Generation of reactive oxygen species in 1-methyl-4-phenylpyridinium (MPP+) treated dopaminergic neurons occurs as an NADPH oxidase-dependent two-wave cascade

<p>Abstract</p> <p>Background</p> <p>Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H₂O₂), are necessary for appropriate responses to immune challenges. In the brain, excess superoxide production predicts neuronal cell loss, suggesting that Parkinson's disease (PD) with its wholesale death of dopaminergic neurons in substantia nigra pars compacta (nigra) may be a case in point. Although microglial NADPH oxidase-produced superoxide contributes to dopaminergic neuron death in an MPTP mouse model of PD, this is secondary to an initial die off of such neurons, suggesting that the initial MPTP-induced death of neurons may be via activation of NADPH oxidase in neurons themselves, thus providing an early therapeutic target.</p> <p>Methods</p> <p>NADPH oxidase subunits were visualized in adult mouse nigra neurons and in N27 rat dopaminergic cells by immunofluorescence. NADPH oxidase subunits in N27 cell cultures were detected by immunoblots and RT-PCR. Superoxide was measured by flow cytometric detection of H₂O₂-induced carboxy-H₂-DCFDA fluorescence. Cells were treated with MPP+ (MPTP metabolite) following siRNA silencing of the Nox2-stabilizing subunit p22^phox, or simultaneously with NADPH oxidase pharmacological inhibitors or with losartan to antagonize angiotensin II type 1 receptor-induced NADPH oxidase activation.</p> <p>Results</p> <p>Nigral dopaminergic neurons <it>in situ</it> expressed three subunits necessary for NADPH oxidase activation, and these as well as several other NADPH oxidase subunits and their encoding mRNAs were detected in unstimulated N27 cells. Overnight MPP+ treatment of N27 cells induced Nox2 protein and superoxide generation, which was counteracted by NADPH oxidase inhibitors, by siRNA silencing of p22^phox, or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H₂O₂production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H₂O₂levels were further increased by 24 hours. This second wave was eliminated by pharmacological inhibitors and a blocker of protein synthesis.</p> <p>Conclusions</p> <p>A two-wave cascade of ROS production is active in nigral dopaminergic neurons in response to neurotoxicity-induced superoxide. Our findings allow us to conclude that superoxide generated by NADPH oxidase present in nigral neurons contributes to the loss of such neurons in PD. Losartan suppression of nigral-cell superoxide production suggests that angiotensin receptor blockers have potential as PD preventatives.</p

Trisomy 21 dysregulates T cell lineages toward an autoimmunity-prone state associated with interferon hyperactivity

Trisomy 21 (T21) causes Down syndrome (DS), a condition characterized by high prevalence of autoimmune disorders. However, the molecular and cellular mechanisms driving this phenotype remain unclear. Building upon our previous finding that T cells from people with DS show increased expression of interferon (IFN)stimulated genes, we have completed a comprehensive characterization of the peripheral T cell compartment in adults with DS with and without autoimmune conditions. CD8+ T cells from adults with DS are depleted of naïve subsets and enriched for differentiated subsets, express higher levels of markers of activation and senescence (e.g., IFN-γ, Granzyme B, PD-1, KLRG1), and overproduce cytokines tied to autoimmunity (e.g., TNF-α). Conventional CD4+ T cells display increased differentiation, polarization toward the Th1 and Th1/17 states, and overproduction of the autoimmunity-related cytokines IL-17A and IL-22. Plasma cytokine analysis confirms elevation of multiple autoimmunity-related cytokines (e.g., TNF-α, IL17A–D, IL-22) in people with DS, independent of diagnosis of autoimmunity. Although Tregs are more abundant in DS, functional assays show that CD8+ and CD4+ effector T cells with T21 are resistant to Treg-mediated suppression, regardless of Treg karyotype. Transcriptome analysis of white blood cells and T cells reveals strong signatures of T cell differentiation and activation that correlate positively with IFN hyperactivity. Finally, mass cytometry analysis of 8 IFN-inducible phosphoepitopes demonstrates that T cell subsets with T21 show elevated levels of basal IFN signaling and hypersensitivity to IFN-α stimulation. Therefore, these results point to T cell dysregulation associated with IFN hyperactivity as a contributor to autoimmunity in DS.Fil: Araya, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Waugh, Katherine A.. University Of Colorado. School Of Medicine.; Estados UnidosFil: Sullivan, Kelly D.. University Of Colorado. School Of Medicine.; Estados UnidosFil: Núñez, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Roselli, Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Smith, Keith P.. University Of Colorado. School Of Medicine.; Estados UnidosFil: Granrath, Ross E.. University Of Colorado. School Of Medicine.; Estados UnidosFil: Rachubinski, Angela L.. University of Colorado; Estados UnidosFil: Enriquez Estrada, Belinda. University Of Colorado. School Of Medicine.; Estados UnidosFil: Butcher, Eric T.. University of Colorado; Estados UnidosFil: Minter, Ross. University of Colorado; Estados UnidosFil: Tuttle, Kathryn D.. University of Colorado; Estados UnidosFil: Bruno, Tullia C.. University Of Colorado. School Of Medicine.; Estados UnidosFil: Maccioni, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Espinosa, Joaquín M.. University Of Colorado. School Of Medicine.; Estados Unido

