/metabolismo
4.
Neuroimage ; 260: 119494, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-35870696

RESUMO

The complex organization of brain regions during development requires a three-dimensional approach to facilitate the visualization and quantification of dynamic changes taking place throughout this important period. Using the tissue clearing method combined with immunohistochemistry, three-dimensional (3D) lightsheet microscopy and a multiresolution registration technique, we provide the first 3D atlases of the main cholinergic (CH) and catecholaminergic (CA) systems in the mouse brain from embryonic day 12 (E12) to post-natal day 8 (P8). We report that in several brain structures, there is a logarithmic scale increase of choline acetyltransferase and tyrosine hydroxylase positive neurons from E18 to P8. In addition, a detailed voxel-wise analysis revealed abrupt modifications in the developmental trajectory of many brain structures during the transition from E18 to P0. Our atlases will not only facilitate developmental studies aimed at quantitatively determining the fate of CH or CA neurons in utero but also be used as an anatomical reference to quantify other neuronal populations present in the annotated regions. In the future, these maps will be a reliable tool to study developmental malformations associated with neurological and psychiatric disorders.


Assuntos
Colina O-Acetiltransferase , Tirosina 3-Mono-Oxigenase , Animais , Encéfalo/metabolismo , Colina O-Acetiltransferase/metabolismo , Colinérgicos , Humanos , Camundongos , Neurônios/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
5.
J Chem Neuroanat ; 124: 102136, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35809809

RESUMO

Senescence is a natural and progressive physiological event that leads to a series of morphophysiological alterations in the organism. The brain is the most vulnerable organ to both structural and functional changes during this process. Dopamine is a key neurotransmitter for the proper functioning of the brain, directly involved in circuitries related with emotions, learning, motivation and reward. One of the main dopamine- producing nuclei is the substantia nigra pars compacta (SNpc), which establish connections with the striatum forming the so-called nigrostriatal pathway. S100B is a calcium binding protein mainly expressed by astrocytes, involved in both intracellular and extracellular processes, and whose expression is increased following injury in the nervous tissue, being a useful marker in altered status of central nervous system. The present study aimed to analyze the impact of senescence on the cells immunoreactive for tyrosine hydroxylase (TH) and S100B along the nigrostriatal pathway of the rat. Our results show an decreased expression of S100B+ cells in SNpc. In addition, there was a significant decrease in TH immunoreactivity in both projection fibers and TH+ cell bodies. In the striatum, a decrease in TH immunoreactivity was also observed, as well as an enlargement of the white matter bundles. Our findings point out that senescence is related to the anatomical and neurochemical changes observed throughout the nigrostriatal pathway.


Assuntos
Dopamina , Tirosina 3-Mono-Oxigenase , Animais , Astrócitos/metabolismo , Corpo Estriado/metabolismo , Dopamina/metabolismo , Ratos , Subunidade beta da Proteína Ligante de Cálcio S100/análise , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/farmacologia , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
6.
J Neural Transm (Vienna) ; 129(10): 1257-1270, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35852604

RESUMO

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in the cell bodies of dopaminergic neurons in the substantia nigra (SN) pars compacta. These neurons are lost in neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. Although it is known that lipids, proteins, and environmental toxins accumulate in NMGs, the function of NMGs has not yet been finally clarified as well as their origin and the synthesis of neuromelanin. We, therefore, isolated NMGs and surrounding SN tissue from control patients by laser microdissection and analyzed the proteomic profile by tandem mass spectrometry. With our improved workflow, we were able to (1) strengthen the regularly reported link between NMGs and lysosomes, (2) detect tyrosine hydroxylase to be highly abundant in NMGs, which may be related to neuromelanin synthesis and (3) indicate a yet undescribed link between stress granules (SGs) and NMGs. Based on our findings, we cautiously hypothesize, that SGs may be the origin of NMGs or form in close proximity to them, potentially due to the oxidative stress caused by neuromelanin-bound metals.


