Implication de la voie de dégradation ubiquitine-dépendante dans la pathologie des maladies de surchage lysosomale

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to- DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation

Molecular signatures and properties of Pitx3-dependent and Pitx3-independent subsets of midbrain dopaminergic neurons

Midbrain dopaminergic (mDA) neurons have been implicated in neurological disorders and Parkinson’s disease (PD), the latter being characterized by progressive degeneration of particular subsets of mDA neurons. For example, mDA neurons localized in ventral substantia nigra compacta (vSNc) display an increased propensity to degenerate in typical PD subjects and animal models, while other subsets in dorsal SNc (dSNc) and VTA are more resistant. The molecular basis for this selective vulnerability remains largely unknown. Using Pitx3-null mice as a PD model for selective mDA cell loss, we performed a genome-wide transcriptome comparison of FACS-purified mDA subpopulations in order to gain insight into their representative gene expression profiles that may explain their respective biology and propensity to degenerate. This strategy first led us to discover an array of novel markers enriched in mDA neurons of vSNc, dSNc and VTA. Secondly, among these mDA subset-enriched markers, we identified those that are likely to be dependent on Pitx3. Third, we provide evidence for a putative subset-specific role of Pitx3 in regulation of primary cilium signaling in Pitx3-dependent mDA neurons of the VTA. Fourth, we identified conserved genomic regulatory regions where Pitx3 binds to mediate its gene regulatory actions. Fifth, we propose survival factors and pathways that are preferentially enriched in mDA neurons of vSNc, and that could participate in degenerative processes. Lastly, we study one such potential vSNc-enriched survival factor, that is Regulator of G-protein Signaling 6 (Rgs6) and demonstrate its implication in adult maintenance of vSNc mDA neurons. Indeed, inactivation of the Rgs6 gene in mice results in late-onset degeneration of vSNc mDA neurons associated with decreased expression of Pitx3 and its target genes. The ensemble of this work opened novel perspectives on the molecular understanding of mDA neuron biology in normal and disease states.Les neurones dopaminergiques du mésencéphale (DAm) ont été impliqués dans des désordres neurologiques et la maladie de Parkinson (MP), cette dernière étant caractérisée par la dégénérescence progressive de sous-populations particulières de neurones DAm. Par exemple, les neurones DAm de la partie ventrale de la substance noire (vSNc) et une faible proportion de ceux de l’aire tegmentée (VTA) montrent une tendance élevée pour la dégénérescence dans les patients typiques de la MP et certains modèles animaux, tandis que les sous-populations de neurones DAm dans la partie dorsale de la SNc (dSNc) et celles du VTA sont généralement plus résistantes. La base moléculaire pour cette vulnérabilité sélective reste largement inconnue. En ayant recours à des souris nulles pour Pitx3 en tant que modèles de la perte neuronale sélective, nous avons entrepris des comparaisons du transcriptome de diverses sous-populations de neurones DAm triés par FACS. Ceci a été fait afin de révéler leur profil d’expression génique qui, selon nous, expliquerait leurs propriétés biologiques ainsi que leur vulnérabilité. Cette stratégie a premièrement permis la découverte de marqueurs moléculaires propres aux neurones DAm de la vSNc, dSNc et du VTA. Deuxièmement, parmi ces marqueurs spécifiques, nous avons identifié ceux dont l’expression dépendrait de Pitx3. Troisièmement, nous avons montré une expression prédominante de la signalisation reliée au cil primaire uniquement dans la sous-population de neurones DAm Pitx3-dépendants du VTA. Quatrièmement, nous avons identifié les profils de liaison du facteur de transcription Pitx3 sur l’ensemble du génome. Cinquièmement, nous avons identifié des facteurs et voies de survie qui sont préférentiellement actives dans les neurones DAm de la vSNc et qui participeraient aux processus de dégénérescence. Enfin, nous avons étudié un de ces facteurs de survie enrichi dans la vSNc, le Regulator of G-protein Signaling 6 (Rgs6), et nous démontrons son implication dans la survie des neurones DAm de la vSNc chez la souris adulte. En effet, l’inactivation du gène Rgs6 chez les souris produit une dégénérescence tardive de ces derniers, en association avec une baisse d’expression de Pitx3 et de ses gènes cibles. L’ensemble de ce travail ouvre de nouvelles perspectives pour la compréhension moléculaire de la biologie des neurones DAm dans l’état normal et la maladie

<i>Rgs6−/−</i> mice as a model for Parkinsonian neurodegeneration.

<p>(A) Rgs6-dependent signaling pathways in vSNc mDA neurons control Pitx3 expression, which itself controls expression of Aldh1a1, TH, Fgf10, Vmat2 and Bdnf. Together, Rgs6 and Pitx3 define a survival pathway in these neurons. The dopamine receptor Drd2 may be a target of Rgs6 action and consistent with this hypothesis, expression of the dopamine transporter DAT is up-regulated in <i>Rgs6^−/−</i> vSNc. (B) Schematic representation of major subsets of SNc mDA neurons present at different ages in midbrains of <i>Rgs6</i>^−/− mice showing progressive degeneration (small pale green) followed by loss of vSNc mDA neurons.</p

Reduced expression of Pitx3 and its target genes in degenerating neurons.

