Alpha-Synuclein Gene Alterations Modulate Tyrosine Hydroxylase in Human iPSC-Derived Neurons in a Parkinson's Disease Animal Model.

Parkinson's disease (PD) caused by SNCA gene triplication (3XSNCA) leads to early onset, rapid progression, and often dementia. Understanding the impact of 3XSNCA and its absence is crucial. This study investigates the differentiation of human induced pluripotent stem cell (hiPSC)-derived floor-plate progenitors into dopaminergic neurons. Three different genotypes were evaluated in this study: patient-derived hiPSCs with 3XSNCA, a gene-edited isogenic line with a frame-shift mutation on all SNCA alleles (SNCA 4KO), and a normal wild-type control. Our aim was to assess how the substantia nigra pars compacta (SNpc) microenvironment, damaged by 6-hydroxydopamine (6-OHDA), influences tyrosine hydroxylase-positive (Th+) neuron differentiation in these genetic variations. This study confirms successful in vitro differentiation into neuronal lineage in all cell lines. However, the SNCA 4KO line showed unusual LIM homeobox transcription factor 1 alpha (Lmx1a) extranuclear distribution. Crucially, both 3XSNCA and SNCA 4KO lines had reduced Th+ neuron expression, despite initial successful neuronal differentiation after two months post-transplantation. This indicates that while the SNpc environment supports early neuronal survival, SNCA gene alterations-either amplification or knock-out-negatively impact Th+ dopaminergic neuron maturation. These findings highlight SNCA's critical role in PD and underscore the value of hiPSC models in studying neurodegenerative diseases.

Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography.

In recent years, the study of extracellular vesicles (EVs) in the context of various diseases has dramatically increased due to their diagnostic and therapeutic potential. Typically, EVs are isolated in vitro from the cell culture of primary cells or cell lines or from bodily fluids. However, these cell culture methods do not represent the whole complexity of an in vivo microenvironment, and bodily fluids contain a high heterogeneous population of vesicles since they originate from different tissues. This highlights the need to develop new methods to isolate EVs directly from tissue samples. In the present study, we established a protocol for isolating EVs from hepatic and adipose tissue of mice, using a combination of ultracentrifugation and iodixanol-sucrose density gradient separation. EV isolation was confirmed with EV protein marker enrichment in Western blot assays, total protein quantification, and transmission electron microscopy. Regarding the liver tissue, we additionally implemented size exclusion chromatography (SEC) to further increase the purity grade of the EVs. The successful isolation of EVs from tissue samples will allow us to uncover a more precise molecular composition and functions, as well as their role in intercellular communication in an in vivo microenvironment.

Changes in the Number and Morphology of Dendritic Spines in the Hippocampus and Prefrontal Cortex of the C58/J Mouse Model of Autism.

Autism spectrum disorder (ASD) has a broad range of neurobiological characteristics, including alterations in dendritic spines, where approximately 90% of excitatory synapses occur. Therefore, changes in their number or morphology would be related to atypical brain communication. The C58/J inbred mouse strain displays low sociability, impaired communication, and stereotyped behavior; hence, it is considered among the animal models suitable for the study of idiopathic autism. Thus, this study aimed to evaluate the dendritic spine differences in the hippocampus and the prefrontal cortex of C58/J mice. We found changes in the number of spines and morphology in a brain region-dependent manner: a subtle decrease in spine density in the prefrontal cortex, higher frequency of immature phenotype spines characterized by filopodia-like length or small morphology, and a lower number of mature phenotype spines with mushroom-like or wide heads in the hippocampus. Moreover, an in silico analysis showed single nucleotide polymorphisms (SNPs) at genes collectively involved in regulating structural plasticity with a likely association with ASD, including MAP1A (Microtubule-Associated Protein 1A), GRM7 (Metabotropic Glutamate Receptor, 7), ANKRD11 (Ankyrin Repeat Domain 11), and SLC6A4 (Solute Carrier Family 6, member 4), which might support the relationship between the C58/J strain genome, an autistic-like behavior, and the observed anomalies in the dendritic spines.