Assuntos
Proteômica , Tirosina 3-Mono-Oxigenase , Humanos , Lisossomos/metabolismo , Melaninas/metabolismo , Proteômica/métodos , Grânulos de Estresse , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
7.
J Chem Neuroanat ; 124: 102135, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35792205

RESUMO

Protein tyrosine phosphatase receptor U (PTPRU) is involved in midbrain patterning during early stages of development and is continuously expressed in the adult midbrain areas where dopaminergic neurons reside in. However, whether PTPRU is also involved in the maintenance and survival of midbrain dopaminergic (mDA) neurons during the late stages of development or in the adult midbrain remains largely unknown. In the present study, Ptpru was ablated by crossing a floxed Ptpru mouse strain with tyrosine hydroxylase (TH)-Cre mice that express Cre recombinase in postmitotic mDA neurons. Conditional ablation of Ptpru in postmitotic mDA neurons resulted in a reduction of somatic and nuclear size in adulthood. However, TH-immunoreactivity of Ptpru-ablated mDA neurons and their projections to the striatum appeared undisturbed. We also investigated the maintenance of several mDA neuronal markers following Ptpru ablation and found no significant changes. Taken together, these findings suggest that PTPRU is involved in regulating the neuronal size of mDA neurons and provided mechanistic insights into the development and maintenance of mDA neurons.


Assuntos
Neurônios Dopaminérgicos , Mesencéfalo , Animais , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/metabolismo , Camundongos , Proteínas Tirosina Fosfatases/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
8.
Transl Psychiatry ; 12(1): 238, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35672280

RESUMO

Dopaminergic (DA) dysfunction is a significant feature in the pathophysiology of schizophrenia. Established developmental risk factors for schizophrenia such as maternal immune activation (MIA) or developmental vitamin D (DVD) deficiency, when modelled in animals, reveal the differentiation of early DA neurons in foetal brains is delayed suggesting this may be a convergent aetiological pathway. Here we have assessed the effects of prenatal hypoxia, another well-known developmental risk factor for schizophrenia, on developing DA systems. Pregnant mice were exposed to a hypoxic environment of 10% oxygen for 48 h from embryonic day 10 (E10) to E12. Embryonic brains were collected and the positioning of mesencephalic cells, expression of DA specification and maturation factors were examined along with the expression of factors that may govern the migration of these neurons. We show that prenatal hypoxia results in a decrease in dopaminergic progenitors retards early DA neuron lateral migration and reduces expression of the receptors known to govern this process. A second time-point, postnatal day 10 (P10) was also examined in order to assess whether prenatal hypoxia alters early presynaptic architecture in the developing striatum. We show reduced expression of tyrosine hydroxylase (TH) in the postnatal striatum along with increases in the density of high-probability DA release sites within TH varicosities. These findings add to the emerging literature showing that multiple epidemiologically validated environmental risk factors for schizophrenia may induce early alterations to develop DA systems. This may represent a possible convergent mechanism in the onset of presynaptic DA dysfunction in patients.


Assuntos
Neurônios Dopaminérgicos , Mesencéfalo , Animais , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Feminino , Humanos , Hipóxia/metabolismo , Mesencéfalo/metabolismo , Camundongos , Gravidez , Tirosina 3-Mono-Oxigenase/metabolismo
9.
J Comp Neurol ; 530(14): 2562-2586, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35715989

RESUMO

Calcium-binding proteins (CBPs) regulate neuronal function in midbrain dopamine (DA)-ergic neurons in mammals by buffering and sensing the intracellular Ca2+ , and vesicular release. In birds, the equivalent set of neurons are important in song learning, directed singing, courtship, and energy balance, yet the status of CBPs in these neurons is unknown. Herein, for the first time, we probe the nature of CBPs, namely, Calbindin-, Calretinin-, Parvalbumin-, and Secretagogin-expressing DA neurons in the ventral tegmental area (VTA) and substantia nigra (SN) in the midbrain of zebra finch, Taeniopygia guttata. qRT-PCR analysis of ventral midbrain tissue fragment revealed higher Calbindin- and Calretinin-mRNA levels compared to Parvalbumin and Secretagogin. Application of immunofluorescence showed CBP-immunoreactive (-i) neurons in VTA (anterior [VTAa], mid [VTAm], caudal [VTAc]), SN (compacta [SNc], and reticulata [SNr]). Compared to VTAa, higher Calbindin- and Parvalbumin-immunoreactivity (-ir), and lower Calretinin-ir were observed in VTAm and VTAc. Secretagogin-ir was highly localized to VTAa. In SN, Calbindin- and Calretinin-ir were higher in SNc, SNr was Parvalbumin enriched, and Secretagogin-ir was not detected. Weak, moderate, and intense tyrosine hydroxylase (TH)-i VTA neurons were demarcated as subtypes 1, 2, and 3, respectively. While subtype 1 TH-i neurons were neither Calbindin- nor Calretinin-i, ∼80 and ∼65% subtype 2 and ∼30 and ∼45% subtype 3 TH-i neurons co-expressed Calbindin and Calretinin, respectively. All TH-i neuronal subtypes co-expressed Parvalbumin with reciprocal relationship with TH-ir. We suggest that the CBPs may determine VTA DA neuronal heterogeneity and differentially regulate their activity in T. guttata.


