Enfermedad de Parkinson: actualización de estudios preclínicos con el uso de células troncales pluripotentes inducidas / Parkinson's disease: An update on preclinical studies of induced pluripotent stem cells

La enfermedad de Parkinson (EP) es la segunda enfermedad neurodegenerativa más común a nivel mundial en adultos mayores. Se caracteriza por la pérdida de neuronas dopaminérgicas (nDAs) en la sustancia nigra pars compacta del mesencéfalo y en algunos casos acompañada de la aparición de cuerpos intracitoplasmáticos de Lewy de -sinucleína, signo patognomónico de la enfermedad. La EP se diagnostica clínicamente por la presencia de alteraciones motoras principalmente, y en la actualidad los tratamientos presentan nula actividad neuroprotectora. Aún no se han establecido las causas exactas de la EP, por lo que en los últimos años se ha buscado el desarrollo de modelos preclínicos más precisos, utilizando células troncales pluripotentes inducidas, permitiendo el estudio de la enfermedad de manera in vitro para generar conocimiento novedoso sobre su patogénesis y el descubrimiento de nuevos posibles blancos terapéuticos o el desarrollo de nuevos fármacos.(AU)

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among adults worldwide. It is characterised by the death of dopaminergic neurons in the substantia nigra pars compacta and, in some cases, presence of intracytoplasmic inclusions of α-synuclein, called Lewy bodies, a pathognomonic sign of the disease. Clinical diagnosis of PD is based on the presence of motor alterations. The treatments currently available have no neuroprotective effect. The exact causes of PD are poorly understood. Therefore, more precise preclinical models have been developed in recent years that use induced pluripotent stem cells. In vitro studies can provide new information on PD pathogenesis and may help to identify new therapeutic targets or to develop new drugs.(AU)

Parkinson's disease: an update on preclinical studies of induced pluripotent stem cells.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among adults worldwide. It is characterised by the death of dopaminergic neurons in the substantia nigra pars compacta and, in some cases, presence of intracytoplasmic inclusions of α-synuclein, called Lewy bodies, a pathognomonic sign of the disease. Clinical diagnosis of PD is based on the presence of motor alterations. The treatments currently available have no neuroprotective effect. The exact causes of PD are poorly understood. Therefore, more precise preclinical models have been developed in recent years that use induced pluripotent stem cells (iPSC). In vitro studies can provide new information on PD pathogenesis and may help to identify new therapeutic targets or to develop new drugs.

Parkinson's disease: An update on preclinical studies of induced pluripotent stem cells. / Enfermedad de Parkinson: actualización de estudios preclínicos con el uso de células troncales pluripotentes inducidas.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among adults worldwide. It is characterised by the death of dopaminergic neurons in the substantia nigra pars compacta and, in some cases, presence of intracytoplasmic inclusions of α-synuclein, called Lewy bodies, a pathognomonic sign of the disease. Clinical diagnosis of PD is based on the presence of motor alterations. The treatments currently available have no neuroprotective effect. The exact causes of PD are poorly understood. Therefore, more precise preclinical models have been developed in recent years that use induced pluripotent stem cells. In vitro studies can provide new information on PD pathogenesis and may help to identify new therapeutic targets or to develop new drugs.

Effects of Mating and Social Exposure on Cell Proliferation in the Adult Male Prairie Vole (Microtus ochrogaster).

Microtus ochrogaster is a rodent with a monogamous reproductive strategy characterized by strong pair bond formation after 6 h of mating. Here, we determine whether mating-induced pair bonding increases cell proliferation in the subventricular zone (SVZ), rostral migratory stream (RMS), and dentate gyrus (DG) of the hippocampus in male voles. Males were assigned to one of the four groups: (1) control: males were placed alone in a clean cage; (2) social exposure to a female (SE m/f): males that could see, hear, and smell a sexually receptive female but where physical contact was not possible, because the animals were separated by an acrylic screen with small holes; (3) social exposure to a male (SE m/m): same as group 2 but males were exposed to another male without physical contact; and (4) social cohabitation with mating (SCM): males that mated freely with a receptive female for 6 h. This procedure leads to pair bond formation. Groups 2 and 3 were controls for social interaction. Male prairie voles were injected with 5-bromo-2'-deoxyuridine (BrdU) during the behavioral tests and were sacrificed 48 h later. Brains were processed to identify the new cells (BrdU-positive) and neuron precursor cells (neuroblasts). Our principal findings are that in the dorsal region of the SVZ, SCM and SE m/f and m/m increase the percentage of neuron precursor cells. In the anterior region of the RMS, SE m/f decreases the percentage of neuron precursor cells, and in the medial region SE m/f and m/m decrease the number of new cells and neuron precursor cells. In the infrapyramidal blade of the subgranular zone of the DG, SE m/m and SCM increase the number of new neuron precursor cells and SE m/m increases the percentage of these neurons. Our data suggests that social interaction, as well as sexual stimulation, leads to pair bonding in male voles modulating cell proliferation and differentiation to neuronal precursor cells at the SVZ, RMS, and DG.

