Microglia-secreted TNF-α affects differentiation efficiency and viability of pluripotent stem cell-derived human dopaminergic precursors.

Disease is a neurodegenerative disorder characterised by the progressive loss of dopaminergic cells of the substantia nigra pars compacta. Even though successful transplantation of dopamine-producing cells into the striatum exhibits favourable effects in animal models and clinical trials; transplanted cell survival is low. Since every transplant elicits an inflammatory response which can affect cell survival and differentiation, we aimed to study in vivo and in vitro the impact of the pro-inflammatory environment on human dopaminergic precursors. We first observed that transplanted human dopaminergic precursors into the striatum of immunosuppressed rats elicited an early and sustained activation of astroglial and microglial cells after 15 days' post-transplant. This long-lasting response was associated with Tumour necrosis factor alpha expression in microglial cells. In vitro, conditioned media from activated BV2 microglial cells increased cell death, decreased Tyrosine hydroxylase-positive cells and induced morphological alterations on human neural stem cells-derived dopaminergic precursors at two differentiation stages: 19 days and 28 days. Those effects were ameliorated by inhibition of Tumour necrosis factor alpha, a cytokine which was previously detected in vivo and in conditioned media from activated BV-2 cells. Our results suggest that a pro-inflammatory environment is sustained after transplantation under immunosuppression, providing a window of opportunity to modify this response to increase transplant survival and differentiation. In addition, our data show that the microglia-derived pro-inflammatory microenvironment has a negative impact on survival and differentiation of dopaminergic precursors. Finally, Tumour necrosis factor alpha plays a key role in these effects, suggesting that this cytokine could be an interesting target to increase the efficacy of human dopaminergic precursors transplantation in Parkinson's Disease.

A familiar study on self-limited childhood epilepsy patients using hIPSC-derived neurons shows a bias towards immaturity at the morphological, electrophysiological and gene expression levels.

BACKGROUND: Self-limited Childhood Epilepsies are the most prevalent epileptic syndrome in children. Its pathogenesis is unknown. In this disease, symptoms resolve spontaneously in approximately 50% of patients when maturity is reached, prompting to a maturation problem. The purpose of this study was to understand the molecular bases of this disease by generating and analyzing induced pluripotent stem cell-derived neurons from a family with 7 siblings, among whom 4 suffer from this disease. METHODS: Two affected siblings and, as controls, a healthy sister and the unaffected mother of the family were studied. Using exome sequencing, a homozygous variant in the FYVE, RhoGEF and PH Domain Containing 6 gene was identified in the patients as a putative genetic factor that could contribute to the development of this familial disorder. After informed consent was signed, skin biopsies from the 4 individuals were collected, fibroblasts were derived and reprogrammed and neurons were generated and characterized by markers and electrophysiology. Morphological, electrophysiological and gene expression analyses were performed on these neurons. RESULTS: Bona fide induced pluripotent stem cells and derived neurons could be generated in all cases. Overall, there were no major shifts in neuronal marker expression among patient and control-derived neurons. Compared to two familial controls, neurons from patients showed shorter axonal length, a dramatic reduction in synapsin-1 levels and cytoskeleton disorganization. In addition, neurons from patients developed a lower action potential threshold with time of in vitro differentiation and the amount of current needed to elicit an action potential (rheobase) was smaller in cells recorded from NE derived from patients at 12 weeks of differentiation when compared with shorter times in culture. These results indicate an increased excitability in patient cells that emerges with the time in culture. Finally, functional genomic analysis showed a biased towards immaturity in patient-derived neurons. CONCLUSIONS: We are reporting the first in vitro model of self-limited childhood epilepsy, providing the cellular bases for future in-depth studies to understand its pathogenesis. Our results show patient-specific neuronal features reflecting immaturity, in resonance with the course of the disease and previous imaging studies.

Cell therapy for Parkinson's disease is coming of age: current challenges and future prospects with a focus on immunomodulation.

