RESUMEN
Animal models for Parkinson's disease (PD) are very useful in understanding the pathogenesis of PD and screening for new therapeutic approaches. The present study compared two commonly used neurotoxininduced mouse models of chronic PD to guide model selection, explore the pathogenesis and mechanisms underlying PD and develop effective treatments. The chronic PD mouse models were established via treatment with rotenone or 1methyl4phenyl1,2,3,6-tetrahydropyridine (MPTP) for 6 weeks. The effects of rotenone and MPTP in the mice were compared by assessing neurobehavior, neuropathology and mitochondrial function through the use of the pole, rotarod and open field tests, immunohistochemistry for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), ionized calciumbinding adapter molecule 1 (Iba1), neuronal nuclear antigen (NeuN) and (p)S129 αsynuclein, immunofluorescence for GFAP, Iba1 and NeuN, western blotting for TH, oxygen consumption, complex I enzyme activity. The locomotor activity, motor coordination and exploratory behavior in both rotenone and MPTP groups were significantly lower compared with the control group. However, behavioral tests were no significant differences between the two groups. In the MPTP group, the loss of dopaminergic (DA) neurons in the substantia nigra (SN) pars compacta, the reduction of the tyrosine hydroxylase content in the SN and striatum and the astrocyte proliferation and microglial activation in the SN were more significant compared with the rotenone group. Notably, mitochondrialdependent oxygen consumption and complex I enzyme activity in the SN were significantly reduced in the rotenone group compared with the MPTP group. In addition, Lewy bodies were present only in SN neurons in the rotenone group. Although no significant differences in neurobehavior were observed between the two mouse models, the MPTP model reproduced the pathological features of PD more precisely in terms of the loss of DA neurons, decreased dopamine levels and neuroinflammation in the SN. On the other hand, the rotenone model was more suitable for studying the role of mitochondrial dysfunction (deficient complex I activity) and Lewy body formation in the SN, which is a characteristic pathological feature of PD. The results indicated that MPTP and rotenone PD models have advantages and disadvantages, therefore one or both should be selected based on the purpose of the study.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedad de Parkinson Secundaria/metabolismo , Enfermedad de Parkinson Secundaria/fisiopatología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Reacción de Prevención/fisiología , Western Blotting , Enfermedad Crónica , Proteínas de Unión al ADN/metabolismo , Neuronas Dopaminérgicas/citología , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunohistoquímica , Ratones Endogámicos C57BL , Actividad Motora/fisiología , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Parkinson Secundaria/inducido químicamente , Rotenona , Sustancia Negra/citología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Our previous investigation showed Wnt signal pathway was significantly activated during DA neuron differentiation of epiblast-derived stem cells. In this study, we next attempt to examine the therapeutic potential of the purified exosomes derived bone marrow mesenchymal stem cells (BMSCs) by administrating exosomes into the rat striatum of parkinson's disease (PD) animal model. Results revealed that the protein levels of interleukin (IL)-6, IL-1ß, tumor necrosis factor-alpha (TNF-α), and reactive oxygen species (ROS) in the substantia nigra of PD rats were down regulated after injection of BMSC induced-Exosomes into the striatum of PD model compared to BMSC quiescent-Exosomes. In addition, the expression of ionized calcium binding adaptor molecule 1 (Iba1) mRNA was significantly decreased, while the expression of tyrosine hydroxylase (TH) mRNA was increased after injection of BMSC induced-Exosomes. Injection of BMSC induced-Exosomes into the striatum rescued the rotation behavior and climbing speed in the PD rats. More importantly, Wnt5a was found to be enriched in BMSC induced Exosomes, which could be effectively transferred to the substantia nigra of PD rats. In conclusion, these findings demonstrated that exosomes isolated during dopaminergic neuron differentiation could rescue the pathogenic features of Parkinson's disease by reshaping the inflammatory microenvironment in the substantia nigra and repairing the injury to DA nerves.