No description available.

Le système d’activation du plasminogène (SAP), initialement décrit dans la circulation sanguine, intervient dans la dégradation des caillots de fibrine (fibrinolyse). Ce système permet de convertir un zymogène inactif (le plasminogène) en enzyme active (la plasmine) via l’action de l’activateur tissulaire du plasminogène (tPA), lui-même inhibé par la serpine inhibitrice de l’activateur du plasminogène de type 1 (PAI-1). Il a été suggéré qu’une dérégulation du SAP dans le système nerveux central (SNC) pouvait être un processus physiopathologique dans la sclérose en plaques (SEP). Cependant, l’origine et les conséquences de cette dérégulation restent peu caractérisées dans cette pathologie. Durant cette thèse, nous avons donc cherché à mieux caractériser la dérégulation du SAP dans la SEP et à déterminer son rôle dans la physiopathologie de cette maladie. Les travaux réalisés au cours de ce doctorat ont permis de montrer qu’une forte surexpression de PAI-1 survient dans le SNC lors des phases symptomatiques de modèles murins de SEP, entraînant une inhibition de la fibrinolyse intraparenchymateuse. Cette altération du SAP est une cause de la formation des zones de lésion, et coïncide temporellement avec les phases symptomatiques des modèles murins de SEP. Les astrocytes réactifs pro-inflammatoires sont les responsables de cette surexpression parenchymateuse délétère de PAI-1. Il apparaît qu’une intervention à visée thérapeutique permettant la restauration de l’activité fibrinolytique intraparenchymateuse, via l’inhibition de PAI-1, s’avère bénéfique dans des modèles animaux de SEP. En conclusion, nos travaux révèlent que la présence d’astrocytes réactifs est à l’origine de la surexpression de PAI-1 engendrant l’inhibition de la fibrinolyse intraparenchymateuse constatée dans la SEP. Ce processus physiopathologique délétère est une des causes menant à la formation de zones lésionnelles dans la SEP. / Degradation of fibrin blood clots (fibrinolysis) is mediated by the plasminogen activation system (PAS), initially discovered in the blood. This system allows the inactive plasminogen conversion into active plasmin by tissue-type plasminogen activator (tPA). The plasminogen activator inhibitor type 1 (PAI-1) is able to inhibit tPA. It has been suggested that in the central nervous system (CNS), a PAS dysregulation could be a physiopathological mechanism of multiple sclerosis (MS). However, this dysregulation cause and consequences are poorly described in this disease. This work aimed to characterise the PAS dysregulation occurring in MS, and to better define its role in MS physiopathology. Our results describe a strong PAI-1 overexpression in the CNS during symptomatic periods in animal models of MS, leading to an intraparenchymal fibrinolysis inhibition. The PAS dysregulation is a cause of lesion formation, and temporally coincides with symptomatic periods in these models. Pro-inflammatory reactive astrocytes overexpress PAI-1. It appears that an increase of parenchymal fibrinolysis by inhibiting PAI-1 reduces EAE severity. To conclude, our results highlight a role of reactive astrocytes in MS, leading to an over-expression of PAI-1 and an impairment of parenchymal fibrinolysis. This physiopathological mechanism is implied in lesion formation in MS.

Thyroid hormones (THs) play a fundamental role in brain function during development and adulthood. THs are essential regulators of neurogenesis, cell maturation and migration as well myelination and synaptogenesis. Neuroglial cells, including astrocytes and microglia are targets of TH and implicated in TH regulation; however, the regulation is not properly understood at the cellular level. In this study, TH regulation was investigated in vitro using human brain cell lines of astrocytes (Svg-P12) and microglia (HMC3). The cells were exposed to TH receptor agonist (triiodothyronine; T3) and inhibitors (amiodarone/1-850), of different concentrations, followed by RNA extraction and quantitative PCR. The gene expression of known TH regulated genes was studied for a better understanding of TH signaling in astrocytes and microglia. All target genes were successfully measured in both cell types. Interestingly, the regulatory effects of TH in astrocytes and microglia exhibited differences. In astrocytes, T3 exposure resulted in an upregulation in gene expression of DDX54 (DEAD-Box Helicase 54) and KLF9 (Krüppel-like factor 9) but did not affect other genes. Also, THR inhibitor exposure resulted in n upregulation in gene expression of DDX54 (DEAD-Box Helicase 54) and KLF9 (Krüppel-like factor 9) but did not affect other genes. Also, THR inhibitor exposure resulted in downregulation in gene expression of KLF9, NES (Nestin), PTGDS (Prostaglandin D2 Synthase) and MAPT (Microtubule Associated Protein Tau). In contrast, none of the TH regulated genes demonstrated a statistical significance in T3-treated microglia compared to control cells. However, THR inhibitor exposure resulted in a downregulation in gene expression of KLF9 and DDX54 and an upregulation of NES, PTGDS and MAPT. The observed differences indicate that TH signaling and regulation is different in microglia and astrocytes. The The differential signaling suggests that T3 does not regulate all of its target genes directly; rather, the regulatory effects of T3 may be exerted through complex mechanisms with other key factors involved. It can be concluded that astrocytes and microglia play important roles as mediators of the effects of THs in CNS development and function. However, further analysis is needed to acknowledge other key factors and TH signaling mechanisms influencing the gene expression in neuroglia.