Assuntos
Tentilhões , Área Tegmentar Ventral , Animais , Calbindina 2/metabolismo , Calbindinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Neurônios Dopaminérgicos/metabolismo , Tentilhões/metabolismo , Mamíferos , Parvalbuminas/metabolismo , Proteína G de Ligação ao Cálcio S100/análise , Proteína G de Ligação ao Cálcio S100/metabolismo , Secretagoginas/metabolismo , Substância Negra , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/metabolismo
10.
Neurochem Res ; 47(8): 2317-2332, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35661962

RESUMO

The effects of prenatal opioid exposure in adult animals has been widely studied, but little is known about the effects of prenatal opioid on adolescents. Most of the risk behaviors associated with drug abuse are initiated during adolescence. The developmental state of the adolescent brain makes it vulnerable to initiate drug use and susceptible to drug-induced brain changes. In this study, pregnant rats were subcutaneously injected with an increasing dose of morphine (5 mg/kg, 7 mg/kg, 10 mg/kg) for 9 days since the gestation day 11. The effects of prenatal morphine (PNM) on learning and memory, anxiety- and depressive- like behavior, morphine induced conditioned place preference (CPP) as well as locomotor sensitization were tested in both adolescent and adult rats. The results showed that: (1) PNM decreased anxiety-like behavior in both adolescent and adult female rats, but not males; (2) PNM decreased depressive-like behavior in adolescent but increased depressive -like behavior in adult females; (3) PNM increased low dose morphine induced locomotor sensitization in females; (4) PNM decreased tyrosine hydroxylase (TH) expression in the prefrontal cortex but decreased dopamine D1 receptor expression in the nucleus-accumbens (NAc) in female rats. These results suggested that PNM altered the emotional and addictive behavior mainly in female rats, with female rats being less anxiety and depressive during adolescence, but more depressive in adult, and more sensitive to low dose morphine induced locomotor activity sensitization, which might be mediated in part by the differential expression of the TH, dopamine D1 receptors in the female brain.


Assuntos
Comportamento Aditivo , Morfina , Efeitos Tardios da Exposição Pré-Natal , Analgésicos Opioides/efeitos adversos , Animais , Comportamento Animal , Condicionamento Clássico , Feminino , Masculino , Morfina/efeitos adversos , Núcleo Accumbens/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal/psicologia , Ratos , Fatores Sexuais , Tirosina 3-Mono-Oxigenase/metabolismo
11.
Beijing Da Xue Xue Bao Yi Xue Ban ; 54(3): 421-426, 2022 Jun 18.
Artigo em Chinês | MEDLINE | ID: mdl-35701117

RESUMO

OBJECTIVE: To explore whether the using of mimetic peptide Gap27, a selective inhibitor of connexin 43 (Cx43), could block the death of dopamine neurons and influence the expression of Cx43 in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease mouse models. METHODS: Eighteen C57BL/6 mice were randomly divided into control group, 6-OHDA group and 6-OHDA+Gap27 group, with 6 mice in each group. Bilateral substantia nigra stereotactic injection was performed. The control group was injected with ascorbate solution, 6-OHDA group was injected with 6-OHDA solution, and 6-OHDA+Gap27 group was injected with 6-OHDA and Gap27 mixed solution. Immuno-histochemical staining was used to detect the number of dopamine neurons, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of Cx43 messenger ribonucleic acid (mRNA), immuno-fluorescence staining was used to detect the distribution of Cx43 protein, the contents of Cx43 protein and Cx43 phosphorylation at serine 368 (Cx43-ps368) in mouse midbrain were detected by Western blot. RESULTS: After injection of 6-OHDA, numerous dopamine neurons in substantia nigra died as Cx43 content increased, Cx43-ps368 content decreased. Mixing Gap27 while injecting 6-OHDA could reduce the number of death dopamine neurons and weaken the changes of Cx43 and Cx43-ps368 content caused by 6-OHDA. The number of tyrosine hydroxylase (TH) immunoreactive positive neurons in 6-OHDA group decreased to 27.7% ± 0.02% of the control group (P < 0.01); The number of TH immunoreactive positive neurons in 6-OHDA+Gap27 group was (1.64±0.16) times higher than that in 6-OHDA group (P < 0.05); The content of total Cx43 protein in 6-OHDA group was (1.44±0.07) times higher than that in 6-OHDA+Gap27 group (P < 0.05) while (1.68±0.07) times higher than that in control group (P < 0.01). In 6-OHDA group, the content of Cx43-ps368 protein and its proportion in total Cx43 protein were significantly lower than that in 6-OHDA+Gap27 group (P < 0.05). CONCLUSION: In 6-OHDA mouse models, mimetic peptide Gap27 played a protective role in reducing the damage to substantia nigra dopamine neurons, which was induced by 6-OHDA. The overexpression of Cx43 protein might have neurotoxicity to dopamine neuron. Meanwhile, decreasing Cx43 protein level and keeping Cx43-ps368 protein level may be the protective mechanisms of Gap27.


