RESUMO
Several efforts to develop new protocols to differentiate in in vitro human mesenchymal stromal cells (hMSCs) into dopamine (DA) neurons have been reported. We have formulated NeuroForsk 2.0 medium containing fibroblast growth factor type beta (FGFb), brain-derived neurotrophic factor (BDNF), melatonin, purmorphamine, and forskolin. We report for the first time that menstrual stromal cells (MenSCs) cultured in NeuroForsk 2.0 medium for 7 days transdifferentiated into DA-like neurons (DALNs) expressing specific DA lineage markers tyrosine hydroxylase-positive cells (TH+) and DA transporter-positive (DAT+) cells and were responsive to DA-induced transient Ca2+ influx. To test the usefulness of this medium, DALNs were exposed to rotenone (ROT), a naturally occurring organic neurotoxin used extensively to chemically induce an in vitro model of Parkinson's disease (PD), which is a movement disorder characterized by the specific loss of DA neurons. We wanted to determine whether ROT induces apoptotic cell death and autophagy pathway under acute or chronic conditions in DALNs. Here, we report that acute ROT exposure induced several molecular changes in DALNS. ROT induced a loss of mitochondrial membrane potential (ΔΨm), high expression of parkin (PRKN), and high colocalization of dynamin-related protein 1 (DRP1) with the mitochondrial translocase of the outer membrane of mitochondria 20 (TOMM20) protein. Acute ROT also induced the appearance of DJ-1Cys106-SO3, as evidenced by the generation of H2O2 and oxidative stress (OS) damage. Remarkably, ROT triggered the phosphorylation of leucine-rich repeat kinase 2 (LRRK2) at residue Ser935 and phosphorylation of α-Syn at residue Ser129, a pathological indicator. ROT induced the accumulation of lipidated microtubule-associated protein 1B-light chain 3 (LC3B), a highly specific marker of autophagosomes. Finally, ROT induced cleaved caspase 3 (CC3), a marker of activated caspase 3 (CASP3) in apoptotic DALNs compared to untreated DANLs. However, the chronic condition was better at inducing the accumulation of lysosomes than the acute condition. Importantly, the inhibitor of the LRRK2 kinase PF-06447475 (PF-475) almost completely blunted ROT-induced apoptosis and reduced ROT-induced accumulation of lysosomes in both acute and chronic conditions in DALNs. Our data suggest that LRRK2 kinase regulated both apoptotic cell death and autophagy in DALNs under OS. Given that defects in mitochondrial complex I activity are commonly observed in PD, ROT works well as a chemical model of PD in both acute and chronic conditions. Therefore, prevention and treatment therapy should be guided to relieve DALNs from mitochondrial damage and OS, two of the most important triggers in the apoptotic cell death of DALNs.
Assuntos
Doença de Parkinson , Rotenona , Humanos , Rotenona/farmacologia , Rotenona/metabolismo , Dopamina/metabolismo , Caspase 3/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Apoptose , Doença de Parkinson/metabolismo , Neurônios Dopaminérgicos/metabolismo , Autofagia , Doença CrônicaRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra and the intraneuronal presence of Lewy bodies (LBs), composed of aggregates of phosphorylated alpha-synuclein at residue Ser129 (p-Ser129α-Syn). Unfortunately, no curative treatment is available yet. To aggravate matters further, the etiopathogenesis of the disorder is still unresolved. However, the neurotoxin rotenone (ROT) has been implicated in PD. Therefore, it has been widely used to understand the molecular mechanism of neuronal cell death. In the present investigation, we show that ROT induces two convergent pathways in HEK-293 cells. First, ROT generates H2O2, which, in turn, either oxidizes the stress sensor protein DJ-Cys106-SH into DJ-1Cys106SO3 or induces the phosphorylation of the protein LRRK2 kinase at residue Ser395 (p-Ser395 LRRK2). Once active, the kinase phosphorylates α-Syn (at Ser129), induces the loss of mitochondrial membrane potential (ΔΨm), and triggers the production of cleaved caspase 3 (CC3), resulting in signs of apoptotic cell death. ROT also reduces glucocerebrosidase (GCase) activity concomitant with the accumulation of lysosomes and autophagolysosomes reflected by the increase in LC3-II (microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate II) markers in HEK-293 cells. Second, the exposure of HEK-293 LRRK2 knockout (KO) cells to ROT displays an almost-normal phenotype. Indeed, KO cells showed neither H2O2, DJ-1Cys106SO3, p-Ser395 LRRK2, p-Ser129α-Syn, nor CC3 but displayed high ΔΨm, reduced GCase activity, and the accumulation of lysosomes and autophagolysosomes. Similar observations are obtained when HEK-293 LRRK2 wild-type (WT) cells are exposed to the inhibitor GCase conduritol-ß-epoxide (CBE). Taken together, these observations imply that the combined development of LRRK2 inhibitors and compounds for recovering GCase activity might be promising therapeutic agents for PD.