Parkinson's disease (PD) is a neurodegenerative disease that affects more than 1% of people over the age of 60. The principal feature of this disease is the progressive loss of dopaminergic neurons (DAn) within the nigrostriatal system, causing the motor symptoms observed in these patients. At present, there is no therapeutic approach with a cytoprotective effect that can prevent DAn cell death or disease progression. Cell replacement therapy began 30 years ago with the objective to compensate for the loss of DAn by transplantation of dopamine-producing cells. The results from these trials have provided proof of concept of safety and efficacy of cell replacement. However, a major limiting factor of this strategy has been the poor survival rate of grafted DAn. An important factor that could cause cell death of DA precursors is the host response to the graft. In this review, we discuss the factors that affect the outcome of cell therapy in PD, with focus on the cell types used and the functional effects of the host immune response on graft survival and differentiation. We also discuss the strategies that may increase the efficacy of cell replacement therapy which target the host immune response.

Chronic Hippocampal Expression of Notch Intracellular Domain Induces Vascular Thickening, Reduces Glucose Availability, and Exacerbates Spatial Memory Deficits in a Rat Model of Early Alzheimer.

The specific roles of Notch in progressive adulthood neurodegenerative disorders have begun to be unraveled in recent years. A number of independent studies have shown significant increases of Notch expression in brains from patients at later stages of sporadic Alzheimer's disease (AD). However, the impact of Notch canonical signaling activation in the pathophysiology of AD is still elusive. To further investigate this issue, 2-month-old wild-type (WT) and hemizygous McGill-R-Thy1-APP rats (Tg(+/-)) were injected in CA1 with lentiviral particles (LVP) expressing the transcriptionally active fragment of Notch, known as Notch Intracellular Domain (NICD), (LVP-NICD), or control lentivirus particles (LVP-C). The Tg(+/-) rat model captures presymptomatic aspects of the AD pathology, including intraneuronal amyloid beta (Aß) accumulation and early cognitive deficits. Seven months after LVP administration, Morris water maze test was performed, and brains isolated for biochemical and histological analysis. Our results showed a learning impairment and a worsening of spatial memory in LVP-NICD- as compared to LVP-C-injected Tg(+/-) rats. In addition, immuno histochemistry, ELISA multiplex, Western blot, RT-qPCR, and 1H-NMR spectrometry of cerebrospinal fluid (CSF) indicated that chronic expression of NICD promoted hippocampal vessel thickening with accumulation of Aß in brain microvasculature, alteration of blood-brain barrier (BBB) permeability, and a decrease of CSF glucose levels. These findings suggest that, in the presence of early Aß pathology, expression of NICD may contribute to the development of microvascular abnormalities, altering glucose transport at the BBB with impact on early decline of spatial learning and memory.

Cell therapy for Parkinson's disease: Functional role of the host immune response on survival and differentiation of dopaminergic neuroblasts.

Parkinson's disease (PD) is a neurodegenerative disorder, whose cardinal pathology is the loss of dopaminergic neurons in the substantia nigra. Current treatments for PD have side effects in the long term and do not halt disease progression or regenerate dopaminergic cell loss. Attempts to compensate neuronal cell loss by transplantation of dopamine-producing cells started more than 30 years ago, leading to several clinical trials. These trials showed safety and variable efficacy among patients. In addition to variability in efficacy, several patients developed graft-induced dyskinesia. Nevertheless, they have provided a proof of concept that motor symptoms could be improved by cell transplantation. Cell transplantation in the brain presents several immunological challenges. The adaptive immune response should be abolished to avoid graft rejection by the host. In addition, the innate immune response will always be present after transplanting cells into the brain. Remarkably, the innate immune response can have dramatic effects on the survival, differentiation and proliferation of the transplanted cells, but has been hardly investigated. In this review, we analyze data on the functional effects of signals from the innate immune system on dopaminergic differentiation, survival and proliferation. Then, we discussed efforts on cell transplantation in animal models and PD patients, highlighting the immune response and the immunomodulatory treatment strategies performed. The analysis of the available data lead us to conclude that the modulation of the innate immune response after transplantation can increase the success of future clinical trials in PD by enhancing cell differentiation and survival. This article is part of a Special Issue entitled SI: PSC and the brain.

Stem cell research in Latin America: update, challenges and opportunities in a priority research area.

