Fetal Neuronal Cells Research Articles

Modulation of Ceramide-Induced Apoptosis in Enteric Neurons by Aryl Hydrocarbon Receptor Signaling: Unveiling a New Pathway beyond ER Stress.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent organic pollutant and a potent aryl hydrocarbon receptor (AHR) ligand, causes delayed intestinal motility and affects the survival of enteric neurons. In this study, we investigated the specific signaling pathways and molecular targets involved in TCDD-induced enteric neurotoxicity. Immortalized fetal enteric neuronal (IM-FEN) cells treated with 10 nM TCDD exhibited cytotoxicity and caspase 3/7 activation, indicating apoptosis. Increased cleaved caspase-3 expression with TCDD treatment, as assessed by immunostaining in enteric neuronal cells isolated from WT mice but not in neural crest cell-specific Ahr deletion mutant mice (Wnt1Cre+/-/Ahrb(fl/fl)), emphasized the pivotal role of AHR in this process. Importantly, the apoptosis in IM-FEN cells treated with TCDD was mediated through a ceramide-dependent pathway, independent of endoplasmic reticulum stress, as evidenced by increased ceramide synthesis and the reversal of cytotoxic effects with myriocin, a potent inhibitor of ceramide biosynthesis. We identified Sptlc2 and Smpd2 as potential gene targets of AHR in ceramide regulation by a chromatin immunoprecipitation (ChIP) assay in IM-FEN cells. Additionally, TCDD downregulated phosphorylated Akt and phosphorylated Ser9-GSK-3β levels, implicating the PI3 kinase/AKT pathway in TCDD-induced neurotoxicity. Overall, this study provides important insights into the mechanisms underlying TCDD-induced enteric neurotoxicity and identifies potential targets for the development of therapeutic interventions.

Aryl hydrocarbon receptor activation affects nitrergic neuronal survival and delays intestinal motility in mice.

Despite progress describing the effects of persistent organic pollutants (POPs) on the central nervous system, the effect of POPs on enteric nervous system (ENS) function remains underexplored. We studied the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a POP, and a potent aryl hydrocarbon receptor (AHR) ligand, on the ENS and intestinal motility in mice. C57Bl/6J mice treated with TCDD (2.4 µg/kg body weight) for 8 weeks (once per week) exhibited significant delay in intestinal motility as shown by reduced stool frequency, prolonged intestinal transit time, and a persistence of dye in the jejunum compared to control mice with maximal dye retention in the ileum. TCDD significantly increased Cyp1a1 expression, an AHR target gene, and reduced the total number of neurons and affected nitrergic neurons in cells isolated from WT mice, but not Ahr-/- mice. In immortalized fetal enteric neuronal cells, TCDD-induced nuclear translocation of AHR as well as increased Cyp1a1 expression. AHR activation did not affect neuronal proliferation. However, AHR activation resulted in enteric neuronal toxicity, specifically, nitrergic neurons. Our results demonstrate that TCDD adversely affects nitrergic neurons and thereby contributes to delayed intestinal motility. These findings suggest that AHR signaling in the ENS may play a role in modulating TCDD-induced gastrointestinal pathophysiology.

First Clinical Report on the Treatment of Parkinson's Disease with Fetal Midbrain Precursor Cells.

Because human fetal ventral mesencephalic tissue grafts provide promising results in ameliorating Parkinson's disease-implicated motor dysfunctions, human fetal midbrain-derived dopamine neuronal precursor cells are considered good candidates for cell-based therapy for Parkinson's disease in that large quantities of cells can be supplied through a good manufacturing practice-compliant system. We conducted a prospective, phase I/IIa, dose-escalation, open-label "first-in-human" clinical trial with fetal neural precursor cells to assess their safety and therapeutic efficacy in patients with idiopathic Parkinson's disease. Fifteen patients were assigned to receive three different doses of cells (4 × 106 , 12 × 106 , and 40 × 106 cells) and completed a 12-month follow-up. The primary outcome was safety, by measuring the presence of grade 3 or higher cells according to National Cancer Institute guidelines and any contaminated cells. Secondary outcomes assessed motor and neurocognitive function, as well as the level of dopamine transporters, by positron emission tomography-computed tomography. Although a pronation-supination and hand/arm movement performance was remarkably enhanced in all three groups (all P < 0.05), the medium- and high-dose-treated groups exhibited significant improvement in Unified Parkinson's Disease Rating Scale Part III only up to 26.16% and 40%, respectively, at 12 months after transplantation without any serious clinical complications or graft-induced dyskinesia in all patients. However, the motor improvements did not correlate with increase in the dopamine transporter on positron emission tomography images. Our results primarily demonstrate the safety and plausible dose-dependent efficacy of human fetal midbrain-derived dopamine neuronal precursor cells for idiopathic Parkinson's disease. © 2023 International Parkinson and Movement Disorder Society.