New transgenic models of Parkinson's disease using genome editing technology. / Nuevos modelos transgénicos para el estudio de la enfermedad de Parkinson basados en sistemas de edición con nucleasas.

INTRODUCTION: Parkinson's disease (PD) is the second most common neurodegenerative disorder. It is characterised by selective loss of dopaminergic neurons in the substantia nigra pars compacta, which results in dopamine depletion, leading to a number of motor and non-motor symptoms. DEVELOPMENT: In recent years, the development of new animal models using nuclease-based genome-editing technology (ZFN, TALEN, and CRISPR/Cas9 nucleases) has enabled the introduction of custom-made modifications into the genome to replicate key features of PD, leading to significant advances in our understanding of the pathophysiology of the disease. CONCLUSIONS: We review the most recent studies on this new generation of in vitro and in vivo PD models, which replicate the most relevant symptoms of the disease and enable better understanding of the aetiology and mechanisms of PD. This may be helpful in the future development of effective treatments to halt or slow disease progression.

Neuroprotective effects of phytochemicals on dopaminergic neuron cultures. / Efecto neuroprotector de fitoquímicos en cultivo de neuronas dopaminérgicas.

INTRODUCTION: Parkinson's disease is a progressive neurodegenerative disorder characterised by a loss of dopaminergic neurons in the substantia nigra pars compacta, which results in a significant decrease in dopamine levels and consequent functional motor impairment. DEVELOPMENT: Although its aetiology is not fully understood, several pathogenic mechanisms, including oxidative stress, have been proposed. Current therapeutic approaches are based on dopamine replacement drugs; these agents, however, are not able to stop or even slow disease progression. Novel therapeutic approaches aimed at acting on the pathways leading to neuronal dysfunction and death are under investigation. CONCLUSIONS: In recent years, such natural molecules as polyphenols, alkaloids, and saponins have been shown to have a neuroprotective effect due to their antioxidant and anti-inflammatory properties. The aim of our review is to analyse the most relevant studies worldwide addressing the benefits of some phytochemicals used in in vitro models of Parkinson's disease.

Central nervous system development-related microRNAs levels increase in the serum of gestational diabetic women during the first trimester of pregnancy.

MicroRNAs are heterochronic molecules important during brain development, which could be altered by gestational diabetes mellitus (GDM). To explore these molecules in maternal serum, we performed an RT-qPCR analysis. Our results revealed the heterochronic character of some neural development-related microRNA in serum samples of pregnant women. In relation to the first trimester, higher levels of miR-183-5p, -200b-3p, and -125-5p in the second trimester, and higher levels of miR-137 in the third trimester, were found. Furthermore, an insult such as GDM led to higher levels of miR-183-5p, -200b-3p, -125-5p, and -1290 relative to the control in the first trimester, which might be related to changes in neurogenesis and cell proliferation. An in silico analysis suggested that increased microRNAs in the second trimester in the control contributed to cell proliferation and neuron differentiation and that the rise in miR-137 in the third trimester led to neuron maturation. In the diabetic, higher levels of the microRNAs in the first trimester suggested alterations in cell proliferation and neuron differentiation. In conclusion, we showed that fetal-related microRNAs can be detected in the serum of pregnant woman and exhibit temporary regulation during pregnancy and that microRNAs involved in cell proliferation and neuron differentiation are upregulated under GDM.

Mating and social exposure induces an opioid-dependent conditioned place preference in male but not in female prairie voles (Microtus ochrogaster).

In rodents, sexual stimulation induces a positive affective state that is evaluated by the conditioned place preference (CPP) test. Opioids are released during sexual behavior and modulate the rewarding properties of this behavior. Prairie voles (Microtus ochrogaster) are a socially monogamous species, in which copulation with cohabitation for 6h induces a pair bond. However, the mating-induced reward state that could contribute to the establishment of the long-term pair bond has not been evaluated in this species. The present study aimed to determine whether one ejaculation or cohabitation with mating for 6h is rewarding for voles. We also evaluated whether this state is opioid dependent. Our results demonstrate that mating with one ejaculation and social cohabitation with mating for 6h induce a CPP in males, while exposure to a sexually receptive female without mating did not induce CPP. In the female vole, mating until one ejaculation, social cohabitation with mating, or exposure to a male without physical interaction for 6h did not induce CPP. To evaluate whether the rewarding state in males is opioid dependent, the antagonist naloxone was injected i.p. The administration of naloxone blocked the rewarding state induced by one ejaculation and by social cohabitation with mating. Our results demonstrate that in the prairie vole, on the basis of the CPP in the testing conditions used here, the stimulation received with one ejaculation and the mating conditions that lead to pair bonding formation may be rewarding for males, and this reward state is opioid dependent.