Stem cell research is attracting wide attention as a promising and fast-growing field in Latin America, as it is worldwide. Many countries in the region have defined Regenerative Medicine as a research priority and a focus of investment. This field generates not only opportunities but also regulatory, technical and operative challenges. In this review, scientists from Uruguay, Mexico, Chile, Brazil and Argentina provide their view on stem cell research in each of their countries. Despite country-specific characteristics, all countries share several issues such as regulatory challenges. Key initiatives of each country to promote stem cell research are also discussed. As a conclusion, it is clear that regional integration should be more emphasized and international collaboration, promoted.

Cell reprogramming and neuronal differentiation applied to neurodegenerative diseases: Focus on Parkinson's disease.

Adult cells from patients can be reprogrammed to induced pluripotent stem cells (iPSCs) which successively can be used to obtain specific cells such as neurons. This remarkable breakthrough represents a new way of studying diseases and brought new therapeutic perspectives in the field of regenerative medicine. This is particular true in the neurology field, where few techniques are amenable to study the affected tissue of the patient during illness progression, in addition to the lack of neuroprotective therapies for many diseases. In this review we discuss the advantages and unresolved issues of cell reprogramming and neuronal differentiation. We reviewed evidence using iPSCs-derived neurons from neurological patients. Focusing on data obtained from Parkinson's disease (PD) patients, we show that iPSC-derived neurons possess morphological and functional characteristics of this disease and build a case for the use of this technology to study PD and other neuropathologies while disease is in progress. These data show the enormous impact that this new technology starts to have on different purposes such as the study and design of future therapies of neurological disease, especially PD.

Current status of stem cells and regenerative medicine research in Argentina.

Since Takahashi and Yamanaka demonstrated for the first time that fully differentiated somatic cells can be reprogrammed to a pluripotent state with a small group of transcription factors a revolution erupted in the regenerative medicine field. New advances showing direct differentiation of mature cells increased the excitement of the field. This work describes the present situation of the field in Argentina and the efforts implemented by science authorities to strengthen and push the field forward.

Differential vulnerability of adult neurogenesis by adult and prenatal inflammation: role of TGF-ß1.

Peripheral inflammation, both during the prenatal period and in adulthood, impairs adult neurogenesis. We hypothesized that, similar to other programming effects of prenatal treatments, only prenatal inflammation causes long-term consequences in adult neurogenesis and its neurogenic niche. To test this, pregnant Wistar rats were subcutaneously injected with lipopolysaccharide (LPS; 0.5 mg/kg) or saline solution every other day from gestational/embryonic day (GD) 14-20. In addition adult animals were injected with a single intraperitoneal saline or LPS injection (1 mg/kg) and the effects on neurogenesis were assessed 7 days later. Alternatively, to evaluate long-term consequences of adult LPS injections, LPS (1 mg/kg) was administered peripherally to adult rats four times every other day, and the effects on neurogenesis were assessed 60 days later. Prenatal and adult LPS treatments reduced adult neurogenesis and provoked specific microglial (but not astroglial) activation in the dentate gyrus (DG). However, only prenatal inflammation-mediated effects were long-lasting (at least 60 days). Moreover, these effects were specific to the DG since the Subventricular Zone (SVZ) and the Rostral Migratory Stream (RMS) were not affected. In addition, these stimuli caused differential effects on the molecular components of the neurogenic niche; only prenatal LPS treatment reduced the local levels of TGF-ß1 mRNA in the DG. Finally, TGF-ß1 exerted its pro-neurogenic effects via the Smad 2/3 pathway in a neural stem cell culture. Taken together, these data add evidence to the duration, regional specificity and dramatic consequences of prenatal immune programming on CNS physiology, compared with the limited response observed in the adult brain.

Interleukin-1ß and tumor necrosis factor-α: reliable targets for protective therapies in Parkinson's Disease?