Assuntos
Doença de Parkinson , Animais , Conexina 43/genética , Conexina 43/metabolismo , Conexina 43/farmacologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oxidopamina/efeitos adversos , Oxidopamina/metabolismo , Doença de Parkinson/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismo , Tirosina 3-Mono-Oxigenase/farmacologia
12.
Int J Mol Sci ; 23(11)2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35682548

RESUMO

Sleep apnea syndrome (SAS), characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia (IH)), is a risk factor for hypertension and insulin resistance. We report a correlation between IH and insulin resistance/diabetes. However, the reason why hypertension is induced by IH is elusive. Here, we investigated the effect of IH on the expression of catecholamine-metabolizing enzymes using an in vitro IH system. Human and mouse neuroblastoma cells (NB-1 and Neuro-2a) were exposed to IH or normoxia for 24 h. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of dopamine ß-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in both NB-1 and Neuro-2a. Western blot showed that the expression of DBH and PNMT in the NB-1 cells was significantly increased by IH. Reporter assays revealed that promoter activities of DBH and PNMT were not increased by IH. The miR-375 level of IH-treated cells was significantly decreased relative to that of normoxia-treated cells. The IH-induced up-regulation of DBH and PNMT was abolished by the introduction of the miR-375 mimic, but not by the control RNA. These results indicate that IH stress increases levels of DBH and PNMT via the inhibition of miR-375-mediated mRNA degradation, potentially playing a role in the emergence of hypertension in SAS patients.


Assuntos
Hipertensão , Resistência à Insulina , MicroRNAs , Neuroblastoma , Animais , Dopamina beta-Hidroxilase/metabolismo , Humanos , Hipóxia/genética , Camundongos , MicroRNAs/genética , Neuroblastoma/genética , Feniletanolamina N-Metiltransferase/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
13.
Int J Mol Sci ; 23(12)2022 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-35743179

RESUMO

Since the 1980s, the concept of dopamine-rich brain centers as clusters of only dopaminergic neurons has been fundamentally revised. It has been shown that, in addition to dopaminergic neurons, most of these centers contain neurons expressing one of the enzymes of dopamine synthesis: tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC). We have obtained convincing evidence that in rats, the hypothalamic periventricular nucleus (PeVN) is one of the largest dopamine-rich centers, containing dopaminergic and monoenzymatic neurons. Indeed, using double immunostaining for TH and AADC, the PeVN was shown to contain almost three thousand dopaminergic and monoenzymatic neurons. According to high-performance liquid chromatography, PeVN contains L-DOPA and dopamine, which, apparently, are synthesized in monoenzymatic TH neurons and bienzymatic neurons, respectively. According to confocal microscopy, neurons (cell bodies, fibers), which were immunopositive only to TH, only to AADC, or both, are in close topographic relationships with each other and with the 3rd ventricle. These data suggest the mutual regulation of the neurons, as well as the delivery of dopamine and L-DOPA to the third ventricle, which is confirmed by their detection in the cerebrospinal fluid. Thus, evidence has been obtained that PeVN is one of the largest dopamine-rich centers of the brain, containing dopaminergic and monoenzymatic neurons.