Bilateral enucleation at birth modifies calcium spike amplitude, but not frequency, in neurons of the somatosensory thalamus and cortex: Implications for developmental cross-modal plasticity.

Bilateral eye enucleation at birth (BE) leads to an expansion of the primary somatosensory cortex (S1) in rat pups. Although increased growth of the somatosensory thalamo-cortical afferents (STCAs) in part explains S1 expansion, timing mechanisms governing S1 formation are also involved. In this work, we begin the search of a developmental clock by intending to document the existence of putative clock neurons in the somatosensory thalamus (VPM) and S1 based upon changes of spontaneous spike amplitude; a biophysical property sensitive to circadian regulation; the latter known to be shifted by enucleation. In addition, we also evaluated whether STCAs growth rate and segregation timing were modified, as parameters the clock might time. We found that spontaneous spike amplitude transiently, but significantly, increased or decreased in VPM and S1 neurons of BE rat pups, respectively, as compared to their control counterparts. The growth rate and segregation timing of STCAs was, however, unaffected by BE. These results support the existence of a developmental clock that ticks differently in the VPM and S1 after BE. This observation, together with the fact that STCAs growth rate and segregation timing is unchanged, suggests that S1 expansion in BE rats may in part be controlled at the cortical level.

Dopamine Released from TiO2 Semicrystalline Lattice Implants Attenuates Motor Symptoms in Rats Treated with 6-Hydroxydopamine.

The motor dysfunction featured by patients aggrieved by Parkinson's disease (PD) results from the reduction of dopamine (DA) availability in the caudate nucleus (CN). Restituting CN DA levels is therefore essential to ameliorate PD motor deficits. In this regard, nanotechnology may offer solutions to restore CN DA availability. DA, however, can be rapidly oxidized into toxic compounds if made available in situ, unprotected. Then, we tested whether a semicrystalline TiO2 lattice, implanted into the CN of 6-hydroxydopamine (6-OHDA)-lesioned, hemiparkinsonian rats, was able to release DA during a time window sufficient to attenuate motor symptoms while protecting it from the ongoing oxidation. Accordingly, implanted semicrystalline TiO2 lattices released incremental amounts of DA into the CN of lesioned rats. Motor symptoms were already attenuated by the 1st month and significantly reduced 2 months after implantation. These effects were specific since TiO2 lattices alone did not modify motor symptoms in lesioned rats. DA-unloaded or -loaded TiO2 lattices did not produce obvious symptoms of systemic or neurological toxicity nor significantly increased CN lipid peroxidation in implanted, lesioned rats at the time of sacrifice. Our results thus support that loaded TiO2 lattices are capable of releasing DA while protecting it from the ongoing oxidation when implanted into the brain. Their implantation does not cause noticeable systemic or local toxicity. On the contrary, they attenuated motor symptoms in hemiparkinsonian rats.

The presence of VEGF and Notch2 during preantral-antral follicular transition in infantile rats: Anatomical evidence and its implications.

Folliculogenesis is a process that depends on angiogenesis, in which VEGF and Notch signaling pathway members are involved. Although this pathway is present in preantral and antral follicular structures during the second stage of folliculogenesis, this association has not been described. Therefore, this study aimed to identify VEGF and Notch2 in ovary structures of infantile rats after induction of follicular development with a gonadotropin stimulus. In order to explore this possibility we analyzed rat ovary morphology from days 10-25 after birth; subsequently, the transition from preantral follicle to an antral stage was analyzed by the induction of follicular development with equine chorionic gonadotropin (eCG) and VEGF and Notch were identified in the rat ovary by fluorescence. The histological analysis revealed that the ovary of a 10-day-old rat has the highest percentage of preantral follicles and based on this a 10IU eCG dose promoted an increase in the number of antral follicles, as well as a decrease in the number of preantral follicles, related to which there was an increase in ovary weight and size. In addition, a higher concentration of circulating estradiol was observed, proliferation of granulosa cells in both follicle groups was stimulated, and the accumulation of VEGF in granulosa and theca cells and in the antral follicle oocyte was increased (p<0.05), whereas the presence of Notch2 was limited to mural granulosa cells, in granulosa cells that formed the cumulus oophorus and in the oocyte of both groups of follicles. The multiple correspondence analysis allowed us to support an association between VEGF and Notch2 during the transition from preantral to antral follicles in the ovary of an infantile rat.