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Exosomas/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Neurogénesis , Enfermedad de Parkinson/terapia , Animales , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Neuronas Dopaminérgicas/citología , Interleucina-6/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Sustancia Negra/citología , Sustancia Negra/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Parkinson disease (PD) prevalence varies by ethnicity. In an earlier study, we replicated the reduced vulnerability to PD in an admixed population, using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-susceptible C57BL/6 J, MPTP-resistant CD-1 and their F1 crossbreds. In the present study, we investigated if the differences have a developmental origin. Substantia nigra was evaluated at postnatal days 2 (P2), P6, P10, P14, P18, and P22. C57BL/6 J mice had smaller nigra and fewer dopaminergic neurons than the CD-1 and crossbreds at P2, which persisted through development. A significant increase in numbers and nigral volume was observed across strains until P14. A drastic decline thereafter was specific to C57BL/6 J. CD-1 and crossbreds retained their numbers from P14 to stabilize with supernumerary neurons at adulthood. The neuronal size increased gradually to attain adult morphology at P10 in the resistant strains, vis-à-vis at P22 in C57BL/6 J. Accordingly, in comparison to C57BL/6 J, the nigra of CD-1 and reciprocal crossbreds possessed cytomorphological features of resilience, since birth. The considerably lesser dopaminergic neuronal loss in the CD-1 and crossbreds was seen at P2 and P14 and thereafter was complemented by attenuated developmental cell death. The differences in programmed cell death were confirmed by reduced TUNEL labelling, AIF, and caspase-3 expression. GDNF expression aligned with the cell death pattern at P2 and P14 in both nigra and striatum. Earlier maturity of nigra and its neurons appears to be better features that reflect as MPTP resistance at adulthood. Thus, variable MPTP vulnerability in mice and also differential susceptibility to PD in humans may arise early during nigral development.
Asunto(s)
Apoptosis , Neuronas/patología , Enfermedad de Parkinson/etiología , Sustancia Negra/patología , Animales , Animales Recién Nacidos/anatomía & histología , Recuento de Células , Susceptibilidad a Enfermedades/patología , Femenino , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad de Parkinson/patología , Sustancia Negra/citologíaRESUMEN
Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.
Asunto(s)
Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Axones/patología , Muerte Celular , Dendritas/metabolismo , Dendritas/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Levodopa/farmacología , Levodopa/uso terapéutico , Masculino , Ratones , Destreza Motora/efectos de los fármacos , NADH Deshidrogenasa/deficiencia , NADH Deshidrogenasa/genética , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/fisiopatología , Fenotipo , Sustancia Negra/citología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
Parkinson's disease (PD), a common neurodegenerative disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. The cause of dopaminergic loss in PD remains unknown for a long time, however, recent reports suggest oxidative stress plays a key role in the pathogenesis of PD. Paraquat (PQ), a widely used herbicide is an oxidative stress inducer that has been implicated as a potential risk factor for the development of PD. Flavonoids are naturally occurring polyphenolic compounds that display a variety of therapeutic properties against oxidative stress. Naringenin (NAR), a natural flavonoid, exhibits neuroprotection against PD-related pathology. However, studies on its neuroprotective role and the underlying mechanisms are scarce, therefore the present study explored the potential neuroprotective role of NAR in PQ-induced