Neuroinflammation has received increased attention as a target for putative neuroprotective therapies in Parkinson's Disease (PD). Two prototypic pro-inflammatory cytokines interleukin-1ß (IL-1) and tumor necrosis factor-α (TNF) have been implicated as main effectors of the functional consequences of neuroinflammation on neurodegeneration in PD models. In this review, we describe that the functional interaction between these cytokines in the brain differs from the periphery (e.g., their expression is not induced by each other) and present data showing predominantly a toxic effect of these cytokines when expressed at high doses and for a sustained period of time in the substantia nigra pars compacta (SN). In addition, we highlight opposite evidence showing protective effects of these two main cytokines when conditions of duration, amount of expression or state of activation of the target or neighboring cells are changed. Furthermore, we discuss these results in the frame of previous disappointing results from anti-TNF-α clinical trials against Multiple Sclerosis, another neurodegenerative disease with a clear neuroinflammatory component. In conclusion, we hypothesize that the available evidence suggests that the duration and dose of IL-1ß or TNF-α expression is crucial to predict their functional effect on the SN. Since these parameters are not amenable for measurement in the SN of PD patients, we call for an in-depth analysis to identify downstream mediators that could be common to the toxic (and not the protective) effects of these cytokines in the SN. This strategy could spare the possible neuroprotective effect of these cytokines operative in the patient at the time of treatment, increasing the probability of efficacy in a clinical setting. Alternatively, receptor-specific agonists or antagonists could also provide a way to circumvent undesired effects of general anti-inflammatory or specific anti-IL-1ß or TNF-α therapies against PD.

Special issue commentary: the changing face of inflammation in the brain.

The study of inflammation in the brain has been extended to include a wide range of conditions, but there remains plenty of argument over semantics and the precise definition of what constitutes inflammation in these pathologies. In this special issue, we sought to highlight the diversity of what is considered to be inflammation in the brain, and we have accepted that the presence of microglia cells with altered morphology remains a useful starting point. However, it is clear that whatever is the molecular expression profile that accompanies an activated microglial cell, it is not static and it is influenced by factors both intrinsic and extrinsic to the brain. This article is part of a Special Issue entitled 'Neuroinflammation in neurodegeneration and neurodysfunction'.

CNS response to a second pro-inflammatory event depends on whether the primary demyelinating lesion is active or resolved.

Interleukin-1ß (IL-1ß) is considered to be one of the most important mediators in the pathogenesis of inflammatory diseases, particularly in neurodegenerative diseases such as multiple sclerosis (MS). MS is a chronic inflammatory disease characterized by demyelination and remyelination events, with unpredictable relapsing and remitting episodes that seldom worsen MS lesions. We proposed to study the effect of a unique component of the inflammatory process, IL-1ß, and evaluate its effect in repeated episodes, similar to the relapsing-remitting MS pathology. Using adenoviral vectors, we developed a model of focal demyelination/remyelination triggered by the chronic expression of IL-1ß. The long-term expression of IL-1ß in the striatum produced blood-brain barrier (BBB) breakdown, demyelination, microglial/macrophage activation, and neutrophil infiltration but no overt neuronal degeneration. This demyelinating process was followed by complete remyelination of the area. This simple model allows us to study demyelination and remyelination independently of the autoimmune and adaptive immune components. Re-exposure to this cytokine when the first inflammatory response was still unresolved generated a lesion with decreased neuroinflammation, demyelination, axonal injury and glial response. However, a second long-term expression of IL-1ß when the first lesion was resolved could not be differentiated from the first event. In this study, we demonstrated that the response to a second inflammatory stimulus varies depending on whether the initial lesion is still active or has been resolved. Considering that anti-inflammatory treatments have shown little improvement in MS patients, studies about the behavior of specific components of the inflammatory process should be taken into account to develop new therapeutic tools.

Pleiotrophin over-expression provides trophic support to dopaminergic neurons in parkinsonian rats.

BACKGROUND: Pleiotrophin is known to promote the survival and differentiation of dopaminergic neurons in vitro and is up-regulated in the substantia nigra of Parkinson's disease patients. To establish whether pleiotrophin has a trophic effect on nigrostriatal dopaminergic neurons in vivo, we injected a recombinant adenovirus expressing pleiotrophin in the substantia nigra of 6-hydroxydopamine lesioned rats. RESULTS: The viral vector induced pleiotrophin over-expression by astrocytes in the substantia nigra pars compacta, without modifying endogenous neuronal expression. The percentage of tyrosine hydroxylase-immunoreactive cells as well as the area of their projections in the lesioned striatum was higher in pleiotrophin-treated animals than in controls. CONCLUSIONS: These results indicate that pleiotrophin over-expression partially rescues tyrosine hydroxylase-immunoreactive cell bodies and terminals of dopaminergic neurons undergoing 6-hydroxydopamine-induced degeneration.