Distinct regulation of hippocampal neuroplasticity and ciliary genes by corticosteroid receptors

Glucocorticoid hormones (GCs) — acting through hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) — are critical to physiological regulation and behavioural adaptation. We conducted genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus to elucidate MR- and GR-regulated genes under circadian variation or acute stress. In a subset of genes, these physiological conditions resulted in enhanced MR and/or GR binding to DNA sequences and associated transcriptional changes. Binding of MR at a substantial number of sites however remained unchanged. MR and GR binding occur at overlapping as well as distinct loci. Moreover, although the GC response element (GRE) was the predominant motif, the transcription factor recognition site composition within MR and GR binding peaks show marked differences. Pathway analysis uncovered that MR and GR regulate a substantial number of genes involved in synaptic/neuro-plasticity, cell morphology and development, behavior, and neuropsychiatric disorders. We find that MR, not GR, is the predominant receptor binding to >50 ciliary genes; and that MR function is linked to neuronal differentiation and ciliogenesis in human fetal neuronal progenitor cells. These results show that hippocampal MRs and GRs constitutively and dynamically regulate genomic activities underpinning neuronal plasticity and behavioral adaptation to changing environments.

Mutual Interactions Between GnRH and Kisspeptin in GnRH- and Kiss-1-Expressing Immortalized Hypothalamic Cell Models.

Kisspeptin and gonadotropin-releasing hormone (GnRH) are central regulators of the hypothalamic-pituitary-gonadal axis and control female reproductive functions. Recently established mHypoA-50 and mHypoA-55 cells are immortalized hypothalamic neuronal cell models that originated from the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) regions of the mouse hypothalamus, respectively. mHypoA-50 or mHypoA-55 cells were stimulated with kisspeptin-10 (KP10) and GnRH, after which the expression of kisspeptin and GnRH was determined. Primary cultures of fetal rat brain cells were also examined. mHypoA-50 and mHypoA-55 cells expressed mRNA for Kiss-1 (which encodes kisspeptin) and GnRH as well as receptors for kisspeptin and GnRH. We found that Kiss-1 mRNA expression was significantly increased in mHypoA-50 AVPV cells by KP10 and GnRH stimulation. Kisspeptin protein expression was also increased by KP10 and GnRH stimulation in these cells. In contrast, GnRH expression was unchanged in mHypoA-50 AVPV cells by KP10 and GnRH stimulation. In mHypoA-55 ARC cells, kisspeptin expression was also significantly increased at the mRNA and protein levels by KP10 and GnRH stimulation; however, GnRH expression was also upregulated by KP10 and GnRH stimulation in these cells. KP10 and estradiol (E2) both increased Kiss-1 gene expression in mHypoA-50 AVPV cells, but combined stimulation with KP10 and E2 did not potentiate their individual effects on Kiss-1 gene expression. On the other hand, E2 did not increase Kiss-1 gene expression in mHypoA-55 ARC cells, and the KP10-induced increase of Kiss-1 gene expression was inhibited in the presence of E2 in these cells. KP10 and GnRH significantly increased c-Fos protein expression in the mHypoA-50 AVPV and mHypoA-55 ARC cell lines. In primary cultures of fetal rat neuronal cells, KP10 significantly increased Kiss-1 gene expression, whereas GnRH significantly increased GnRH gene expression. We found that kisspeptin and GnRH affected Kiss-1- and GnRH-expressing hypothalamic cells and modulated Kiss-1 and/or GnRH gene expression with a concomitant increase in c-Fos protein expression. A mutual- or self-regulatory system might be present in Kiss-1 and/or GnRH neurons in the hypothalamus.