parkinsonism in SH-SY5Y cells and rat model. The effect of NAR on PQ-induced cellular toxicity was determined by measuring cell viability, oxidative stress, ATP levels and the same effect was determined by assessing behavioral, biochemical, immunohistochemical, qRT-PCR and Western blot in rat model. NAR treatment in SH-SY5Y cells resulted in increased cell viability, reduced oxidative stress, elevated mitochondrial membrane potential, and higher cellular ATP levels. In rats, NAR treatment resulted in significant neuroprotection against PQ-induced behavioral deficits, oxidative stress, mitochondrial dysfunction, and astrocytosis. NAR treatment significantly modulated PQ-induced mRNA expressions of DRD2, DAT, LRRK2, SNCA, ß-catenin, caspase-3, BDNF genes. NAR treatment increased TH protein expression and modulated its immunoreactivity in rat striatum. Also, GFAP decreased in response to NAR treatment. So, in the present study, NAR exhibits neuroprotection against PQ-induced neurotoxicity and neurodegeneration indicating its novel therapeutic potential against PD.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Flavanonas/farmacología , Herbicidas/efectos adversos , Fármacos Neuroprotectores , Paraquat/efectos adversos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Adenosina Trifosfato/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas Wistar , Sustancia Negra/citología , Sustancia Negra/patologíaRESUMEN
In Parkinson's disease (PD), substantia nigra (SN) dopaminergic (DA) neurons degenerate, while related ventral tegmental area (VTA) DA neurons remain relatively unaffected. Here, we present a methodology that directs the differentiation of mouse and human pluripotent stem cells toward either SN- or VTA-like DA lineage and models their distinct vulnerabilities. We show that the level of WNT activity is critical for the induction of the SN- and VTA-lineage transcription factors Sox6 and Otx2, respectively. Both WNT signaling modulation and forced expression of these transcription factors can drive DA neurons toward the SN- or VTA-like fate. Importantly, the SN-like lineage enriched DA cultures recapitulate the selective sensitivity to mitochondrial toxins as observed in PD, while VTA-like neuron-enriched cultures are more resistant. Furthermore, a proteomics approach led to the identification of compounds that alter SN neuronal survival, demonstrating the utility of our strategy for disease modeling and drug discovery.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Degeneración Nerviosa/genética , Enfermedad de Parkinson/genética , Células Madre Pluripotentes/metabolismo , Sustancia Negra/metabolismo , Área Tegmental Ventral/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Neuronas Dopaminérgicas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Modelos Neurológicos , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Células Madre Pluripotentes/citología , Factores de Transcripción SOXD/genética , Factores de Transcripción SOXD/metabolismo , Sustancia Negra/citología , Área Tegmental Ventral/citologíaRESUMEN
A growing body of evidence indicates an association between flavin-containing monooxygenase (FMO) and neurodegeneration, including Parkinson's disease (PD); however, the details of this association are unclear. We previously showed that the level of Fmo1 mRNA is decreased in an in vitro rotenone model of parkinsonism. To further explore the potential involvement of FMO1 deficiency in parkinsonism, we generated Fmo1 knockout (KO) mice and examined the survival of dopaminergic neurons and relative changes. Fmo1 KO mice exhibited loss of tyrosine hydroxylase-positive neurons, decreased levels of tyrosine hydroxylase and Parkin proteins, and increased levels of pro-inflammatory cytokines (IL1ß and IL6) in the nigrostriatal region. Moreover, the protein levels of PTEN induced kinase 1 (PINK1) and p62, and the Microtubule associated protein 1 light chain 3 (LC3)-II/I ratio were not significantly altered in Fmo1 KO mice (P > 0.05). FMO1 deficiency promotes neuroinflammation in dopaminergic neurons in mice, thus may plays a potential pathological role in dopaminergic neuronal loss. These findings may provide new insight into the pathogenesis of PD.