Prenatal inflammation impairs adult neurogenesis and memory related behavior through persistent hippocampal TGFß1 downregulation.

Prenatal exposure to inflammatory stimuli is known to influence adult brain function. In addition, adult hippocampal neurogenesis is impaired by a local pro-inflammatory microenvironment. On this basis, we hypothesized that a pro-inflammatory insult during gestation would have negative effects on adult neurogenesis in the offspring. Pregnant Wistar rats received subcutaneous injections of lipopolysaccharide (LPS; 0.5mg/kg) or saline every other day from gestational day 14 to 20. The adult offspring prenatally treated with LPS showed a decrease in the proliferating cells and the newborn neurons of the dentate gyrus. Furthermore, prenatal LPS treatment impaired performance in the neurogenesis-dependent novel object recognition test. Maternal care was impaired by prenatal LPS administration but did not contribute to the effects of prenatal LPS on adult neurogenesis. Persistent microglial activation and downregulated expression of transforming growth factor beta-1 (TGFß(1)) occurred specifically in the adult hippocampus of animals treated prenatally with LPS. Importantly, chronic hippocampal TGFß(1) overexpression restored neurogenesis as well as recognition memory performance to control levels. These findings demonstrate that prenatal inflammation triggered by LPS impairs adult neurogenesis and recognition memory. Furthermore, we provide a model of reduced adult neurogenesis with long-lasting defined alterations in the neurogenic niche. Finally, we show that the expression of a single cytokine (TGFß(1)) in the hippocampus can restore adult neurogenesis and its related behavior, highlighting the role of TGFß(1) in these processes.

Patients beware: commercialized stem cell treatments on the web.

A report by the International Society for Stem Cell Research (ISSCR)'s Task Force on Unproven Stem Cell Treatments outlines development of resources for patients, their families, and physicians seeking information on stem cell treatments.

Overexpression of IL-1beta by adenoviral-mediated gene transfer in the rat brain causes a prolonged hepatic chemokine response, axonal injury and the suppression of spontaneous behaviour.

Acute brain injury induces early and transient hepatic expression of chemokines, which amplify the injury response and give rise to movement of leukocytes into the blood and subsequently the brain and liver. Here, we sought to determine whether an ongoing injury stimulus within the brain would continue to drive the hepatic chemokine response and how it impacts on behaviour and CNS integrity. We generated chronic IL-1beta expression in rat brain by adenoviral-mediated gene transfer, which resulted in chronic leukocyte recruitment, axonal injury and prolonged depression of spontaneous behaviour. IL-1beta could not be detected in circulating blood, but a chronic systemic response was established, including extended production of hepatic and circulating chemokines, leukocytosis, liver damage, weight loss, decreased serum albumin and marked liver leukocyte recruitment. Thus, hepatic chemokine synthesis is a feature of active chronic CNS disease and provides an accessible target for the suppression of CNS inflammation.

Model based analysis of real-time PCR data from DNA binding dye protocols.

BACKGROUND: Reverse transcription followed by real-time PCR is widely used for quantification of specific mRNA, and with the use of double-stranded DNA binding dyes it is becoming a standard for microarray data validation. Despite the kinetic information generated by real-time PCR, most popular analysis methods assume constant amplification efficiency among samples, introducing strong biases when amplification efficiencies are not the same. RESULTS: We present here a new mathematical model based on the classic exponential description of the PCR, but modeling amplification efficiency as a sigmoidal function of the product yield. The model was validated with experimental results and used for the development of a new method for real-time PCR data analysis. This model based method for real-time PCR data analysis showed the best accuracy and precision compared with previous methods when used for quantification of in-silico generated and experimental real-time PCR results. Moreover, the method is suitable for the analyses of samples with similar or dissimilar amplification efficiency. CONCLUSION: The presented method showed the best accuracy and precision. Moreover, it does not depend on calibration curves, making it ideal for fully automated high-throughput applications.

Neurogenic niche modulation by activated microglia: transforming growth factor beta increases neurogenesis in the adult dentate gyrus.