Roles of intracerebral activin, inhibin, and follistatin in the regulation of Kiss-1 gene expression: Studies using primary cultures of fetal rat neuronal cells

Hypothalamic kisspeptin, encoded by the Kiss-1 gene, governs the hypothalamic-pituitary-gonadal axis by directly regulating the release of gonadotropin-releasing hormone. In this study, we examined the roles of activin, inhibin, and follistatin in the regulation of Kiss-1 gene expression using primary cultures of fetal rat neuronal cells, which express the Kiss-1 gene and kisspeptin. Stimulation with activin significantly increased Kiss-1 gene expression in these cultures by 2.02 ± 0.39-fold. In contrast, a significant decrease in Kiss-1 gene expression was observed with inhibin A and follistatin treatment. Inhibin B did not modulate Kiss-1 gene expression. Activin, inhibin, and follistatin were also expressed in fetal rat brain cultures and their expression was controlled by estradiol (E2). The inhibin α, βA, and βB subunits were upregulated by E2. Similarly, follistatin gene expression was significantly increased by E2 in these cells. Our results suggest the possibility that activin, inhibin, and follistatin expressed in the brain participate in the E2-induced feedback control of the hypothalamic-pituitary-gonadal axis.

Neurturin-containing laminin matrices support innervated branching epithelium from adult epithelial salispheres

Cell transplantation of autologous adult biopsies, grown ex vivo as epithelial organoids or expanded as spheroids, are proposed treatments to regenerate damaged branching organs. However, it is not clear whether transplantation of adult organoids or spheroids alone is sufficient to initiate a fetal-like program of branching morphogenesis in which coordinated branching of multiple cell types including nerves, mesenchyme and blood vessels occurs. Yet this is an essential concept for the regeneration of branching organs such as lung, pancreas, and lacrimal and salivary glands. Here, we used factors identified from fetal organogenesis to maintain and expand adult murine and human epithelial salivary gland progenitors in non-adherent spheroid cultures, called salispheres. These factors stimulated critical developmental pathways, and increased expression of epithelial progenitor markers such as Keratin5, Keratin14, FGFR2b and KIT. Moreover, physical recombination of adult salispheres in a laminin-111 extracellular matrix with fetal salivary mesenchyme, containing endothelial and neuronal cells, only induced branching morphogenesis when neurturin, a neurotrophic factor, was added to the matrix. Neurturin was essential to improve neuronal survival, axon outgrowth, innervation of the salispheres, and resulted in the formation of branching structures with a proximal-distal axis that mimicked fetal branching morphogenesis, thus recapitulating organogenesis. Epithelial progenitors were also maintained, and developmental differentiation programs were initiated, showing that the fetal microenvironment provides a template for adult epithelial progenitors to initiate branching and differentiation. Further delineation of secreted and physical cues from the fetal niche will be useful to develop novel regenerative therapies that instruct adult salispheres to resume a developmental-like program in vitro and to regenerate branching organs in vivo.

Congenital Zika virus infection as a silent pathology with loss of neurogenic output in the fetal brain.

Zika virus (ZIKV) is a flavivirus with teratogenic effects on the fetal brain, but the spectrum of brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model, we demonstrate that ZIKV injury to the fetal brain was significant even in the absence of microcephaly and is clinically challenging to detect. A common and subtle injury pattern was identified including: 1) periventricular T2-hyperintense foci and loss of fetal non-cortical brain volume, 2) injury to the ependymal epithelium with underlying gliosis, and 3) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially significant teratogenic effects of congenital ZIKV infection even without microcephaly. All children exposed to ZIKV in utero should be followed long-term for neurocognitive deficits, regardless of head size at birth.