Asunto(s)
Neuronas Dopaminérgicas/patología , Enfermedades Neuroinflamatorias/inmunología , Oxigenasas/deficiencia , Enfermedad de Parkinson/inmunología , Sustancia Negra/patología , Animales , Neuronas Dopaminérgicas/inmunología , Neuronas Dopaminérgicas/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Enfermedades Neuroinflamatorias/patología , Oxigenasas/genética , Enfermedad de Parkinson/patología , Proteínas Quinasas/análisis , Proteínas Quinasas/metabolismo , Proteína Sequestosoma-1/análisis , Proteína Sequestosoma-1/metabolismo , Sustancia Negra/citología , Sustancia Negra/inmunología , Sustancia Negra/metabolismo , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/metabolismo , Ubiquitina-Proteína Ligasas/análisis , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
p62/SQSTM1 is a multifunctional, cytoplasmic protein with fundamental roles in autophagy and antioxidant responses. Here we showed that p62 translocated from the cytoplasm to the nucleus in nigral dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrid (MPTP)-induced mouse model of Parkinson's disease (PD). We found that p62 was physically associated with glycogen synthase kinase (GSK)-3ß, a serine/threonine protein kinase implicated in dopaminergic neurodegeneration in PD, and that MPTP treatment promoted dissociation of the complex in mice. Conditional knockout of GSK-3 prevented nuclear translocation of p62, suggesting that this translocation was detrimental to dopaminergic neurons. p62 knockout mice were used to investigate the role of p62 in MPTP-induced neuronal death. Knockout of p62 aggravated neuronal injury induced by MPTP intoxication, suggesting that p62 plays an important role in dopaminergic cell survival in stress conditions. Together, our data demonstrate that GSK-3 mediates nuclear translocation of p62 during MPTP-induced parkinsonian neurodegeneration. These findings shed new light on the role of the cytoplasmic-nuclear shuttling of p62 and the mechanism underlying GSK-3-depedent neuronal death in PD pathogenesis.
Asunto(s)
Neuronas Dopaminérgicas/patología , Glucógeno Sintasa Quinasa 3/metabolismo , Trastornos Parkinsonianos/patología , Proteína Sequestosoma-1/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/efectos de los fármacos , Humanos , Masculino , Ratones , Trastornos Parkinsonianos/inducido químicamente , Sustancia Negra/citología , Sustancia Negra/patologíaRESUMEN
Midbrain dopamine (DA) neurons are slow pacemakers that maintain extracellular DA levels. During the interspike intervals, subthreshold slow depolarization underlies autonomous pacemaking and determines its rate. However, the ion channels that determine slow depolarization are unknown. Here we show that TRPC3 and NALCN channels together form sustained inward currents responsible for the slow depolarization of nigral DA neurons. Specific TRPC3 channel blockade completely blocked DA neuron pacemaking, but the pacemaking activity in TRPC3 knock-out (KO) mice was perfectly normal, suggesting the presence of compensating ion channels. Blocking NALCN channels abolished pacemaking in both TRPC3 KO and wild-type mice. The NALCN current and mRNA and protein expression are increased in TRPC3 KO mice, indicating that NALCN compensates for TRPC3 currents. In normal conditions, TRPC3 and NALCN contribute equally to slow depolarization. Therefore, we conclude that TRPC3 and NALCN are two major leak channels that drive robust pacemaking in nigral DA neurons.
Asunto(s)
Relojes Biológicos/fisiología , Neuronas Dopaminérgicas/fisiología , Canales Iónicos/genética , Proteínas de la Membrana/genética , Neuronas/fisiología , Sustancia Negra/fisiología , Canales Catiónicos TRPC/genética , Potenciales de Acción , Animales , Relojes Biológicos/genética , Neuronas Dopaminérgicas/citología , Femenino , Masculino , Ratones , Ratones Noqueados , Sustancia Negra/citologíaRESUMEN
Encompassing live cell imaging and morphometrics at the microscopical level, we showed here, for the first time, protection of neuronal-like cells by the novel drug candidate, SKIP, against the Parkinson's disease-related neurotoxin, rotenone. Mechanistically, rotenone disrupted microtubule dynamics, which SKIP partially repaired through microtubule end-binding proteins, coupled with increasing neurite branch length. Given the previous association of rotenone toxicity with increased dopaminergic cell death hallmarking Parkinson's disease, we chose an established rat model of 6-hydroxydopamine (6-OHDA) toxicity to initially evaluate SKIP in vivo. SKIP pretreatment showed protection against nigral dopaminergic cell degeneration and improved motor behavior in the forelimb asymmetry test. With Parkinson's disease being a major neurodegenerative disorder, afflicting millions of people globally, and with disease modification challenges, SKIP may hold promise for future therapeutic development.