Escherichia coli-induced temporal and differential secretion of heat-shock protein 70 and interleukin-1ß by human fetal membranes in a two-compartment culture system.

INTRODUCTION: Escherichia coli is recognized as an etiological bacteria associated with chorioamnionitis and the preterm premature rupture of fetal membranes. This pathological condition induces pro-inflammatory cytokines and degradative metalloproteinases, which are considered biological markers secreted in an acute stage of infection. Heat-shock proteins (HSPs) are an important component of the innate immunity response and are found in different pathological conditions. They have not been previously measured in human fetal membranes in response to infectious conditions. We hypothesized that the choriodecidual tissue and amniotic epithelium secreted temporal and differential Hsp-60, Hsp-70, and interleukin (IL)-1ß mediated by E. coli infection. METHODS: Fetal membranes were mounted in a two-compartment culture system and infected with two passes of live E. coli at different doses (10², 104, 105, and 106 colony-forming units (CFU)/mL) and intervals of incubation (3, 6, and 24 h). The culture medium was collected, and Hsp-60, Hsp-70, and IL-1ß were assessed using the enzyme-linked immunosorbent assay (ELISA) method. RESULTS: After 3 and 6 h of infection, E. coli induced an increase in Hsp-70 secretion in the choriodecidual tissue. However, after 24 h of incubation, Hsp-70 was downregulated and we observed an increase in IL-1ß secretion. By contrast, E. coli induced a lower Hsp-60 secretion in the amnion compared to Hsp-70. DISCUSSION: Human fetal membranes responded actively to E. coli infection, with an increase in Hsp-70 during the first hours of infection. After 24 h, there was an increase in the liberation of IL-1ß.

Altered levels of brain neurotransmitter from new born rabbits with intrauterine restriction.

Fetal intrauterine growth restriction generates chronic hypoxia due to placental insufficiency. Despite the hemodynamic process of blood flow, redistributions are taking place in key organs such as the fetal brain during intrauterine growth restriction, in order to maintain oxygen and nutrients supply. The risk of short- and long-term neurological effects are still present in hypoxic offspring. Most studies previously reported the effect of hypoxia on the levels of a single neurotransmitter, making it difficult to have a better understanding of the relationship among neurotransmitter levels and the defects reported in products that suffer intrauterine growth restriction, such as motor development, coordination and execution of movement, and the learning-memory process. The aim of this study was to evaluate the levels of gamma-aminobutyric acid, glutamate, dopamine and serotonin in three structures of the brain related to the above-mentioned function such as the cerebral cortex, the striatum, and the hippocampus in the chronic hypoxic newborn rabbit model. Our results showed a significant increase in glutamate and dopamine levels in all studied brain structures and a significant decrease in gamma-aminobutyric acid levels but only in the striatum, suggesting that the imbalance on the levels of several neurotransmitters could be involved in new born brain damage due to perinatal hypoxia.

Behavioral characterization of non-copulating male mice.

Non-copulating (NC) males are those animals that do not mate in spite of repeated testing with sexually receptive females. They have been observed in several species including rats and mice. The present experiment was designed to perform a detailed behavioral characterization of NC male mice. Thus, we evaluated their sexual incentive motivation for a sexually receptive female or a sexually active male, olfactory preference for volatile and non-volatile odors from females or males, and olfactory discrimination between female and male volatile odors and food related odors (milk versus vinegar). We compared the activity of the accessory olfactory system (AOS) in copulating (C) and NC males in response to estrous bedding using the induction of Fos-immunoreactivity (Fos-IR) as a measure of neuronal activation. We also determined if estradiol or dopamine treatment could induce sexual behavior in NC males. Finally, we compared the testis weight and the number of penile spines in C, NC, and gonadectomized males. In the sexual incentive motivation test C males spend significantly more time in the female incentive zone than in the male incentive zone. On the other hand, NC males spend the same amount of time in both incentive zones. In tests of olfactory preference, NC males spent less time investigating estrous odors than C males. As well, NC males discriminate urine from conspecifics but they spend less time smelling these odors than C males. In addition, no increase in Fos expression is observed in NC males when they are exposed to odors from estrous females. Our data also suggest that the deficits observed in NC males are not due to lower circulating levels of gonadal hormones, because estradiol supplementation does not induce sexual behavior in these animals, and their testis weight and the number of penile spines are normal. The results suggest that NC males are not sexually motivated by the receptive females and their odors.

Histamine is required during neural stem cell proliferation to increase neuron differentiation.