Assuntos
Dopamina , Levodopa , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Encéfalo/metabolismo , Neurônios Dopaminérgicos/metabolismo , Ratos , Tirosina 3-Mono-Oxigenase/metabolismo
14.
Neuroreport ; 33(8): 336-344, 2022 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-35594436

RESUMO

OBJECTIVES: The translation elongation factor-1, alpha-2 (eEF1A2) plays an important role in protein synthesis. Mutations in this gene have been described in individuals with neurodevelopmental disorders. Here, we silenced the expression of eEFA2 in human SH-SY5Y neuroblastoma cells and observed its roles in neuronal proliferation and differentiation upon induction with retinoic acid. METHODS: eEF1A2 were silenced using siRNA transfection. Cell proliferation was qualitatively evaluated by Ki-67 immunocytochemistry. Neuronal differentiation was induced with retinoic acid for 3, 5, 7 and 10 days. Neurite length was measured. The expression of microtubule-associated protein 2 (MAP2) was analyzed by western blotting. Tyrosine hydroxylase expression was visualized by immunofluorescence. Cytotoxicity to a neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and western blotting of cleaved caspase-3. RESULTS: eEF1A2 knockdown suppressed the proliferative activity of undifferentiated SH-SY5Y cells as shown by decreased Ki-67 immunostaining. Upon retinoic acid-induction, differentiated neurons with eEF1A2 knockdown exhibited shorter neurite length than untransfected cells, which was associated with the reduction of tyrosine hydroxylase and suppression of MAP2 at 10 days of differentiation. eEF1A2 knockdown decreased the survival of neurons, which was clearly observed in undifferentiated and short-term differentiated cells. Upon treatment with MPP+, cells with eEF1A2 knockdown showed a further reduction in cell survival and an increase of cleaved caspase-3 protein. CONCLUSIONS: Our results suggest that eEF1A2 may be required for neuronal proliferation and differentiation of SH-SY5Y cells. Increased cell death susceptibility against MPP+ in eEF1A2-knockdown neurons may imply the neuroprotective role of eEF1A2.


Assuntos
Proliferação de Células , Neuroblastoma , Crescimento Neuronal , Fator 1 de Elongação de Peptídeos , 1-Metil-4-fenilpiridínio/toxicidade , Caspase 3/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Antígeno Ki-67/metabolismo , Neuroblastoma/metabolismo , Crescimento Neuronal/genética , Neurônios/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Tretinoína/metabolismo , Tretinoína/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismo
15.
Exp Anim ; 71(3): 399-410, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35584940

RESUMO

We previously showed that a diet containing calcium carbonate causes impairments in spatial and recognition memory in mice. In this study, we investigated the effects of calcium carbonate supplementation on motor function. Motor function was determined using different tests that have been used to analyze different aspects of Parkinsonism. A catalepsy test for akinesia; a muscular strength assessment, pole test, beam-walking test, and gait analysis for motor coordination and balance assessment; and an open-field test for locomotor activity assessment were performed. The mice were fed diets containing 0.6% or 1.0% calcium carbonate for eight weeks, after which they were evaluated for motor functions. The diets containing calcium carbonate caused significant motor dysfunction, as revealed by the different tests, although the spontaneous locomotor activity did not change. Calcium carbonate supplementation decreased the dopamine content in the basal ganglia, including the striatum and substantia nigra, and the number of tyrosine hydroxylase-positive neurons in the substantia nigra. In addition, administration of L-dopa led to at least a partial recovery of motor dysfunction, suggesting that calcium carbonate supplementation causes motor dysfunction by decreasing the dopamine content in the basal ganglia. These results suggest that mice with calcium carbonate-induced motor dysfunction may be useful as a new animal model for Parkinson's disease and Huntington's disease.


Assuntos
Carbonato de Cálcio , Suplementos Nutricionais , Dopamina , Transtornos Motores , Animais , Carbonato de Cálcio/efeitos adversos , Suplementos Nutricionais/efeitos adversos , Modelos Animais de Doenças , Camundongos , Transtornos Motores/induzido quimicamente , Doença de Parkinson , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
16.
eNeuro ; 9(3)2022.
Artigo em Inglês | MEDLINE | ID: mdl-35580986

RESUMO

The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH)-positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with Fluoro-Gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP- and TH-/VIP+ neurons. The DAT+/TH-/VIP+ subpopulation would be non-DA noncanonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH-/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP- and TH-/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.