Asunto(s)
Antiparkinsonianos/farmacología , Microtúbulos/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Animales , Antiparkinsonianos/uso terapéutico , Línea Celular Tumoral , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Masculino , Microtúbulos/metabolismo , Oxidopamina/toxicidad , Enfermedad de Parkinson/etiología , Ratas , Ratas Sprague-Dawley , Rotenona/toxicidad , Sustancia Negra/citologíaRESUMEN
In Parkinson's disease, the depletion of iron-rich dopaminergic neurons in nigrosome 1 of the substantia nigra precedes motor symptoms by two decades. Methods capable of monitoring this neuronal depletion, at an early disease stage, are needed for early diagnosis and treatment monitoring. Magnetic resonance imaging (MRI) is particularly suitable for this task due to its sensitivity to tissue microstructure and in particular, to iron. However, the exact mechanisms of MRI contrast in the substantia nigra are not well understood, hindering the development of powerful biomarkers. In the present report, we illuminate the contrast mechanisms in gradient and spin echo MR images in human nigrosome 1 by combining quantitative 3D iron histology and biophysical modeling with quantitative MRI on post mortem human brain tissue. We show that the dominant contribution to the effective transverse relaxation rate (R2*) in nigrosome 1 originates from iron accumulated in the neuromelanin of dopaminergic neurons. This contribution is appropriately described by a static dephasing approximation of the MRI signal. We demonstrate that the R2* contribution from dopaminergic neurons reflects the product of cell density and cellular iron concentration. These results demonstrate that the in vivo monitoring of neuronal density and iron in nigrosome 1 may be feasible with MRI and provide directions for the development of biomarkers for an early detection of dopaminergic neuron depletion in Parkinson's disease.
Asunto(s)
Neuronas Dopaminérgicas/química , Hierro/análisis , Imagen por Resonancia Magnética/métodos , Sustancia Negra/citología , Anciano de 80 o más Años , Biofisica , Ferritinas/análisis , Humanos , Masculino , Melaninas/análisis , Persona de Mediana Edad , Modelos Neurológicos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Programas Informáticos , Sustancia Negra/químicaRESUMEN
Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by the accumulation of pathogenic phosphorylated α-synuclein in oligodendrocytes. In brains affected by MSA, severe astrogliosis is also observed, but its precise role in MSA pathogenesis remains largely unknown. Recently, the stimulator of interferon genes (STING) pathway and type I interferons, its downstream molecules, have been reported to be involved in the neurodegenerative process and to be activated in astrocytes. This study aimed to investigate the role of the STING pathway in the pathogenesis of MSA using postmortem brains. Samples used for immunohistochemical analysis included 6 cases of MSA parkinsonism type (MSA-P), 6 cases of MSA cerebellar type (MSA-C), and 7 age-matched controls. In MSA-P cases, astrocytes immunopositive for STING and TANK-binding kinase 1 (TBK1), its downstream molecule, were abundantly observed in the putamen and the substantia nigra. Moreover, these molecules colocalized with glial fibrillary acidic protein (GFAP) in reactive astrocytes, and the density of STING-positive astrocytes correlated with that of GFAP-positive reactive astrocytes in the brains of patients with MSA-P. These results suggest that the upregulated expression of STING pathway-related proteins in astrocytes and the subsequent inflammation may contribute to the pathogenesis in MSA-P and could provide novel therapeutic targets for the treatment of MSA.