The co-expression of Neogenin with SOX2 in hippocampal neurons

Dementia has been shown to be closely related with neuronal degeneration and/or a decrease in the activity of neural stem cells in many brain regions, including the hippocampus. It has been recently established that Neogenin is involved in the cell fate determination by regulating Oct3/4, SOX and Nanog, notable embryonic cell markers, expressions in pre-implantation mouse embryos. Further, Neogenin expression at both mRNA and protein levels is manifest in many brain regions in mice, but it remains unclear whether Neogenin expression is prerequisite for the maintenance of neural stem cells, particularly, playing a critical role in the hippocampus, a brain region known to be involved in memory generation and consolidation. Here, we provide evidence that supports that Neogenin is implicated in the maintenance of neural stem cells in the hippocampus by enhancing PCNA expressions. We have performed RT-PCR analysis, Western blotting, and immunohistochemistry with fetal rat brain tissues at E18 for Neogenin mRNA and protein profiling. Neuronal cells obtained from the hippocampus were subjected to FACS analysis for the identification of Neogenin-positive and/or neuronal stem cell marker-positive cells. Western blotting results showed that Neogenin expression was higher in the hippocampal region compared to the cortical region. FACS analysis results indicated that a significant population of fetal rat neuronal cells exhibiting Neogenin expression also displayed SOX2 expression, implying co-expression of Neogenin and SOX2 in the hippocampus. Next, we investigated the role of Neogenin through gain- and loss-of-function studies with cultured rat hippocampal neurons. Neogenin down-regulation by small hairpin RNAs led to a dramatic decrease in SOX2 expression while its up-regulation by overexpression caused an increase in PCNA expression, a cell proliferation marker, compared with none-transfected cells. From this study, we propose a model whereby Neogenin could maintain neural stem cell population and control cell proliferation.

Bacterial Peptidoglycan Traverses the Placenta to Induce Fetal Neuroproliferation and Aberrant Postnatal Behavior

Maternal infection during pregnancy is associated with adverse outcomes for the fetus, including postnatal cognitive disorders. However, the underlying mechanisms are obscure. We find that bacterial cell wall peptidoglycan (CW), a universal PAMP for TLR2, traverses the murine placenta into the developing fetal brain. In contrast to adults, CW-exposed fetal brains did not show any signs of inflammation or neuronal death. Instead, the neuronal transcription factor FoxG1 was induced, and neuroproliferation leading to a 50% greater density of neurons in the cortical plate was observed. Bacterial infection of pregnant dams, followed by antibiotic treatment, which releases CW, yielded the same result. Neuroproliferation required TLR2 and was recapitulated in vitro with fetal neuronal precursor cells and TLR2/6, but not TLR2/1, ligands. The fetal neuroproliferative response correlated with abnormal cognitive behavior in CW-exposed pups following birth. Thus, the bacterial CW-TLR2 signaling axis affects fetal neurodevelopment and may underlie postnatal cognitive disorders.

Best practice for passaging murine embryonic enteric neuronal cell line before differentiation.

The enteric nervous system (ENS) is a complex network of neurons in the gut, regulating many local, vital functions of the gastro-intestinal tract. The ENS is also part of the bidirectional gut-brain axis. The murine immorto fetal enteric neuronal (IM-FEN) cell line was chosen as a model to study enteric neurons. This cell line can be differentiated into cells with a neuronal phenotype, although they do not produce action potentials in vitro. It was concluded that the differentiation process in our laboratory was successful, based on positive staining for neuronal proteins. Proliferating IM-FEN cells have an unstable growth rate in our laboratory. An indicator of growth rate was calculated, and this indicator was found to be related to seeding density and number of days in culture, and was unrelated to person culturing, previous overconfluency or passage number. The indicator of growth rate was also unrelated to successful use of differentiated cells in follow-up experiments. We recommend the following conditions for optimal culture of IM-FEN cells. Keep cells in culture until 80 % confluent before passaging, seed cells at a density of 0.0133 million cells per cm2, and anticipate on unstable growth rates and the risk for overconfluency.

Anti-inflammatory and neuroprotective effects of GILZ analogs. (THER7P.951)

Abstract Increased activation of the p65 subunit of nuclear factor kappa-B (NF-κB) upregulate inflammatory and apoptotic factors that mediate neuroinflammation and neurodegeneration in chronic neurological diseases, suggesting that activated p65 is an excellent therapeutic target. Glucocorticoid induced leucine zipper (GILZ) is a p65 interacting protein, that sequesters activated p65 in the cytoplasm and suppresses NF-κB. The GILZ:p65 interaction is localized to the proline rich region at the carboxy terminus of GILZ. Previously we reported that a synthetic peptide (GILZ-P) derived from the p65 binding domain of GILZ ameliorated experimental encephalomyelitis. To determine the potential application of GILZ-P in humans, we evaluated the toxicity and efficacy of the peptide drug in mature human neuroblastoma cells, macrophage like THP-1 cells and fetal neuronal cells. Treatment with GILZ-P at a wide range of concentrations commonly used for peptide drugs was non-toxic as determined by the cell viability and apoptosis assays in THP-1 cells. Functionally GILZ-P suppressed pro-inflammatory cytokine secretion by proteolipid protein or amyloid-β (Aβ1-42) stimulated mature neuroblastoma cells and suppressed glutamate secretion by activated macrophages by inhibiting nuclear translocation of p65. Collectively, GILZ-P exhibits therapeutic potential for chronic diseases wherein persistent inflammation precipitates neurodegeneration such as progressive multiple sclerosis and Alzheimer’s disease.

