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How neurons in the CNS can maintain cellular identity over an entire lifespan remains largely unknown. Here we show that long-term maintenance of identity in differentiated dopaminergic and serotonergic neurons is critically reliant on the Polycomb repressive complex 2 (PRC2). Deletion of the obligate PRC2-component, Eed, in these neurons, resulted in global loss of H3K27me3, followed by a gradual activation of genes harbouring both H3K27me3 and H3K9me3 modifications. Notably, H3K9me3 was also lost at these PRC2-targets prior to gene activation. Neuronal survival was not compromised, instead there was a reduction in subtype specific gene expression as well as a progressive impairment of dopaminergic or serotonergic neuronal function leading to behavioural deficits characteristic of Parkinson’s disease (PD) or mood disorders, respectively. Single cell analysis revealed an unexpected subtype specific vulnerability to loss of PRC2-repression in dopamine neurons of the substantia nigra, the neurons primarily affected in PD. Taken together, our study reveals that a PRC2-dependent non-permissive chromatin state is essential to maintain subtype identity and function of dopaminergic and serotonergic neurons.

Cell replacement is a long-standing and realistic goal for the treatment of Parkinsonʼs disease (PD). Cells for transplantation can be obtained from fetal brain tissue or from stem cells. However, after transplantation, dopamine (DA) neurons are seen to be a minor component of grafts, and it has remained difficult to determine the identity of other cell types. Here, we report analysis by single-cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts from human embryonic stem cells (hESCs) and fetal tissue after functional maturation in a pre-clinical rat PD model. We show that neurons and astrocytes are major components in both fetal and stem cell-derived grafts. Additionally, we identify a cell type closely resembling a class of recently identified perivascular-like cells in stem cell-derived grafts. Thus, this study uncovers previously unknown cellular diversity in a clinically relevant cell replacement PD model. What happens to cells on engrafting into the brain in animal models to treat Parkinson’s disease is unclear. Here, the authors use scRNA-seq to examine ventral midbrain (VM)-patterned human embryonic stem cells after functional maturation in a pre-clinical rat model for Parkinson’s disease and identify perivascular-like cells.

Dopamine (DA) neurons of the midbrain are at risk to become affected by mitochondrial damage over time and mitochondrial defects have been frequently reported in Parkinson’s disease (PD) patients. However, the causal contribution of adult-onset mitochondrial dysfunction to PD remains uncertain. Here, we developed a mouse model lacking Mitofusin 2 (MFN2), a key regulator of mitochondrial network homeostasis, in adult midbrain DA neurons. The knockout mice develop severe and progressive DA neuron-specific mitochondrial dysfunction resulting in neurodegeneration and parkinsonism. To gain further insights into pathophysiological events, we performed transcriptomic analyses of isolated DA neurons and found that mitochondrial dysfunction triggers an early onset immune response, which precedes mitochondrial swelling, mtDNA depletion, respiratory chain deficiency and cell death. Our experiments show that the immune response is an early pathological event when mitochondrial dysfunction is induced in adult midbrain DA neurons and that neuronal death may be promoted non-cell autonomously by the cross-talk and activation of surrounding glial cells., Author summary Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by progressive loss of dopamine (DA)-producing neurons and strongly compromised motor performance. Multiple observations suggest that DA neurons are particularly prone to acquire mitochondrial damage in adult life. This acquired mitochondrial dysfunction likely impairs DA neuron function and contributes to cell death. To study the consequences of adult-onset mitochondrial dysfunction in DA neurons, we generated a conditional activatable knockout mouse model lacking Mitofusin 2, a key regulator of mitochondrial homeostasis. This animal model allows the induction of mitochondrial dysfunction selectively in adult DA neurons and leads to motor defects and the typical pattern of neurodegeneration seen in PD. By studying gene expression in isolated DA neurons at early disease stages and by using in situ approaches on brain sections, we report an early onset of an inflammatory response. Inflammation is present already when the mutant DA neurons display the first signs of mitochondrial fragmentation and precedes the onset of respiratory chain dysfunction and neurodegeneration. The inflammatory response in DA neurons and activation of surrounding glia thus likely exacerbates or drives the neurodegenerative process in this animal model of adult-onset PD.