Effect of ovine luteinizing hormone (oLH) charge isoforms on VEGF and cAMP production.

Although an increase in VEGF expression and synthesis in association with LH has been established; it is unknown if all LH isoforms act similarly. This study evaluated the production of cAMP and VEGF among LH isoforms in two in vitro bioassays. The LH was obtained from hypophyses and the group of isoforms was isolated by chromatofocusing. cAMP production was assessed using the in vitro bioassay of HEK-293 cells and VEGF production was evaluated in granulosa cells. Immunological activity was measured with a homologous RIA. Immunoactivity and bioactivity for each isoform were compared against a standard, by estimating the IC50 and the EC50. The basic isoforms were more immunoactive than the standard. The neutral and the moderately acidic had an immunological activity similar to the standard. The acidic isoform was the least immunoreactive. cAMP production at the EC50 dose was similar among the basic isoforms, the moderately acidic and the standard; for the neutral and the acidic, the EC50 dose was higher. It was observed that compared with the control, VEGF production at the lowest LH dose was no different in the standard and each isoform. In the intermediate dose, a positive response was caused in the standard and the neutral and basic isoforms. Although the acidic isoform showed a dose-dependent response, it was not significant relative to the control. In conclusion, the basic isoform generated the greatest cAMP and VEGF production, similar to the reference standard, and the acidic the smallest.

Efecto de la exposición al pesticida rotenona sobre el desarrollo del sistema dopaminérgico nigro-estriatal en ratas / Developmental effects of rotenone exposure on the rat nigro-striatal dopaminergic system

Rotenone is a pesticide used in Mexico, despite the experimental evidence showing dopaminergic neurons degeneration induced by this compound, which may lead to a psychomotor impairment. However, the possible effects of rotenone on the offspring when they are indirectly exposed through their mothers are still unknown. In this study rotenone was administered to female rats during pregnancy and nursing, in order to assess its effects on the offspring's dopaminergic neurons in the substantia nigra, as well as on motor coordination at 30 or 60 postnatal days. Six groups of pregnant Wistar rats were used: an intact control group, a vehicle group injected with the rotenone solvent, and four groups injected subcutaneously with the following doses of rotenone: 0.2, 0.4, 0.6, and 1 mg/kg/day. In a parallel experiment, the offspring of other groups of dams treated with rotenone 1 mg/kg/day, or controls vehicle-treated, were used to evaluate motor coordination at 30 and 60 postnatal days. Rotenone treated dams showed a significant lower amount of dopaminergic neurons in the substantia nigra, but only with the 1 mg/kg dose. This effect was also observed in the offspring but at all doses of rotenone tested, either at 30 or 60 postnatal days. Furthermore, the offspring of rotenone exposed dams significantly increased the time in which they accomplished the motor coordination test, compared to the offspring of control dams. These data indicate that rotenone is able to damage the dopaminergic neurons of the offspring though their mothers. This effect requires lower rotenone doses than in adult rats. The reduced number of dopaminergic neurons at early stages of life enhances the risk of developing disorders related to the brains' dopaminergic system.