Background: Norepinephrine and glutamate are among several neurotransmitters implicated in the neuropathology of major depressive disorder (MDD). Glia deficits have also been demonstrated in people with MDD, and glia are critical modulators of central glutamatergic transmission. We studied glia in men with MDD in the region of the brain (locus coeruleus; LC) where noradrenergic neuronal cell bodies reside and receive glutamatergic input. Methods: The expression of 3 glutamate-related genes (SLC1A3, SLC1A2, GLUL) concentrated in glia and a glia gene (GFAP) were measured in postmortem tissues from men with MDD and from paired psychiatrically healthy controls. Initial gene expression analysis of RNA isolated from homogenized tissue (n = 9-10 pairs) containing the LC were followed by detailed analysis of gene expressions in astrocytes and oligodendrocytes (n = 6-7 pairs) laser captured from the LC region. We assessed protein changes in GFAP using immunohistochemistry and immunoblotting (n = 7-14 pairs). Results: Astrocytes, but not oligodendrocytes, demonstrated robust reductions in the expression of SLC1A3 and SLC1A2, whereas GLUL expression was unchanged. GFAP expression was lower in astrocytes, and we confirmed reduced GFAP protein in the LC using immunostaining methods. Limitations: Reduced expression of protein products of SLC1A3 and SLC1A2 could not be confirmed because of insufficient amounts of LC tissue for these assays. Whether gene expression abnormalities were associated with only MDD and not with suicide could not be confirmed because most of the decedents who had MDD died by suicide. Conclusion: Major depressive disorder is associated with unhealthy astrocytes in the noradrenergic LC, characterized here by a reduction in astrocyte glutamate transporter expression. These findings suggest that increased glutamatergic activity in the LC occurs in men with MDD.

No description available.

Background: The anterior cingulate cortex (ACC) is a brain area involved in modulating behavior associated with social interaction, disruption of which is a core feature of autism spectrum disorder (ASD). Functional brain imaging studies demonstrate abnormalities of the ACC in ASD as compared to typically developing control patients. However, little is known regarding the cellular basis of these functional deficits in ASD. Pyramidal neurons in the ACC are excitatory glutamatergic neurons and key cellular mediators of the neural output of the ACC. This study was designed to investigate the potential role of ACC pyramidal neurons in ASD brain pathology. Methods: Postmortem ACC tissue from carefully matched ASD and typically developing control donors was obtained from two national brain collections. Pyramidal neurons and surrounding astrocytes were separately collected from layer III of the ACC by laser capture microdissection. Isolated RNA was subjected to reverse transcription and endpoint PCR to determine gene expression levels for 16 synaptic genes relevant to glutamatergic neurotransmission. Cells were also collected from the prefrontal cortex (Brodmann area 10) to examine those genes demonstrating differences in expression in the ACC comparing typically developing and ASD donors. Results: The level of NTRK2 expression was robustly and significantly lower in pyramidal neurons from ASD donors as compared to typically developing donors. Levels of expression of GRIN1, GRM8, SLC1A1, and GRIP1 were modestly lower in pyramidal neurons from ASD donors, but statistical significance for these latter genes did not survive correction for multiple comparisons. No significant expression differences of any genes were found in astrocytes laser captured from the same neocortical area. In addition, expression levels of NTRK2 and other synaptic genes were normal in pyramidal neurons laser captured from the prefrontal cortex. Conclusions: These studies demonstrate a unique pathology of neocortical pyramidal neurons of the ACC in ASD. NTRK2 encodes the tropomyosin receptor kinase B (TrkB), transmission through which neurotrophic factors modify differentiation, plasticity, and synaptic transmission. Reduced pyramidal neuron NTRK2 expression in the ACC could thereby contribute to abnormal neuronal activity and disrupt social behavior mediated by this brain region.