Midbrain dopamine (mDA) neurons constitute a heterogenous group of cells that have been intensely studied, not least because their degeneration causes major symptoms in Parkinson’s disease. Understanding the diversity of mDA neurons – previously well characterized anatomically – requires a systematic molecular classification at the genome-wide gene expression level. Here, we use single cell RNA sequencing of isolated mouse neurons expressing the transcription factor Pitx3, a marker for mDA neurons. Analyses include cells isolated during development up until adulthood and the results are validated by histological characterization of newly identified markers. This identifies seven neuron subgroups divided in two major branches of developing Pitx3-expressing neurons. Five of them express dopaminergic markers, while two express glutamatergic and GABAergic markers, respectively. Analysis also indicate evolutionary conservation of diversity in humans. This comprehensive molecular characterization will provide a valuable resource for elucidating mDA neuron subgroup development and function in the mammalian brain., Midbrain dopamine (mDA) neurons are significantly associated with Parkinson’s disease and yet there is no systematic molecular classification of these heterogenous group of cells. Here authors use single cell RNA sequencing of isolated mouse neurons expressing the transcription factor Pitx3 (broad mDA neuronal marker) to identify and characterize seven neuron subgroups divided in two major branches of developing Pitx3-expressing neurons.

Stem cell engineering and grafting of mesencephalic dopamine (mesDA) neurons is a promising strategy for brain repair in Parkinson's disease (PD). Refinement of differentiation protocols to optimize this approach will require deeper understanding of mesDA neuron development. Here, we studied this process using transcriptome-wide single-cell RNA sequencing of mouse neural progenitors expressing the mesDA neuron determinant Lmx1a. This approach resolved the differentiation of mesDA and neighboring neuronal lineages and revealed a remarkably close relationship between developing mesDA and subthalamic nucleus (STN) neurons, while also highlighting a distinct transcription factor set that can distinguish between them. While previous hESC mesDA differentiation protocols have relied on markers that are shared between the two lineages, we found that application of these highlighted markers can help to refine current stem cell engineering protocols, increasing the proportion of appropriately patterned mesDA progenitors. Our results, therefore, have important implications for cell replacement therapy in PD.

Since the pioneering studies using fetal cell transplants in Parkinson’s disease (PD), brain repair by cell replacement has remained a long-standing and realistic goal for the treatment of neurodegenerative disorders including PD. Authentic and functional midbrain dopamine (DA) neurons can now be generated from human pluripotent stem cells (hPSCs) via a floor plate intermediate1,2, and these cell preparations are both safe and functional when transplanted to animal models of PD3. However, although resulting grafts from fetal brain tissue and hPSCs contain large numbers of desired DA neurons, these therapeutic cells are a minor component of the grafts. Moreover, the cellular composition of the graft has remained difficult to assess due to limitations in histological methods that rely on pre-conceived notions concerning cell types. Here, we used single cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts from ventral midbrain (VM)-patterned human embryonic stem cells (hESCs) and VM fetal tissue after long-term survival and functional maturation in a pre-clinical rat model of PD. The analyses revealed that while both cell preparations gave rise to neurons and astrocytes, oligodendrocytes were only detected in grafts of fetal tissue. On the other hand, a cell type closely resembling a class of newly identified perivascular-like cells was identified as a unique component of hESC-derived grafts. The presence of these cells was confirmed in transplants from three different hESC lines, as well as from iPSCs. Thus, these experiments have addressed one of the major outstanding questions in the field of cell replacement in neurological disease by revealing graft composition and differences between hESC- and fetal cell-derived grafts, which can have important implications for clinical trials.

Cell replacement is a long-standing and realistic goal for the treatment of Parkinsonʼs disease (PD). Cells for transplantation can be obtained from fetal brain tissue or from stem cells. However, after transplantation, dopamine (DA) neurons are seen to be a minor component of grafts, and it has remained difficult to determine the identity of other cell types. Here, we report analysis by single-cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts from human embryonic stem cells (hESCs) and fetal tissue after functional maturation in a pre-clinical rat PD model. We show that neurons and astrocytes are major components in both fetal and stem cell-derived grafts. Additionally, we identify a cell type closely resembling a class of recently identified perivascular-like cells in stem cell-derived grafts. Thus, this study uncovers previously unknown cellular diversity in a clinically relevant cell replacement PD model., What happens to cells on engrafting into the brain in animal models to treat Parkinson’s disease is unclear. Here, the authors use scRNA-seq to examine ventral midbrain (VM)-patterned human embryonic stem cells after functional maturation in a pre-clinical rat model for Parkinson’s disease and identify perivascular-like cells.