Transient but not permanent benefit of neuronal progenitor cell therapy after traumatic brain injury: potential causes and translational consequences.

Background: Numerous studies have reported a beneficial impact of neural progenitor cell transplantation on functional outcome after traumatic brain injury (TBI) during short and medium follow-up periods. However, our knowledge regarding long-term functional effects is fragmentary while a direct comparison between local and systemic transplantation is missing so far.Objectives: This study investigated the long-term (12 week) impact of human fetal neuronal progenitor cell (hNPC) transplantation 24 h after severe TBI in rats.Methods: Cells were either transplanted stereotactically (1 × 105) into the putamen or systemically (5 × 105) via the tail vein. Control animals received intravenous transplantation of vehicle solution.Results: An overall functional benefit was observed after systemic, but not local hNPC transplantation by area under the curve analysis (p < 0.01). Surprisingly, this effect vanished during later stages after TBI with all groups exhibiting comparable functional outcomes 84 days after TBI. Investigation of cell-mediated inflammatory processes revealed increasing microglial activation and macrophage presence during these stages, which was statistically significant after systemic cell administration (p < 0.05). Intracerebral hNPC transplantation slightly diminished astrogliosis in perilesional areas (p < 0.01), but did not translate into a permanent functional benefit. No significant effects on angiogenesis were observed among the groups.Conclusion: Our results suggest the careful long-term assessment of cell therapies for TBI, as well as to identify potential long-term detrimental effects of such therapies before moving on to clinical trials. Moreover, immunosuppressive protocols, though widely used, should be rigorously assessed for their applicability in the respective setup.

Single-Cell Suspension Methodology Favors Survival and Vascularization of Fetal Striatal Grafts in the YAC128 Mouse Model of Huntington's Disease

Cell replacement therapies have yielded variable and short-lived benefits in Huntington's disease (HD) patients. This suboptimal outcome is likely due to the fact that graft survival is compromised long term because grafts are subjected to a host's microglial inflammatory response, to a lack of adequate trophic support, and possibly to cortical excitotoxicity. However, graft demise may also relate to more straightforward issues such as cell preparation methodology (solid grafts vs. cell suspension). Indeed, we recently reported that solid grafts are poorly revascularized in HD patients transplanted 9 and 12 years previously. To evaluate whether methodological issues relating to cell preparation may have an impact on graft viability, we implanted green fluorescent protein (GFP(+)) single-cell suspensions of fetal striatal neuronal cells into the striatum of YAC128 HD mice. Postmortem evaluation yielded comparable graft survival in YAC128 mice and their wild-type littermates (noncarrier) at 1 and 3 months posttransplantation. Additionally, the degrees of graft revascularization in the YAC128 and noncarrier mice were similar, with both capillaries and large-caliber vessels observable within the grafted tissue. Furthermore, GFP(+) cells interacted well with host blood vessels, indicating integration of the donor cells within the recipient brain. These observations, combined with our recent report of poor revascularization of solid grafts in the HD-transplanted patients, suggest that the success of cell transplantation can be improved by optimizing methodological aspects relating to cell preparation.