Assuntos
Núcleo Dorsal da Rafe , Substância Cinzenta Periaquedutal , Tonsila do Cerebelo/metabolismo , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina , Neurônios Dopaminérgicos/metabolismo , Núcleo Dorsal da Rafe/metabolismo , Camundongos , Substância Cinzenta Periaquedutal/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Peptídeo Intestinal Vasoativo
17.
Pest Manag Sci ; 78(9): 3859-3870, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35524967

RESUMO

BACKGROUND: Tyrosine hydroxylase (TH), a melanin synthesis pathway enzyme hydroxylating tyrosine into 3,4-dihydroxyphenylalanine, is involved in the pigmentation and sclerotization of insect cuticles. However, the role of TH in 28-spotted potato ladybeetle (Henosepilachna vigintioctopunctata), an emerging pest of the solanaceous crops has been explored to a limited extent. In this study, we integrated dietary RNA interference (RNAi) and hematoxylin and eosin (H&E) staining with various bioassays to analyze the role of tyrosine hydroxylase (HvTH) throughout the developmental processes of Henosepilachna vigintioctopunctata. RESULTS: The results revealed that ingestion of dsHvTH led to cuticle tanning impairment, arrested larval feeding in the first and second instars of Henosepilachna vigintioctopunctata, and subsequently resulted in 100% mortality. The H&E staining assays revealed that dsHvTH prevented new abdominal cuticle formation. A pharmacological study using 3-iodo-tyrosine (3-IT), a HvTH inhibitor, disrupted larval-larval-pupal cuticle tanning during the third-fourth instar larval development and eventually failed to pupate. Similarly, dsHvTH fed to fourth instars hindered larval-pupal-adult cuticle tanning, and the eclose adults were 100% malformed. Ingestion of dsHvTH or 3-IT significantly down-regulated HvTH, HvDDC, Hvebony, and Hvlaccase2 expression and reduced dopamine levels. Finally, HvTH silencing in adult females substantially reduced the offspring hatching rates. CONCLUSIONS: The collective results of the study suggested that HvTH plays conserved roles in larval-pupal-adult cuticle melanization and sclerotization while exhibiting a novel function in Henosepilachna vigintioctopunctata reproduction. © 2022 Society of Chemical Industry.


Assuntos
Besouros , Solanum tuberosum , Animais , Besouros/metabolismo , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva , Pupa , Interferência de RNA , Reprodução , Solanum tuberosum/metabolismo , Tirosina/genética , Tirosina/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
18.
J Parkinsons Dis ; 12(5): 1463-1478, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35527562

RESUMO

BACKGROUND: Parkinson's disease involves aberrant aggregation of the synaptic protein alpha-synuclein (aSyn) in the nigrostriatal tract. We have previously shown that proSAAS, a small neuronal chaperone, blocks aSyn-induced dopaminergic cytotoxicity in primary nigral cultures. OBJECTIVE: To determine if proSAAS overexpression is neuroprotective in animal models of Parkinson's disease. METHODS: proSAAS- or GFP-encoding lentivirus was injected together with human aSyn-expressing AAV unilaterally into the substantia nigra of rats and motor asymmetry assessed using a battery of motor performance tests. Dopamine neuron survival was assessed by nigral stereology and striatal tyrosine hydroxylase (TH) densitometry. To examine transsynaptic spread of aSyn, aSyn AAV was injected into the vagus of mice in the presence of AAVs encoding either GFP or proSAAS; the spread of aSyn-positive neurites into rostral nuclei was quantified following immunohistochemistry. RESULTS: Coinjection of proSAAS-encoding lentivirus profoundly reduced the motor asymmetry caused by unilateral nigral AAV-mediated human aSyn overexpression. This was accompanied by significant amelioration of the human aSyn-induced loss of both nigral TH-positive cells and striatal TH-positive terminals, demonstrating clear proSAAS-mediated protection of the nigrostriatal tract. ProSAAS overexpression reduced human aSyn protein levels in nigra and striatum and reduced the loss of TH protein in both regions. Following vagal administration of human aSyn-encoding AAV, the number of human aSyn-positive neurites in the pons and caudal midbrain was considerably reduced in mice coinjected with proSAAS-, but not GFP-encoding AAV, supporting proSAAS-mediated blockade of transsynaptic aSyn transmission. CONCLUSION: The proSAAS chaperone may represent a promising target for therapeutic development in Parkinson's disease.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Camundongos , Neuroproteção , Doença de Parkinson/terapia , Ratos , Roedores/metabolismo , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/metabolismo
19.
Mov Disord ; 37(7): 1394-1404, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35579496