α-synuclein, a protein enriched in Lewy bodies and highly implicated in neurotoxicity in Parkinson's disease, is distributed both at nerve terminals and in the cell nucleus. Here we show that a nuclear derivative of α-synuclein induces more pronounced changes at the gene expression level in mouse primary dopamine (DA) neurons compared to a derivative that is excluded from the nucleus. Moreover, by RNA sequencing we analyzed the extent of genome-wide effects on gene expression resulting from expression of human α-synuclein in primary mouse DA neurons. The results implicated the transcription factor Nurr1 as a key dysregulated target of α-synuclein toxicity. Forced Nurr1 expression restored the expression of hundreds of dysregulated genes in primary DA neurons expressing α-synuclein, and therefore prompted us to test the possibility that Nurr1 can be pharmacologically targeted by bexarotene, a ligand for the retinoid X receptor that forms heterodimers with Nurr1. Although our data demonstrated that bexarotene was ineffective in neuroprotection in ratsin vivo, the results revealed that bexarotene has the capacity to coregulate subsets of Nurr1 target genes including the receptor tyrosine kinase subunit Ret. Moreover, bexarotene was able to restore dysfunctional Ret-dependent neurotrophic signaling in α-synuclein-overexpressing mouse DA neurons. These data highlight the role of the Nurr1–Ret signaling pathway as a target of α-synuclein toxicity and suggest that retinoid X receptor ligands with appropriate pharmacological properties could have therapeutic potential in Parkinson's disease.SIGNIFICANCE STATEMENTHow α-synuclein, a protein enriched in Lewy bodies in Parkinson's disease, is causing neuropathology in dopamine neurons remains unclear. This study elucidated how α-synuclein is influencing gene expression and how Nurr1, a transcription factor known to protect dopamine neurons against α-synuclein toxicity, can counteract these effects. Moreover, given the protective role of Nurr1, this study also investigated how Nurr1 could be pharmacologically targeted via bexarotene, a ligand of Nurr1's heterodimerization partner retinoid X receptor (RXR). The results showed that RXR ligands could increase neurotrophic signaling, but provided a mixed picture of its potential in a Parkinson's disease rat modelin vivo. However, this study clearly emphasized Nurr1's neuroprotective role and indicated that other RXR ligands could have therapeutic potential in Parkinson's disease.

Objective: To reveal specific gene activation in nitric oxide (NO)-related inflammation we studied differential gene expression in inflammatory bowel disease (IBD). Methods. Total RNA was isolated from 20 biopsies of inflamed mucosa from Crohn's disease (CD) and ulcerative colitis (UC) patients each as well as from six controls, labeled with 32P-dCTP and hybridized to a human NO gene array. Significant genes were analyzed for functional gene interactions and heatmaps generated by hierarchical clustering. A selection of differentially expressed genes was further evaluated with immunohistochemical staining. Results. Significant gene expression differences were found for 19 genes in CD and 23 genes in UC compared to controls, both diseases with high expression of ICAM1 and IL-8. Correlation between microarray expression and corresponding protein expression was significant (r 0.47, p 0.002). Clustering analysis together with functional gene interaction analysis revealed clusters of coregulation and coexpression in CD and UC: transcripts involved in angiogenesis, inflammatory response mediated by the transcription factor hypoxia-inducible factor 1, and tissue fibrosis. Also, a fourth cluster with transcripts regulated by the transcription factor Sp1 was found in UC. Conclusions. Expression analysis in CD and UC revealed disease-specific regulation of NO-related genes, which might be involved in perpetuating inflammatory disease activity in IBD.