Asunto(s)
Astrocitos/metabolismo , Proteínas de la Membrana/metabolismo , Atrofia de Múltiples Sistemas/inmunología , Putamen/patología , Sustancia Negra/patología , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Masculino , Proteínas de la Membrana/análisis , Persona de Mediana Edad , Atrofia de Múltiples Sistemas/patología , Putamen/citología , Putamen/inmunología , Transducción de Señal/inmunología , Sustancia Negra/citología , Sustancia Negra/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity. However, existing inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homolog of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro and in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents induces a reversible ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at presynaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.
Asunto(s)
Dopamina/metabolismo , Proteínas de Insectos/genética , Optogenética/métodos , Rodopsina/genética , Potenciales Sinápticos , Animales , Células Cultivadas , Culicidae , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/fisiología , Células HEK293 , Humanos , Proteínas de Insectos/metabolismo , Locomoción , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Rodopsina/metabolismo , Sustancia Negra/citología , Sustancia Negra/fisiologíaRESUMEN
Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant α-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.
Asunto(s)
Neuronas Dopaminérgicas/citología , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , Sustancia Negra/citología , Tirosina 3-Monooxigenasa/metabolismo , alfa-Sinucleína/metabolismo , Animales , Neuronas Dopaminérgicas/metabolismo , Femenino , Ratas , Ratas Sprague-Dawley , Sustancia Negra/metabolismoRESUMEN
Parkinson's disease is associated with the loss of dopamine (DA) neurons in ventral mesencephalon. We have previously reported that no single neurotrophic factor we tested protected DA neurons from the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP+) in dissociated cultures isolated from the P0 rat substantia nigra, but that a combination of five neurotrophic factors was protective. We now report that cerebral DA neurotrophic factor (CDNF) and a variant of neurturin (NRTN), N4, were also not protective when provided alone but were protective when added together. In cultures isolated from the substantia nigra, MPP+ (10 µM) decreased tyrosine hydroxylase-positive cells to 41.7 ± 5.4% of vehicle control. Although treatment of cultures with 100 ng/ml of either CDNF or N4 individually before and after toxin exposure did not significantly increase survival in MPP+-treated cultures, when the two trophic factors were added together at 100 ng/ml each, survival of cells was increased 28.2 ± 6.1% above the effect of MPP+ alone. In cultures isolated from the ventral tegmental area, another DA rich area, a higher dose of MPP+ (1 mM) was required to produce an EC50 in TH-positive cells but, as in the substantia nigra, only the combination of CDNF and N4 (100 ng/ml each) was successful at increasing the survival of these cells compared to MPP+ alone (by 22.5 ± 3.5%). These data support previous findings that CDNF and N4 may be of therapeutic value for treatment of PD, but suggest that they may need to be administered together.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Mesencéfalo/metabolismo , Factores de Crecimiento Nervioso/farmacología , Fármacos Neuroprotectores/farmacología , Neurturina/farmacología , 1-Metil-4-fenilpiridinio , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cricetulus , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Humanos , Nomifensina/farmacología , Ratas Sprague-Dawley , Sustancia Negra/citología , Tritio/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Área Tegmental Ventral/citologíaRESUMEN
The netrin-1/DCC ligand/receptor pair has key roles in central nervous system (CNS) development, mediating axonal, and neuronal navigation. Although expression of netrin-1 and DCC is maintained in the adult brain, little is known about their role in mature neurons. Notably, netrin-1 is highly expressed in the adult substantia nigra, leading us to investigate a role of the netrin-1/DCC pair in adult nigral neuron fate. Here, we show that silencing netrin-1 in the adult substantia nigra of mice induces DCC cleavage and a significant loss of dopamine neurons, resulting in motor deficits. Because loss of adult dopamine neurons and motor impairments are features of Parkinson's disease (PD), we studied the potential impact of netrin-1 in different animal models of PD. We demonstrate that both overexpression of netrin-1 and brain administration of recombinant netrin-1 are neuroprotective and neurorestorative in mouse and rat models of PD. Of interest, we observed that netrin-1 levels are significantly reduced in PD patient brain samples. These results highlight the key role of netrin-1 in adult dopamine neuron fate, and the therapeutic potential of targeting netrin-1 signaling in PD.