Вплив введення кріоконсервованих фетальних нервових клітин на прооксидантно-антиоксидантну рівновагу в щурів із експериментальною краніоскелетною травмою

In the response to cranio-skeletal injury, complicated by bleeding, the content of primary and secondary products of lipid peroxidation (LPO) in liver homogenate significantly increased, and achieved a maximum level to the 14th day of the experiment and was significantly higher than the control level during 25 days of observation. Within the 3rd–14th days the accumulation of LPO products occured on the background of significant decrease of superoxide dismutase (SOD) activity, without any changes in catalase activity and compensatory increase of ceruloplasmin content to the 3rd day of the experiment. From 14th to 25th day following treatment with cryopreserved fetal neuronal cells we observed less pronounced deviations of lipid peroxidation in terms of content of primary LPO products (diene conjugates), and content of secondary LPO products (TBA-active) from the 3rd to 7th day. Catalase content was reduced through the 3rd–14th days of the experiment, increased by the 25th day; from the 14th day we observed a protective effect of cryopreserved fetal neuronal cells in terms of SOD activity. There was virtually no correcting effect found in blood serum ceruloplasmin content. Probl Cryobiol Cryomed 2014; 24(1):67–74 .

Transplantation of β-Endorphin Neurons into the Hypothalamus Promotes Immune Function and Restricts the Growth and Metastasis of Mammary Carcinoma

Neurobehavioral stress has been shown to promote tumor growth and progression and dampen the immune system. In this study, we investigated whether inhibiting stress hormone production could inhibit the development of mammary carcinoma and metastasis in a rat model of breast carcinogenesis. To enhance β-endorphin (BEP), the endogenous opioid polypeptide that boosts immune activity and decreases stress, we generated BEP neurons by in vitro differentiation from fetal neuronal stem cells and transplanted them into the hypothalami of rats subjected to breast carcinogenesis. BEP-transplanted rats displayed a reduction in mammary tumor incidence, growth, malignancy rate, and metastasis compared with cortical cells-transplanted rats. BEP neuron transplants also reduced inflammation and epithelial to mesenchymal transition in the tumor tissues. In addition, BEP neuron transplants increased peripheral natural killer (NK) cell and macrophage activities, elevated plasma levels of antiinflammatory cytokines, and reduced plasma levels of inflammatory cytokines. Antimetastatic effects along with stimulation of NK cells and macrophages could be reversed by treatment with the opiate antagonist naloxone, the β-receptor agonist metaproterenol, or the nicotine acetylcholine receptor antagonist methyllycaconitine. Together, our findings establish a protective role for BEP against the growth and metastasis of mammary tumor cells by altering autonomic nervous system activities that enhance innate immune function.

Long-Term Benefit of Human Fetal Neuronal Progenitor Cell Transplantation in a Clinically Adapted Model after Traumatic Brain Injury

Experimental human fetal neural progenitor cell (hfNPC) transplantation has proven to be a promising therapeutic approach after traumatic brain injury (TBI). However, the long-term efficacy and safety, which are both highly important for clinical translation of this approach, have thus far not been investigated. This study investigated the effect of local (L, 1 × 10(5) cells) and systemic (S, 5 × 10(5) cells) administration of PKH-26-labeled pre-differentiated hfNPCs over a period of 12 weeks, beginning 24 h after severe controlled cortical impact TBI in Sprague-Dawley rats. Accelerating rotarod testing revealed a trend toward functional improvement beginning 1 week after transplantation, and persisting until the end of the experiment. The traumatic lesion volume as quantified by magnetic resonance imaging was smaller in both treatment groups compared to control (C) animals (C = 54.50 mm(3), L = 32 mm(3), S = 37.50 mm(3)). Correspondingly, neuronal (NeuN) staining showed increased neuronal survival at the border of the lesion in both transplanted groups (S = 92.4%; L = 87.2%; 72.5%). Histological analysis of the brain compartments revealed transiently increased angiogenesis and reduced astroglial reaction during the first 4 weeks post-transplantation. PKH-26-positive cells were detected exclusively after local transplantation without any evidence of tumor formation. However, graft differentiation was seen only in very rare cases. In conclusion, transplantation of hfNPCs improved the long-term functional outcome after TBI, diminished trauma lesion size, and increased neuronal survival in the border zone of the lesion. This therapeutic effect was not likely due to cell replacement, but was associated with transiently increased angiogenesis and reduced astrogliosis.