Telomere shortening is observed in peripheral mononuclear cells from patients with major depressive disorder (MDD). Whether this finding and its biological causes impact the health of the brain in MDD is unknown. Brain cells have differing vulnerabilities to biological mechanisms known to play a role in accelerating telomere shortening. Here, two glia cell populations (oligodendrocytes and astrocytes) known to have different vulnerabilities to a key mediator of telomere shortening, oxidative stress, were studied. The two cell populations were separately collected by laser capture micro-dissection of two white matter regions shown previously to demonstrate pathology in MDD patients. Cells were collected from brain donors with MDD at the time of death and age-matched psychiatrically normal control donors (N=12 donor pairs). Relative telomere lengths in white matter oligodendrocytes, but not astrocytes, from both brain regions were significantly shorter for MDD donors as compared to matched control donors. Gene expression levels of telomerase reverse transcriptase were significantly lower in white matter oligodendrocytes from MDD as compared to control donors. Likewise, the gene expression of oxidative defence enzymes superoxide dismutases (SOD1 and SOD2), catalase (CAT) and glutathione peroxidase (GPX1) were significantly lower in oligodendrocytes from MDD as compared to control donors. No such gene expression changes were observed in astrocytes from MDD donors. These findings suggest that attenuated oxidative stress defence and deficient telomerase contribute to telomere shortening in oligodendrocytes in MDD, and suggest an aetiological link between telomere shortening and white matter abnormalities previously described in MDD.

Background: The anterior cingulate cortex (ACC) is a brain area involved in modulating behavior associated with social interaction, disruption of which is a core feature of autism spectrum disorder (ASD). Functional brain imaging studies demonstrate abnormalities of the ACC in ASD as compared to typically developing control patients. However, little is known regarding the cellular basis of these functional deficits in ASD. Pyramidal neurons in the ACC are excitatory glutamatergic neurons and key cellular mediators of the neural output of the ACC. This study was designed to investigate the potential role of ACC pyramidal neurons in ASD brain pathology. Methods: Postmortem ACC tissue from carefully matched ASD and typically developing control donors was obtained from two national brain collections. Pyramidal neurons and surrounding astrocytes were separately collected from layer III of the ACC by laser capture microdissection. Isolated RNA was subjected to reverse transcription and endpoint PCR to determine gene expression levels for 16 synaptic genes relevant to glutamatergic neurotransmission. Cells were also collected from the prefrontal cortex (Brodmann area 10) to examine those genes demonstrating differences in expression in the ACC comparing typically developing and ASD donors. Results: The level of NTRK2 expression was robustly and significantly lower in pyramidal neurons from ASD donors as compared to typically developing donors. Levels of expression of GRIN1, GRM8, SLC1A1, and GRIP1 were modestly lower in pyramidal neurons from ASD donors, but statistical significance for these latter genes did not survive correction for multiple comparisons. No significant expression differences of any genes were found in astrocytes laser captured from the same neocortical area. In addition, expression levels of NTRK2 and other synaptic genes were normal in pyramidal neurons laser captured from the prefrontal cortex. Conclusions: These studies demonstrate a unique pathology of neocortical pyramidal neurons of the ACC in ASD. NTRK2 encodes the tropomyosin receptor kinase B (TrkB), transmission through which neurotrophic factors modify differentiation, plasticity, and synaptic transmission. Reduced pyramidal neuron NTRK2 expression in the ACC could thereby contribute to abnormal neuronal activity and disrupt social behavior mediated by this brain region.