Asunto(s)
Receptor DCC/metabolismo , Netrina-1/genética , Netrina-1/metabolismo , Enfermedad de Parkinson/genética , Sustancia Negra/citología , Animales , Muerte Celular , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Regulación hacia Abajo , Femenino , Silenciador del Gen , Humanos , Masculino , Ratones , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Ratas , Transducción de Señal , Sustancia Negra/metabolismoRESUMEN
The quinone derivative of the non-psychotropic cannabinoid cannabigerol (CBG), so-called VCE-003.2, has been recently investigated for its neuroprotective properties in inflammatory models of Parkinson's disease (PD) in mice. Such potential derives from its activity at the peroxisome proliferator-activated receptor-γ (PPAR-γ). In the present study, we investigated the neuroprotective properties of VCE-003.2 against the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA), in comparison with two new CBG-related derivatives, the cannabigerolic acid quinone (CBGA-Q) and its sodium salt CBGA-Q-Salt, which, similarly to VCE-003.2, were found to be active at the PPAR-γ receptor, but not at the cannabinoid CB1 and CB2 receptors. First, we investigated their cytoprotective properties in vitro by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-003.2 at a concentration of 20 µM, which was not reversed by the blockade of PPAR-γ receptors with GW9662, supporting its activity at an alternative site (non-sensitive to classic antagonists) in this receptor. We also found CBGA-Q and CBGA-Q-Salt being cytoprotective in this cell assay, but their effects were completely eliminated by GW9662, thus indicating that they are active at the canonical site in the PPAR-γ receptor. Then, we moved to in vivo testing using mice unilaterally lesioned with 6-OHDA. Our data confirmed that VCE-003.2 administered orally (20 mg/kg) preserved tyrosine hydroxylase (TH)-positive nigral neurons against 6-OHDA-induced damage, whereas it completely attenuated the astroglial (GFAP) and microglial (CD68) reactivity found in the substantia nigra of lesioned mice. Such neuroprotective effects caused an important recovery in the motor deficiencies displayed by 6-OHDA-lesioned mice in the pole test and the cylinder rearing test. We also investigated CBGA-Q, given orally (20 mg/kg) or intraperitoneally (10 mg/kg, i.p.), having similar benefits compared to VCE-003.2 against the loss of TH-positive nigral neurons, glial reactivity and motor defects caused by 6-OHDA. Lastly, the sodium salt of CBGA-Q, given orally (40 mg/kg) to 6-OHDA-lesioned mice, also showed benefits at behavioral and histopathological levels, but to a lower extent compared to the other two compounds. In contrast, when given i.p., CBGA-Q-Salt (10 mg/kg) was poorly active. We also analyzed the concentrations of dopamine and its metabolite DOPAC in the striatum of 6-OHDA-lesioned mice after the treatment with the different compounds, but recovery in the contents of both dopamine and DOPAC was only found after the treatment with VCE-003.2. In summary, our data confirmed the neuroprotective potential of VCE-003.2 in 6-OHDA-lesioned mice, which adds to its previous activity found in an inflammatory model of PD (LPS-lesioned mice). Additional phytocannabinoid derivatives, CBGA-Q and CBGA-Q-Salt, also afforded neuroprotection in 6-OHDA-lesioned mice, but their effects were lower compared to VCE-003.2, in particular in the case of CBGA-Q-Salt. In vitro studies confirmed the relevance of PPAR-γ receptors for these effects.