Assuntos
Glucosilceramidase , Doença de Parkinson , Humanos , Glucosilceramidase/genética , Rotenona/farmacologia , Rotenona/metabolismo , Células HEK293 , Peróxido de Hidrogênio/metabolismo , alfa-Sinucleína/metabolismo , Doença de Parkinson/metabolismo , Lisossomos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismoRESUMO
Parkinson disease (PD) is a chronic neurodegenerative disorder characterized by a progressive loss of dopamine neurons in the substantia nigra pars compacta (SNpc). In the last years, a growing interest to study the relationship between metabolic dysfunction and neurodegenerative disease like PD has emerged. This study aimed to evaluate the occurrence of possible changes in metabolic homeostasis due to intranigral rotenone administration, a neurotoxin that damages dopaminergic neurons leading to motor impairments mimicking those that happen in PD. Male Wistar rats were distributed into two groups: sham (n = 10) or rotenone (n = 10). Sham group received, bilaterally, within the SNpc, 1 µL of vehicle dimethyl-sulfoxide (DMSO) and the experimental group was bilaterally injected with 1 µL of rotenone (12 µg/µL). Twenty-four hours after the stereotaxic surgeries, the animals underwent the open field test followed by subsequent peripheral blood and cerebrospinal fluid (CSF) samples collection for biochemical testing. The results showed that rotenone was able to replicate the typical motor behavior impairment seen in the disease, i.e., decrease in locomotion (P = 0.05) and increase in immobility (P = 0.01) with a strong correlation (r = - 0.85; P < 0.0001) between them. In addition, it was demonstrated that this model is able to decrease plasmatic total-cholesterol (P = 0.04) and HDL-cholesterol (P = 0.007) potentially impacting peripheral metabolism. Hence, it was revealed a potential ability to reproduce relevant metabolic dysfunctions like hyperglycemia which could be explained by acute and systemic mitochondrial rotenone toxicity and SNpc nigral toxicity. Such mechanisms may still be responsible for the potential occurrence of CSF-hyperglycemia (d = 0.7). Since intranigral rotenone is an early phase model of PD, the present results open a new road for studies aiming to investigate metabolic changes in PD.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Ratos , Animais , Masculino , Doença de Parkinson/metabolismo , Rotenona/toxicidade , Rotenona/metabolismo , Ratos Wistar , Doenças Neurodegenerativas/metabolismo , Neurônios Dopaminérgicos/metabolismo , Colesterol/metabolismo , Modelos Animais de DoençasRESUMO
Hyposmia is found in Parkinsonian patients decades before the onset of motor disorders. The same occurs with sleep disorders, especially infuencing rapid eye movement (REM) sleep, which affect a large percentage of people who have Parkinson's disease. These two disturbances presumably are closely related to a dopaminergic dysfunction. Therefore, we propose that selective lesions, induced by rotenone, of the periglomerular neurons within the olfactory bulb or of the nigrostriatal pathway could result in hyposmia. In addition, we hypothesized that REM sleep deprivation (REMSD) could have potential to generate a synergistic olfactory impairment in both lesion paradigms. The results indicated that rotenone-induced nigrostriatal lesions in female Wistar rats were associated with odor preference changes, similar to hedonic tone impairment, but without a supposed potentiation triggered by REMSD. The nigrostriatal injury negatively affected olfaction performance, which was counteracted, functionally, by REMSD. However, injury to periglomerular neurons was less influenced by REMSD, as olfactory performance was restored after rebound sleep. We conclude that female rats present a pattern of olfactory discrimination/preference that is dependent on the activities of the nigrostriatal and the main olfactory pathways.