Histamine in the adult central nervous system (CNS) acts as a neurotransmitter. This amine is one of the first neurotransmitters to appear during development reaching its maximum concentration simultaneously with neuron differentiation peak. This suggests that HA plays an important role in neurogenesis. We have previously shown that HA is able to increase neuronal differentiation of neural stem cells (NSCs) in vitro, by activating the histamine type 1 receptor. However the mechanism(s) by which HA has a neurogenic effect on NSCs has not been explored. Here we explore how HA is able to increase neuron phenotype. Cortex neuroepithelium progenitors were cultured and at passage two treatments with 100 µM HA were given during cell proliferation and differentiation or only during differentiation. Immunocytochemistry was performed on differentiated cultures to detect mature neurons. To explore the expression of certain important transcriptional factors involved on asymmetric cell division and commitment, RT-PCR and qRT-PCR were performed. Results indicate that HA is required during cell proliferation in order to increase neuron differentiation and suggest that this amine increases neuron commitment during the proliferative phase probably by rising prospero1 and neurogenin1 expression.

An experimental mixed bacterial infection induced differential secretion of proinflammatory cytokines (IL-1ß, TNFα) and proMMP-9 in human fetal membranes.

Overall, 1-4% of all births in the US are complicated by choriamnionitis. Choriamnionitis is a polymicrobial infection most often due to ascending genital microbes which, in over 65% of positive amniotic fluid cultures, involves two or more organisms. In this study, we determine the cytokines expression (IL-1ß, TNFα) and prometalloproteinase activation (proMMP-2 and proMMP-9) after double o single infection an in vitro model of human fetal membranes. Fetal membranes at term were mounted in the Transwell culture system and after 24 h of infection, choriodecidual, and amnion media was collected. IL-1ß and TNFα were evaluated by ELISA, whereas proMMP-9 and proMMP-2 were determined by substrate gel zymography. The choriodecidual and amnion compartments actively respond to the infectious process, which induced the secretion of IL-1ß, TNFα, and proMMP-9 after either mixed or single infection. The proMMP-2 secretion profile was the same after all experimental conditions. There was no synergy between Streptococcus agalactiae and Escherichia coli for inducing the secretion of inflammatory factors or degradative metalloproteinase. In conclusion, these two bacteria could initiate different pathways to induce chorioamnioitis.

Progesterone increases dopamine neurone number in differentiating mouse embryonic stem cells.

Progesterone participates in the regulation of several functions in mammals, including brain differentiation and dopaminergic transmission, but the role of progesterone in dopaminergic cell differentiation is unknown. We investigated the effects of progesterone on dopaminergic differentiation of embryonic stem cells using a five-stage protocol. Cells were incubated with different progesterone concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Progesterone added at 1, 10 and 100 nm during stage 4 increased the number of dopamine neurones at stage 5 by 72%, 80% and 62%, respectively, compared to the control group. The administration of progesterone at stage 5 did not induce significant changes in the number of dopamine neurones. These actions were not mediated by the activation of intracellular progesterone receptors because RU 486 did not block the positive effects of progesterone on differentiation to dopaminergic neurones. The results obtained suggest that progesterone should prove useful with respect to producing higher proportions of dopamine neurones from embryonic stem cells in the treatment of Parkinson's disease.

Comparative analysis of immunoreactive cells for androgen receptors and oestrogen receptor alpha in copulating and non-copulating male rats.

In some species, including gerbils, guinea pigs, mice, rams and rats, some apparently normal males fail to mate. These kinds of animals have been named 'noncopulating (NC)'. The cause of this behavioural deficit is unknown. The present study aimed to determine whether NC male rats have alterations in the amount of androgen (AR) and oestrogen receptor alpha (ERalpha) in a neuronal circuit important for the control of male sexual behaviour; the vomeronasal projection pathway. We evaluated the number of AR and ERalpha immunoreactive (AR-IR and ERalpha-IR) cells in the accessory olfactory bulb (AOB), the bed nucleus of the stria terminalis (BNST), the anterior-dorsal medial amygdala (MeAD), the posterior dorsal amygdala (MePD) and the medial preoptic area (MPOA). The results demonstrate that the number of AR-IR cells in NC males was significantly higher compared to copulating (C) males in the MePD, but no significant differences were found in any of the other structures analysed. ERalpha-IR cells were more abundant in NC than in C males in the MeAD and the MePD. However, in the MPOA the number of ERalpha-IR cells was significantly reduced in NC males. No significant differences were found in the AOB or in the BNST. A similar pattern of results was observed when regions within these structures that are activated by Fos expression, on mating or exposure to sexually relevant cues were analysed. The differences in the number of AR and ER in particular brain areas could be associated with alterations in sexual behaviour as well as partner and olfactory preference for receptive females seen in NC male rats.