Asunto(s)
Antiparkinsonianos/uso terapéutico , Cannabinoides/química , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Quinonas/química , Animales , Antiparkinsonianos/síntesis química , Antiparkinsonianos/farmacología , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Oxidopamina/toxicidad , PPAR gamma/antagonistas & inhibidores , PPAR gamma/metabolismo , Enfermedad de Parkinson/etiología , Sustancia Negra/citología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
Parkinson's disease (PD) is an incurable age-linked neurodegenerative disease with characteristic movement impairments that are caused by the progressive loss of dopamine-containing neurons (DAn) within the substantia nigra pars compacta. It has been suggested that misfolded protein aggregates together with neuroinflammation and glial reactivity, may impact nerve cell function, leading to neurodegeneration and diseases, such as PD. However, not many studies have been able to examine the role of human glial cells in the pathogenesis of PD. With the advent of induced pluripotent stem cell (iPSC) technology, it is now possible to reprogram human somatic cells to pluripotency and to generate viable human patient-specific DA neurons and glial cells, providing a tremendous opportunity for dissecting cellular and molecular pathological mechanisms occurring at early stages of PD. This reviews will report on recent work using human iPSC and 3D brain organoid models showing that iPSC technology can be used to recapitulate PD-relevant disease-associated phenotypes, including protein aggregation, cell death or loss of neurite complexity and deficient autophagic vacuoles clearance and focus on the recent co-culture systems that are revealing new insights into the complex interactions that occur between different brain cell types during neurodegeneration. Consequently, such advances are the key to improve our understanding of PD pathology and generate potential targets for new therapies aimed at curing PD patients.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuroglía/metabolismo , Enfermedad de Parkinson/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Técnicas de Cultivo de Célula/métodos , Neuronas Dopaminérgicas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Neuroglía/citología , Organoides/citología , Organoides/metabolismo , Enfermedad de Parkinson/patología , Sustancia Negra/citología , Sustancia Negra/metabolismoRESUMEN
α-Synuclein is expressed at high levels at presynaptic terminals, but defining its role in the regulation of neurotransmission under physiologically relevant conditions has proven elusive. We report that, in vivo, α-synuclein is responsible for the facilitation of dopamine release triggered by action potential bursts separated by short intervals (seconds) and a depression of release with longer intervals between bursts (minutes). These forms of presynaptic plasticity appear to be independent of the presence of ß- and γ-synucleins or effects on presynaptic calcium and are consistent with a role for synucleins in the enhancement of synaptic vesicle fusion and turnover. These results indicate that the presynaptic effects of α-synuclein depend on specific patterns of neuronal activity.
Asunto(s)
Dopamina/metabolismo , Neuronas/metabolismo , Sustancia Negra/metabolismo , alfa-Sinucleína/metabolismo , Anestésicos por Inhalación/farmacología , Animales , Señalización del Calcio , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Femenino , Isoflurano/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neurotransmisores/metabolismo , Sustancia Negra/citología , Vesículas Sinápticas/metabolismo , alfa-Sinucleína/genética , gamma-Sinucleína/metabolismoRESUMEN
BACKGROUND: The underlying mechanism of viral infection as a risk factor for Parkinson's disease (PD), the second most common neurodegenerative disease, remains unclear. OBJECTIVE: We used Mac-1-/- and gp91phox-/- transgene animal models to investigate the mechanisms by which poly I:C, a mimic of virus double-stranded RNA, induces PD neurodegeneration. METHOD: Poly I:C was stereotaxically injected into the substantia nigra (SN) of wild-type (WT), Mac-1-knockout (Mac-1-/-) and gp91 phox-knockout (gp91 phox-/-) mice (10 µg/µl), and nigral dopaminergic neurodegeneration, α-synuclein accumulation and neuroinflammation were evaluated. RESULT: Dopaminergic neurons in the nigra and striatum were markedly reduced in WT mice after administration of poly I:C together with abundant microglial activation in the SN, and the expression of α-synuclein was also elevated. However, these pathological changes were greatly dampened in Mac-1-/- and gp91 phox-/- mice. CONCLUSIONS: Our findings demonstrated that viral infection could result in the activation of microglia as well as NADPH oxidase, which may lead to neuron loss and the development of Parkinson's-like symptoms. Mac-1 is a key receptor during this process.