RESUMO

BACKGROUND: Viral induction of neurological syndromes has been a concern since parkinsonian-like features were observed in patients diagnosed with encephalitis lethargica subsequent to the 1918 influenza pandemic. Given the similarities in the systemic responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with those observed after pandemic influenza, there is a question whether a similar syndrome of postencephalic parkinsonism could follow coronavirus disease 2019 infection. OBJECTIVE: The goal of this study was to determine whether prior infection with SARS-CoV-2 increased sensitivity to a mitochondrial toxin known to induce parkinsonism. METHODS: K18-hACE2 mice were infected with SARS-CoV-2 to induce mild-to-moderate disease. After 38 days of recovery, mice were administered a non-lesion-inducing dose of the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and euthanized 7 days later. Subsequent neuroinflammation and substantia nigra pars compacta (SNpc) dopaminergic (DA) neuron loss were determined and compared with SARS-CoV-2 or MPTP alone. RESULTS: K18-hACE2 mice infected with SARS-CoV-2 or MPTP showed no SNpc DA neuron loss after MPTP. In mice infected and recovered from SARS-CoV-2 infection, MPTP induced a 23% or 19% greater loss of SNpc DA neurons than SARS-CoV-2 or MPTP, respectively (P < 0.05). Examination of microglial activation showed a significant increase in the number of activated microglia in both the SNpc and striatum of the SARS-CoV-2 + MPTP group compared with SARS-CoV-2 or MPTP alone. CONCLUSIONS: Our observations have important implications for long-term public health, given the number of people who have survived SARS-CoV-2 infection, as well as for future public policy regarding infection mitigation. However, it will be critical to determine whether other agents known to increase risk for PD also have synergistic effects with SARS-CoV-2 and are abrogated by vaccination. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Assuntos
COVID-19 , Influenza Humana , Transtornos Parkinsonianos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , Animais , COVID-19/complicações , Modelos Animais de Doenças , Dopamina , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Transtornos Parkinsonianos/induzido quimicamente , SARS-CoV-2 , Tirosina 3-Mono-Oxigenase/metabolismo
20.
Sci Rep ; 12(1): 7610, 2022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35534594

RESUMO

In addition to well characterized motor symptoms, visual disturbances are increasingly recognized as an early manifestation in Parkinson's disease (PD). A better understanding of the mechanisms underlying these changes would facilitate the development of vision tests which can be used as preclinical biomarkers to support the development of novel therapeutics for PD. This study aims to characterize the retinal phenotype of a mouse model of dopaminergic dysfunction and to examine whether these changes are reversible with levodopa treatment. We use a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD to characterize the neurotoxic effects of MPTP on in vivo retinal function (electroretinography, ERG), retinal structure (optical coherence tomography, OCT) and retinal dopaminergic cell number (tyrosine hydroxylase immunohistochemistry, IHC) at two time points (21 and 45 days) post MPTP model induction. We also investigate the effect of levodopa (L-DOPA) as a proof-of-principle chronic intervention against MPTP-induced changes in the retina. We show that MPTP decreases dopaminergic amacrine cell number (9%, p < 0.05) and that a component of the ERG that involves these cells, in particular oscillatory potential (OP) peak timing, was significantly delayed at Day 45 (7-13%, p < 0.01). This functional deficit was paralleled by outer plexiform layer (OPL) thinning (p < 0.05). L-DOPA treatment ameliorated oscillatory potential deficits (7-13%, p < 0.001) in MPTP animals. Our data suggest that the MPTP toxin slows the timing of inner retinal feedback circuits related to retinal dopaminergic pathways which mirrors findings from humans with PD. It also indicates that the MPTP model causes structural thinning of the outer retinal layer on OCT imaging that is not ameliorated with L-DOPA treatment. Together, these non-invasive measures serve as effective biomarkers for PD diagnosis as well as for quantifying the effect of therapy.


Assuntos
Intoxicação por MPTP , Doença de Parkinson , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Levodopa/farmacologia , Levodopa/uso terapêutico , Intoxicação por MPTP/complicações , Intoxicação por MPTP/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Retina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
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