La rotenona es un pesticida utilizado en México a pesar de que se ha demostrado experimentalmente que produce una degeneración de las neuronas dopaminérgicas, y puede derivar en deterioro psicomotor. Sin embargo, no existen estudios de la exposición indirecta a rotenona a través de las madres en el efecto que produzca sobre su descendencia. Nosotros administramos rotenona a ratas durante la gestación y la lactancia para evaluar las alteraciones producidas sobre las neuronas dopaminérgicas y la coordinación motora de sus crías, a los 30 o 60 días posnatales. Para cuantificar las neuronas inmunorreactivas a tirosina hidroxilasa de la sustancia nigra, se inyectaron subcutáneamente seis grupos de hembras Wistar: intactas (control), con solvente de rotenona (vehículo) y cuatro grupos con rotenona en dosis: 0.2, 0.4, 0.6 y 1.0 mg/kg/día. En un experimento paralelo, las crías de otros grupos de hembras tratadas con rotenona 1 mg/kg/día o controles fueron evaluados en la prueba de coordinación motora a los 30 y 60 días posnatales. Las madres tratadas con 1 mg/kg de rotenona tuvieron menos neuronas dopaminérgicas en la sustancia nigra. Dicho efecto se observó también en las crías, pero con todas las dosis de rotenona utilizadas, tanto a los 30 como a los 60 días posnatales. Además, la exposición indirecta a rotenona aumentó significativamente el tiempo que requirieron las crías para ejecutar la prueba de coordinación motora. Estos datos indican que la rotenona es capaz de inducir daño en las neuronas dopaminérgicas de las crías cuando son expuestas a través de sus madres. Este efecto en las crías se observa con dosis menores de rotenona que en ratas adultas. Por lo tanto, los individuos indirectamente expuestos a rotenona podrían tener menos neuronas dopaminérgicas desde etapas tempranas de la vida, lo que aumenta el riesgo de desarrollar trastornos relacionados con el sistema dopaminérgico.

Cortistatin modulates memory evocation in rats.

The neurochemical control of learning depends on several neurotransmitters, hormones, and neuropeptides. Cortistatin is a neuropeptide with sleep-modulating properties that regulates memory consolidation and evocation. Several reports have suggested that learning processes are expressed under diurnal variations; therefore, it seems that the efficiency to solve learning tasks is related to the arousal state. Although we know that cortistatin modulates learning, we do not know whether its effect is subjected to diurnal variations. Hence, we evaluated memory evocation and the sleep-waking cycle along the day. Additionally, we evaluated the effect of cortistatin on motor control and cyclic adenosine monophosphate (cAMP) concentration. Performance of rats was better at 01:00 h than at 13:00 h to solve the Barnes maze. Cortistatin impaired memory evocation, increased rapid-eye-movement (REM) sleep, and decreased wakefulness at 01:00 h, whereas increasing it at 13:00 h. Cortistatin blunts cAMP concentration and impairs motor control at 13:00 h. These results support further a cortistatin modulatory role in the memory process.

Aspectos moleculares de la demencia asociada con el SIDA / Molecular aspects of dementia associated with AIDS

Se ha observado una variedad de trastornos neuropsiquiátricos entre 20 y 30 por ciento de los pacientes que padecen del síndrome de inmunodeficiencia adquirida (SIDA). Debido a que las neuronas no se infectan directamente con el virus de la inmunodeficiencia adquirida (HIV), las manifestaciones fisiopatológicas de la demencia asociada con el SIDA (ADC) podrían estar relacionadas con mecanismos indirectos. La glucoproteína 120 de la envoltura viral (gp 120) derivada del VIH parece desempeñar un papel importante en el desarrollo de la ADC. Una cantidad cada vez mayor de experimentos han indicado que las concentraciones nanomolares de la gp120 derivada del VIH produce muerte neuronal in vivo, en tanto que las concentraciones picomolares matan a las neuronas in vitro. Los datos recientes sugieren que para inducir el daño neuronal, los receptores a citocinas y el CD4+ desempeñan un papel muy importante en la activación de eventos intracelulares que conducen a un incremento del Ca++ intracelular con la participación de los canales NMDA, lo que dispara los eventos intracelulares y la apoptosis. Entender el mecanismo del daño neuronal desde un punto de vista molecular y conductual, probablemente nos proporcionará el conocimiento para encontrar el tratamiento y la manera de prevenir esta complicación clínica. En esta revisión se incluye también el desarrollo de modelos animales para el estudio del mecanismo fisiopatológico de la demencia asociada con el SIDA