Effects of Neonatal Neural Progenitor Cell Implantation on Adult Neuroanatomy and Cognition in the Ts65Dn Model of Down Syndrome

As much of the aberrant neural development in Down syndrome (DS) occurs postnatally, an early opportunity exists to intervene and influence life-long cognitive development. Recent success using neural progenitor cells (NPC) in models of adult neurodegeneration indicate such therapy may be a viable option in diseases such as DS. Murine NPC (mNPC, C17.2 cell line) or saline were implanted bilaterally into the dorsal hippocampus of postnatal day 2 (PND 2) Ts65Dn pups to explore the feasibility of early postnatal treatment in this mouse model of DS. Disomic littermates provided karyotype controls for trisomic pups. Pups were monitored for developmental milestone achievement, and then underwent adult behavior testing at 14 weeks of age. We found that implanted mNPC survived into adulthood and migrated beyond the implant site in both karyotypes. The implantation of mNPC resulted in a significant increase in the density of dentate granule cells. However, mNPC implantation did not elicit cognitive changes in trisomic mice either neonatally or in adulthood. To the best of our knowledge, these results constitute the first assessment of mNPC as an early intervention on cognitive ability in a DS model

GFP+ cells found in areas outside of the hippocampus.

<p>A. Half of trisomic mice had GFP+ cells in the entorhinal cortex (arrows). B. GFP+ cells were found near the lateral ventricles in the septum (arrows). C. GFP+ mNPC also were found in the CC and again in the underlying septal areas (arrows). rf, rhinal fissure; lv, lateral ventricles; CC, corpus callosum. Scale Bars in A = 100 µm and in B–C = 200 µm.</p

Organizational differences in the granule cells of the DG.

<p>Untreated disomic brains (A) had a lower density of granule cells, with a correspondingly larger cell diameter. Similar results were reported for untreated trisomic mice (C). In both groups, the granule cell layer appears less organized in that there was more space between the cells (arrows in A) and randomness to their location within the layer. In contrast, in the Disomic/mNPC mice (B) and Trisomic/mNPC mice (D), both of which had smaller cell diameters and a tendency towards a higher cell density, have a more organized appearance. The granule layers of these groups have tightly aligned cells, which sometimes appear to be in rows and columns (arrow heads in B). Scale bars in A–D are equal to each other and represent 100 µm.</p

Novel alternations did not differ but mNPC treatment increased Trisomic performance above chance performance.

<p>Overall, trisomic mice showed significantly fewer novel alternations compared to disomic mice (* p<0.05). Treatment with either saline or mNPC did not significantly change the number of novel alternations in either karyotype compared to their respective controls. However, trisomic mice treated with mNPC alternated significantly above chance alone (solid line), while untreated and saline treated trisomic mice did not. Mean ± SEM shown. * Significant main effect of karyotype. ∧ Novel alternations were significantly above chance.</p

Trisomic mice continued to drink significantly more CS on second exposure than disomic mice.

<p>There was no significant difference in degree of preference during first exposure between disomic and trisomic animals. During the second exposure disomic mice had a greater avoidance of the CS than the trisomic animals (* p<0.05). Treatment did not preferentially affect CTA learning and affected both karyotypes to the same degree. Mean ± SEM shown.</p

All groups recognized the novel object.

<p>All groups exhibited a preference for the novel object, as indicated by a DI above zero. While there is apparent variability, the differences in the DI between groups did not reach significance (p>0.05). Mean ± SEM shown.</p

Treatment with mNPC significantly increased the density of the granule cells and decreased their diameter.

<p>A. There was no difference in the density of granule cells between disomic and trisomic mice overall. However, treatment with mNPC significantly increased the density of the granule cells (p<0.05). While this effect is more clearly observable in the disomic/NPC group (#), the interaction between treatment and karyotype suggests that mNPC effect was present in both karyotypes. B. Animals implanted with mNPC had granule cells with significantly smaller cell soma than untreated or saline treated animals (p<0.05). Again, this is best observed in the disomic group (#) although statistically both mNPC groups were affected. Mean ± SEM shown.</p

Interferon hyperactivity impairs cardiogenesis in Down syndrome via downregulation of canonical Wnt signaling

Summary: Congenital heart defects (CHDs) are frequent in children with Down syndrome (DS), caused by trisomy of chromosome 21. However, the underlying mechanisms are poorly understood. Here, using a human-induced pluripotent stem cell (iPSC)-based model and the Dp(16)1Yey/+ (Dp16) mouse model of DS, we identified downregulation of canonical Wnt signaling downstream of increased dosage of interferon (IFN) receptors (IFNRs) genes on chromosome 21 as a causative factor of cardiogenic dysregulation in DS. We differentiated human iPSCs derived from individuals with DS and CHDs, and healthy euploid controls into cardiac cells. We observed that T21 upregulates IFN signaling, downregulates the canonical WNT pathway, and impairs cardiac differentiation. Furthermore, genetic and pharmacological normalization of IFN signaling restored canonical WNT signaling and rescued defects in cardiogenesis in DS in vitro and in vivo. Our findings provide insights into mechanisms underlying abnormal cardiogenesis in DS, ultimately aiding the development of therapeutic strategies