<p>(A) Double immunofluorescence staining against TH (green) and Pitx3 (red) in SNc of control and 1 y-old <i>Rgs6</i>^−/− mice that display dysmorphic mDA neurons. Arrowheads indicate Pitx3-negative neurons of dSNc. Scale bar 20 µm. (B) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Vmat2 (red) in SNc of <i>WT</i> and <i>Rgs6</i>^−/− mice. Scale bar 20 µm. (C) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Bdnf (red) in SNc and VTA of <i>WT</i> and <i>Rgs6</i>^−/− mice. Scale bar 20 µm. (D) Double immunofluorescence staining against TH (green) and Pitx3, Aldh1a1, Fgf10 (red) in control and 1 y-old <i>Rgs6</i>^−/− mice that display dysmorphic mDA neurons. Arrowheads indicate unaffected dSNc neurons and arrows point to affected vSNc neurons. Scale bar 100 µm.</p

Unilateral loss of Pitx3-positive dopaminergic neurons in ventral SNc of <i>Rgs6</i>^−/− mice.

<p>(A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing less SNc TH+ neurons on one side of <i>Rgs6</i>^−/− mice at 1 year of age compared to sib control. Sections are identified with Bregma position. Scale bar 400 µm. (B) Number of TH+ cells in SNc and VTA of TH-stained coronal sections across midbrain (every 30 µm). Cell counts are represented as means +/− S.D. (***p<0.005). (C) Nissl staining of vSNc sections contiguous to A shows fewer cell bodies and abnormal elongated neurons in <i>Rgs6</i>^−/− mice compared to control. Scale bar 100 µm. (D) Double immunofluorescence staining for TH (green) and Pitx3 (red) on sections contiguous to A showing a marked loss of TH+Pitx3+ cells in <i>Rgs6^−/−</i> mice. Scale bar 100 µm.</p

<i>Rgs6</i> is Required for Adult Maintenance of Dopaminergic Neurons in the Ventral Substantia Nigra

<div><p>Parkinson disease (PD) is characterized by the preferential, but poorly understood, vulnerability to degeneration of midbrain dopaminergic (mDA) neurons in the ventral substantia nigra compacta (vSNc). These sensitive mDA neurons express Pitx3, a transcription factor that is critical for their survival during development. We used this dependence to identify, by flow cytometry and expression profiling, the negative regulator of G-protein signaling Rgs6 for its restricted expression in these neurons. In contrast to <i>Pitx3^−/−</i> mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of <i>Rgs6</i>^−/− mutant mice begin to exhibit unilateral signs of degeneration at around 6 months of age, and by one year cell loss is observed in a fraction of mice. Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network. The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10. These low TH neurons also express markers of increased dopamine signaling, namely increased DAT and phospho-Erk1/2 expression. The late onset degeneration may reflect the protective action of Rgs6 against excessive DA signaling throughout life. Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.</p></div

Unilateral degeneration of vSNc neurons in a subset of <i>Rgs6−/−</i> mice.

<p>(A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing dysmorphic TH+ neurons (low TH staining, TH^low, inset) in ventral SNc of <i>Rgs6</i>^−/− mice. The dSNc and VTA mDA neurons are unaffected (strong/normal TH staining, inset). Scale bar 200 µm. (B) Triple staining for TH (red), Dapi (blue) and Fluoro-Jade C (FJC, green) showing presence of degenerating TH^low cells in vSNc of 1 y-old <i>Rgs6</i>^−/− mice (middle panels) and not in control vSNc (upper panels) or in dorsal SNc (lower panels). Scale bar 20 µm. (C) Bilateral cell counts of FJC⁺ mDA neurons in SNc and VTA of coronal sections from control and 1 y-old <i>Rgs6</i>^−/− mice. (D) Co-immunostaining for TH (green) and LC3B (red) in vSNc of <i>WT</i> and <i>Rgs6</i>^−/− mice. Scale bar 20 µm. Arrowheads indicate unaffected neurons while arrows point to TH^low cells. (E) Co-immunostaining for TH (green) and phosphorylated p27^Kip1 (phospho-p27, red) in vSNc of <i>WT</i> and <i>Rgs6</i>^−/− mice. Scale bar 20 µm.</p

Expression of familial PD genes is altered in degenerating neurons of vSNc.

<p>Double immunofluorescence staining against TH (green) and (A) DJ-1 (red) or (B) Pink1 (red) or (C) Lrrk2 (red) in SNc of control and 1 y-old <i>Rgs6</i>^−/− mice that display dysmorphic TH^low mDA neurons. Arrowheads indicate unaffected neurons while arrows point to TH^low cells. Scale bar 20 µm.</p