Adult neural stem cells (NSC) proliferate and differentiate depending on the composition of the cellular and molecular niche in which they are immersed. Until recently, microglial cells have been ignored as part of the neurogenic niche. We studied the dynamics of NSC proliferation and differentiation in the dentate gyrus of the hippocampus (DG) and characterized the changes of the neurogenic niche in adrenalectomized animals (ADX). At the cellular level, we found increased NSC proliferation and neurogenesis in the ADX animals. In addition, a morphologically distinct subpopulation of NSC (Nestin+/GFAP-) with increased proliferating profile was detected. Interestingly, the number of microglial cells at stages 2 and 3 of activation correlated with increased neurogenesis (r2 = 0.999) and the number of Nestin-positive cells (r2 = 0.96). At the molecular level, transforming growth factor beta (TGF-beta) mRNA levels were increased 10-fold in ADX animals. Interestingly, TGF-beta levels correlated with the amount of neurogenesis detected (r2 = 0.99) and the number of stage 2 and 3 microglial cells (r2 = 0.94). Furthermore, blockade of TGF-beta biological activity by administration of an anti-TGF-beta type II receptor antibody diminished the percentage of 5-bromo-2'-deoxyuridine (BrdU)/PSA-NCAM-positive cells in vivo. Moreover, TGF-beta was able to promote neurogenesis in NSC primary cultures. This work supports the idea that activated microglial cells are not pro- or anti-neurogenic per se, but the balance between pro- and anti-inflammatory secreted molecules influences the final effect of this activation. Importantly, we identified an anti-inflammatory cytokine, TGF-beta, with neurogenic potential in the adult brain.

Neuronal differentiation in the adult hippocampus recapitulates embryonic development.

In the adult hippocampus and olfactory bulb, neural progenitor cells generate neurons that functionally integrate into the existing circuits. To understand how neuronal differentiation occurs in the adult hippocampus, we labeled dividing progenitor cells with a retrovirus expressing green fluorescent protein and studied the morphological and functional properties of their neuronal progeny over the following weeks. During the first week neurons had an irregular shape and immature spikes and were synaptically silent. Slow GABAergic synaptic inputs first appeared during the second week, when neurons exhibited spineless dendrites and migrated into the granule cell layer. In contrast, glutamatergic afferents were detected by the fourth week in neurons displaying mature excitability and morphology. Interestingly, fast GABAergic responses were the latest to appear. It is striking that neuronal maturation in the adult hippocampus follows a precise sequence of connectivity (silent --> slow GABA --> glutamate --> fast GABA) that resembles hippocampal development. We conclude that, unlike what is observed in the olfactory bulb, the hippocampus maintains the same developmental rules for neuronal integration through adulthood.

Differential effects of interleukin-1beta on neurotoxicity, cytokine induction and glial reaction in specific brain regions.

An appropriate inflammatory response is crucial for the maintenance of tissue homeostasis. The inflammatory responses seen in the brain parenchyma differ from those elicited in the periphery, ventricles and meninges. However, although an inflammatory component has been associated with many CNS diseases, the differences among parenchymal inflammatory responses in different brain regions have not yet been fully elucidated. Here, we performed a systematic comparison of the effects of a common pro-inflammatory stimulus, IL-1beta, on the hippocampus, substantia nigra, striatum and cortex. We determined various responses, including cytokine mRNA induction, glial activation, immune cell infiltration and changes in neuronal integrity, in both injected and adjacent regions 1 and 6 days after the injection of IL-1beta. We found that the mRNA for TGF-beta was up-regulated in a region-specific manner after IL-1beta administration. Contrary to its response in the periphery, IL-1alpha showed no detectable induction in the tested parenchymal regions. In addition, cytokine induction was also sometimes observed in regions distant from the site of injection. Interestingly, IL-1beta-mediated neurodegeneration was observed in the dentate gyrus of the hippocampus, but not in the other tested regions. The cellular recruitment mediated by IL-1 beta injection consisted mainly of polymorphonuclear cells (PMN), which correlated with IL-1betamRNA induction even in regions far from the injection site. These results indicate that various parenchymal regions show different inflammatory responses and neurodegeneration in response to IL-1beta. Taken together, our results provide a more precise picture of regional inflammation in the brain and should provide a basis for differential interpretation of results based on regional inflammatory differences.