Neuroinflammation is prevalent in neurodegenerative diseases and plays a significant role in the central nervous system (CNS) innate immunity, which is the body’s first line of defense mechanisms against invading pathogens and injuries to maintain homeostasis. However, in neurodegenerative diseases, neuroinflammation becomes persistent alongside the subsequent damage to nearby neurons and affects CNS-resident immune glial cells, such as microglia and astrocytes. Accumulating evidence suggests that neuroinflammation is mainly characterized by the excessive activation of glial cells, thus causing abnormal changes in their microenvironment and release soluble factors that can promote or inhibit neuroinflammation. Currently, there is no effective treatment to cure these progressive neurological disorders. Therefore, it is critical to understand how neuroinflammation affects astroglia cell function and their biomechanical properties that change their behavior throughout disease progression. Astrocytes are the most predominant glial cell in the CNS and are critical in the development and maintenance of neuroinflammatory disorders. To date, very little is known regarding the role and specific function of Piezo1 mechanosensitive ion channel (MSC) in the CNS. Recently, Piezo1 expression was found to be upregulated in Lipopolysaccharide (LPS)-induced neuroinflammation in mouse astrocyte cultures. However, it is unknown whether the aberrant mechanical environment in astrocytes interplay with the mechanosensory function of Piezo1 and its current activity in neuroinflammatory conditions. In this study, we investigated Piezo1 mechanosensitive ionic currents by performing in vitro patch-clamp electrophysiology and calcium imaging. Our preliminary studies revealed that astrocytes derived from the mouse cerebellum stimulated with LPS or Piezo1 agonist, Yoda1, increased Ca2+ influx and further augmented when treated concurrently. We also found that electrophysiology recordings showed changes in mechanosensitive ionic currents and were comparable with our calcium imaging data indicating that MSCs are involved in neuroinflammation. Therefore, we postulated that Piezo1, a non-selective cation MSC that opens in response to mechanical force is a key mechanosensor involved in neuroinflammation by altered mechanical signals in C8-S astrocytes. Using an in vitro system of Mouse C8-S (Astrocyte type II clone), the goal of this study was to investigate if neuroinflammatory conditions upregulate Piezo1 calcium influx and current activity. We show that astrocytic Piezo1 regulates mechanotransducive release of ATP by controlling the mechanically induced calcium influx and current activation in LPS-induced astrocytes. Additionally, Piezo1 antagonist, GsMTx4 and Piezo1 siRNA significantly reduced the LPS-induced current, indicating that Piezo1 is involved in neuroinflammation. Our findings demonstrate that the activity of Piezo1 stimulated by neuroinflammatory conditions may be significant for the development of therapeutics to prevent or treat neuroinflammatory disorders and diseases.

The glial-lymphatic or glymphatic fluid clearance system promotes the exchange of interstitial fluid (ISF) and cerebrospinal fluid through the arterial perivascular spaces into the brain. This process is facilitated in part by aquaporin-4 (AQP4) water channels located primarily on astrocyte end feet abutting endothelial cells of the blood brain barrier. Changes in expression levels or mislocalization of AQP4 from astrocytic end feet to the soma can lead to decreased ISF flow leading to buildup of extracellular waste products like hyperphosphorylated Tau (pTau). pTau accumulation is a neuropathological hallmark in Alzheimer’s disease (AD) and in some people with human immunodeficiency virus (HIV). Approximately 50% of people with HIV (PWH) suffer from HIV-associated neurocognitive disorders (HAND), which is a spectrum disorder linked to cognitive and motor decline in PWH. Limited studies have shown that in HIV CNS infection that expression levels of AQP4 in brain homogenates from the mid-frontal gyrus of PWH with symptomatic HAND were significantly increased compared to those with asymptomatic HAND, which raises the question if AQP4 function and subcellular localization may contribute to cognitive status. Studies in other neuroinflammatory diseases have shown dysregulation of AQP4 through the adenosine A2aR (A2aR) signaling. A2aR activation leads to PKA/PKC-mediated inhibitory phosphorylation of AQP4 (Ser180, Ser276) that is proposed to contribute to channel internalization, mislocalization and decreased expression. In addition, common single nucleotide polymorphisms in aqp4 have been associated with more rapid cognitive decline some neurodegenerative diseases. Therefore, it is possible that common mutations in aqp4, subcellular mislocalization, dysfunction, expression levels or post-translational modifications contribute to HAND. Therefore, the cognitive changes we see in HAND maybe due to changes in AQP4 may contribute by decreasing clearance of toxic aberrant proteins and HIV mechanistically alters AQP4 in part via dysregulation of A2aR. / Biomedical Sciences