Immune response after striatal engraftment of fetal neuronal cells in patients with Huntington’s disease: Consequences for cerebral transplantation programs

Objectives: Striatal transplantation of human fetal neurones as a therapeutic approach to treat Huntington’s disease is under investigation. The allogeneic graft material raises questions of immune response and immunosuppressive treatment. No data is available on how the human brain reacts immunologically over time to neuronal transplantation. Methods: We measured post-engraftment immune responses as assessed by the development of anti-human leukocyte antigen (HLA)-antibodies to allogeneic intrastriatal transplantation of fetal neurons in Huntington’s disease patients. Results: Out of 10 patients without HLA-antibodies before engraftment, five developed HLA-antibodies of class I and II. The time between transplantation and development of HLA-antibodies was variable, with detection either immediately after engraftment, during immunosuppressive therapy or months to years after immunosuppressive drugs were discontinued. There are no typical changes in magnetic resonance imaging that distinguish the different immune behaviors. Positive clinical responses to engraftment might not be reflected in the 18F-deoxyglucose positron emission tomography images as expected. Conclusions: There is indeed an immune response from the human brain, but the time between transplantation and development of anti-HLA-antibodies is variable and unpredictable. A long-term, post-engraftment immunosuppressant therefore cannot be justified. The impact of the emergence of HLA-antibodies on graft survival and clinical efficacy cannot be ascertained at this stage. Given the projected clinical trials of cell transplantation in neurodegenerative disorders, the findings are of practical significance. (Clin. Exp. Neuroimmunol. doi: 10.1111/j.1759-1961.2011.00018.x, May 2011)

Neuroprotection by Minocycline Caused by Direct and Specific Scavenging of Peroxynitrite

Minocycline prevents oxidative protein modifications and damage in disease models associated with inflammatory glial activation and oxidative stress. Although the drug has been assumed to act by preventing the up-regulation of proinflammatory enzymes, we probed here its direct chemical interaction with reactive oxygen species. The antibiotic did not react with superoxide or (•)NO radicals, but peroxynitrite (PON) was scavenged in the range of ∼1 μm minocycline and below. The interaction of pharmacologically relevant minocycline concentrations with PON was corroborated in several assay systems and significantly exceeded the efficacy of other antibiotics. Minocycline was degraded during the reaction with PON, and the resultant products lacked antioxidant properties. The antioxidant activity of minocycline extended to cellular systems, because it prevented neuronal mitochondrial DNA damage and glutathione depletion. Maintenance of neuronal viability under PON stress was shown to be solely dependent on direct chemical scavenging by minocycline. We chose α-synuclein (ASYN), known from Parkinsonian pathology as a biologically relevant target in chemical and cellular nitration reactions. Submicromolar concentrations of minocycline prevented tyrosine nitration of ASYN by PON. Mass spectrometric analysis revealed that minocycline impeded nitrations more effectively than methionine oxidations and dimerizations of ASYN, which are secondary reactions under PON stress. Thus, PON scavenging at low concentrations is a novel feature of minocycline and may help to explain its pharmacological activity.

Electrophysiological characterization of postnatal enteric neuron cell line

Conditionally immortalized fetal and postnatal enteric neuronal cell lines (IM‐FEN and IM‐PEN) have been developed from the H‐2kb‐tsA58 mouse (immortomouse), however, their electrophysiological properties are unknown. The goal of the present study was to characterize the electrophysiological properties of IM‐PEN. We confirmed the neuronal background of the cell line with positive staining of neuron specific β‐III tubulin. The cells exhibited depolarized resting membrane potentials of ‐24.7 ± 4.7mV (n=6) and input resistances of 672 ± 29MΩ. Depolarizing pulses in current clamp failed to yield action potentials from rest or from hyperpolarized holding potentials (n= 6). This was consistent with the absence of inward Na+ or Ca2+ currents to depolarizing pulses (n= 5). In voltage clamp, step depolarization from Vh ‐60 mV to +30 mV in 10 mV steps produced time‐independent currents which were blocked by replacing external chloride. These currents were not blocked by the Icl,swell blocker, DCPIB (10μM) (n= 3), however, niflumic acid (1μM and 500 uM), a chloride channel blocker resulted in increased outward currents and a significant shift in the reversal potential from −37.3 ± 2.8mV to −53.0 ± 3.6mV (n= 14). The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) at 100μM increased the outward current in 3 cells. External application of acetylcholine (500μM) (n= 4), nicotine (1mM) (n= 3), and serotonin (10μM) (n= 3), were ineffective. These studies confirm the neuronal origin of IM‐PEN which have a depolarized resting membrane potential due to high internal chloride concentration. Supported by NIH DA007027.