Aim To compare the therapeutic effect of α2 and α4 integrin-blocking antibodies to conventional inflammatory bowel disease drugs methotrexate, 5-aminosalicylic acid and azathioprine in the dextran sulphate sodium mouse colitis model. Methods Colitis was induced in balb/c mice with 2.5–3.0% dextran sulphate sodium. Treatment was given daily for 7 days after the onset of colitis, by rectal installation. Clinical signs of disease were assessed daily using a disease activity index. After 19 days, all animals were killed and colon samples collected for histological grading and mRNA/protein analysis. All treatment groups were compared with an untreated control group and a treatment group receiving dextran sulphate sodium alone to monitor the potential degree of clinical remission. Results Treatment with anti-α2 antibodies and methotrexate reduced the body weight loss. At the end of treatment, anti-α2 antibodies reduced rectal bleeding, while methotrexate reduced the disease activity index score. Histological evaluation showed that anti-α2 antibodies, methotrexate, 5-aminosalicylic acid and azathioprine treatment reduced the acute inflammation; methotrexate was the only treatment with effect on the crypt score. Compared with the dextran sulphate sodium alone group, the methotrexate group showed down-regulation of IL-1β at the mRNA level, while the anti-α2 antibody group displayed decreased protein expression of iNOS and IL-1β. Conclusions Specific blocking of extravascular trafficking of leucocytes with α2-antibodies could be a new beneficial drug target in inflammatory bowel disease.

Neuronal networks originating in the hypothalamic arcuate nucleus play fundamental roles in the control of energy balance. Neuropeptide Y (NPY)-producing neurons in the arcuate nucleus stimulate food intake, whereas arcuate nucleus neurons that release the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) potently reduce food intake. Relatively little attention has been focused on classical neurotransmitters in regulation of food intake. Here, we have investigated the potential presence of acetylcholine (ACh) in NPY- and POMC-containing neuronal populations of the arcuate nucleus. Antisera to proteins required for cholinergic neurotransmission, including choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT), were employed in double-labeling immunohistochemical experiments. In colchicine-treated rats, ChAT- and VAChT-immunopositive cell bodies were located in the ventral aspect of the arcuate nucleus. ChAT and VAChT immunoreactivities were demonstrated in alpha-MSH- and cocaine- and amphetamine-regulated transcript (CART)-containing cell bodies of the arcuate nucleus, whereas cell bodies containing NPY or agouti-related peptide (AGRP) were distinct from VAChT-immunoreactive neuronal perikarya. VAChT immunoreactivity was also present in a large number of alpha-MSH-containing nerve fiber varicosities throughout the central nervous system. In the commissural part of the nucleus tractus solitarius, no alpha-MSH-containing cell bodies were found to have ChAT or VAChT immunoreactivity. The presence of markers for cholinergic neurotransmission in a subpopulation of hypothalamic POMC/CART neurons suggests co-release of ACh with peptides derived from the POMC precursor and CART. The results indicate a role for ACh in control of energy balance, mediating the effects of peripheral hormones such as leptin and insulin.

Background: Neuropeptide S (NPS) is expressed by gastrointestinal (GI) enteroendocrine cells and macrophages in rat and man. Polymorphisms of the NPS receptor are linked to increased risk of inflammatory bowel disease as well as motor and sensory disturbances of the gut, suggesting a role for NPS in GI disorders. Further knowledge of NPS effects on motility and inflammation is needed. Methods: Studies of motility were carried out in rats with electrodes implanted in the small bowel. NPS was infused IV for 60 min and effects on myoelectrical activity were recorded. Motility effects of NPS were further studied as luminal pressure changes in anaesthetized rats where the proximal small intestine with intact blood supply was perfused with saline for motility recordings. Tissue samples were obtained from rats for evaluation of gene expression and protein elaboration of inflammatory biomarkers. Muscle strips of the human stomach, small intestine and colon were used for pharmacological analysis in organ baths of motility responses to NPS. Localization of the NPS receptor was done with fluorescent-protein tagging using Cy3-NPS. Plasma levels of NPS were measured using ELISA in 5 healthy and 14 IBD patients before and after meal. Results: In conscious rats, NPS 1 nmol kg-1min-1 increased irregular spiking, 4 nmol kg-1min-1 reduced spiking and increased the MMC cycle length (P=0.005). In anesthetized rats, NPS 0.01-1 nmol kg-1min-1 dose-dependently reduced small bowel motility (P

Gastrin Receptor Antagonist YF476 : Effects on Gastric Acid Secretion, Regulatory Peptides and Receptor Gene Expression in Rats