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Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta, which results in a prominent reduction of striatal dopamine levels leading to motor alterations. The mechanisms underlying neurodegeneration in PD remain unknown. Here, we generated an induced pluripotent stem cell line from dermal fibroblasts of a Mexican patient diagnosed with sporadic PD (UNAMi002-A) and another cell line from dermal fibroblasts of a patient carrying the point mutation c.1423delC in PINK1 (UNAMi003-A). These patient-derived iPS cell lines offer the possibility of modeling PD and understanding the mechanisms that contribute to dopamine neuron loss.

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RESUMO

Human embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DANs), and Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson's disease (PD) patients. Here, we obtained well-characterized DANs from hESCs and transplanted them into two parkinsonian monkeys to assess their behavioral and imaging changes. DANs from hESCs expressed dopaminergic markers, generated action potentials, and released dopamine (DA) in vitro. These neurons were transplanted bilaterally into the putamen of parkinsonian NHPs, and using magnetic resonance imaging techniques, we calculated the fractional anisotropy (FA) and mean diffusivity (MD), both employed for the first time for these purposes, to detect in vivo axonal and cellular density changes in the brain. Likewise, positron-emission tomography scans were performed to evaluate grafted DANs. Histological analyses identified grafted DANs, which were quantified stereologically. After grafting, animals showed signs of partially improved motor behavior in some of the HALLWAY motor tasks. Improvement in motor evaluations was inversely correlated with increases in bilateral FA. MD did not correlate with behavior but presented a negative correlation with FA. We also found higher 11C-DTBZ binding in positron-emission tomography scans associated with grafts. Higher DA levels measured by microdialysis after stimulation with a high-potassium solution or amphetamine were present in grafted animals after ten months, which has not been previously reported. Postmortem analysis of NHP brains showed that transplanted DANs survived in the putamen long-term, without developing tumors, in immunosuppressed animals. Although these results need to be confirmed with larger groups of NHPs, our molecular, behavioral, biochemical, and imaging findings support the integration and survival of human DANs in this pre-clinical PD model.

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BACKGROUND: Traumatic spinal cord injury (SCI) is a disabling condition that affects millions of people around the world. Currently, no clinical treatment can restore spinal cord function. Comparison of molecular responses in regenerating to non-regenerating vertebrates can shed light on neural restoration. The axolotl (Ambystoma mexicanum) is an amphibian that regenerates regions of the brain or spinal cord after damage. METHODS: In this study, we compared the transcriptomes after SCI at acute (1-2 days after SCI) and sub-acute (6-7 days post-SCI) periods through the analysis of RNA-seq public datasets from axolotl and non-regenerating rodents. RESULTS: Genes related to wound healing and immune responses were upregulated in axolotls, rats, and mice after SCI; however, the immune-related processes were more prevalent in rodents. In the acute phase of SCI in the axolotl, the molecular pathways and genes associated with early development were upregulated, while processes related to neuronal function were downregulated. Importantly, the downregulation of processes related to sensorial and motor functions was observed only in rodents. This analysis also revealed that genes related to pluripotency, cytoskeleton rearrangement, and transposable elements (e.g., Sox2, Krt5, and LOC100130764) were among the most upregulated in the axolotl. Finally, gene regulatory networks in axolotls revealed the early activation of genes related to neurogenesis, including Atf3/4 and Foxa2. CONCLUSIONS: Immune-related processes are upregulated shortly after SCI in axolotls and rodents; however, a strong immune response is more noticeable in rodents. Genes related to early development and neurogenesis are upregulated beginning in the acute stage of SCI in axolotls, while the loss of motor and sensory functions is detected only in rodents during the sub-acute period of SCI. The approach employed in this study might be useful for designing and establishing regenerative therapies after SCI in mammals, including humans.

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Central nervous system (CNS) infections including meningitis and encephalitis, resulting from the blood-borne spread of specific microorganisms, provoke nervous tissue damage due to the inflammatory process. Moreover, different pathologies such as sepsis can generate systemic inflammation. Bacterial lipopolysaccharide (LPS) induces the release of inflammatory mediators and damage molecules, which are then released into the bloodstream and can interact with structures such as the CNS, thus modifying the blood-brain barrier's (BBB´s) and blood-cerebrospinal fluid barrier´s (BCSFB´s) function and inducing aseptic neuroinflammation. During neuroinflammation, the participation of glial cells (astrocytes, microglia, and oligodendrocytes) plays an important role. They release cytokines, chemokines, reactive oxygen species, nitrogen species, peptides, and even excitatory amino acids that lead to neuronal damage. The neurons undergo morphological and functional changes that could initiate functional alterations to neurodegenerative processes. The present work aims to explain these processes and the pathophysiological interactions involved in CNS damage in the absence of microbes or inflammatory cells.

Assuntos

RESUMO

Parkinson's disease (PD) is a neurodegenerative disease caused by progressive loss of dopaminergic neurons in the substantia nigra pars compacta, which results in motor alterations. The exact mechanisms underlying the dopaminergic neurodegeneration in PD are still unknown. Here, we generated a human induced pluripotent stem cell (iPSC) line from dermal fibroblasts of a Mexican patient diagnosed with sporadic PD. The generated iPS cell line (UNAMi001-A) express pluripotency markers, maintain a normal karyotype and display the ability to differentiate into all three germ layers. This is the first iPSC line from a Mexican patient and will be useful for PD modeling.

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Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins ßIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

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The neuromuscular junction (NMJ) is a specialized structure that works as an interface to translate the action potential of the presynaptic motor neuron (MN) in the contraction of the postsynaptic myofiber. The design of appropriate experimental models is essential to have efficient and reliable approaches to study NMJ development and function, but also to generate conditions that recapitulate distinct features of diseases. Initial studies relied on the use of tissue slices maintained under the same environment and in which single motor axons were difficult to trace. Later, MNs and muscle cells were obtained from primary cultures or differentiation of progenitors and cocultured as monolayers; however, the tissue architecture was lost. Current approaches include self-assembling 3D structures or the incorporation of biomaterials with cells to generate engineered tissues, although the incorporation of Schwann cells remains a challenge. Thus, numerous investigations have established different NMJ models, some of which are quite complex and challenging. Our review summarizes the in vitro models that have emerged in recent years to coculture MNs and skeletal muscle, trying to mimic the healthy and diseased NMJ. We expect our review may serve as a reference for choosing the appropriate experimental model for the required purposes of investigation.

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During midbrain development, dopamine neuron differentiation occurs before birth. Epigenetic processes such as DNA methylation and demethylation as well as post-translational modification of histones occur during neurogenesis. Here, we administered histamine (HA) into the brain of E12 embryos in vivo and observed significant lower immunoreactivity of Lmx1a+ and Tyrosine Hydroxylase (TH)+ cells, with parallel decreases in the expression of early (Lmx1a, Msx1) and late (Th) midbrain dopaminergic (mDA) genes. With MeDIP assays we found that HA decreases the percentage of 5-methylcytosine of Pitx3 and Th, without changes in 5-hydroxymethylcytosine. Additionally, HA treatment caused a significant increase in the repressive epigenetic modifications H3K9me3 in Pitx3 and Th, and also more H3K27me3 marks in Th. Furthermore, HA has a long-term effect on the formation of the nigrostriatal and mesolimbic/mesocortical pathways, since it causes a significant decrease in midbrain TH immunoreactivity, as well as alterations in dopaminergic neuronal fibers, and significant lower TH-positive area in the forebrain in whole-mount stainings. These findings suggest that HA diminishes dopaminergic gene transcription by altering several epigenetic components related to DNA and histone modifications, which affects mDA neuron progression during development.