Depletion of astrocytic transglutaminase 2 improves injury outcomes

Abstract

Astrocytes play an indispensable role in maintaining a healthy, functional neural network in the central nervous system (CNS). A primary function of CNS astrocytes is to support the survival and function of neurons. In response to injury, astrocytes take on a reactive phenotype, which alters their molecular functions. Reactive astrocytes have been reported to be both beneficial and harmful to the CNS recovery process subsequent to injury. Understanding the molecular processes and regulatory proteins that determine the extent to which an astrocyte hinders or supports neuronal survival is important within the context of CNS repair. One protein that plays a role in modulating cellular survival is transglutaminase 2 (TG2). Global deletion of TG2 results in beneficial outcomes subsequent to in vivo ischemic brain injury. Ex vivo studies have also implicated TG2 as a negative regulator of astrocyte viability subsequent to injury. In this study we show that knocking down TG2 in astrocytes significantly increases their ability to protect neurons from oxygen glucose deprivation (OGD)/reperfusion injury. To begin to understand how deletion of TG2 in astrocytes improves their ability to protect neurons from injury, we performed transcriptome analysis of wild type and TG2 astrocytes. TG2 deletion resulted in alterations in genes involved in extracellular matrix remodeling, cell adhesion and axon growth/guidance. In addition, the majority of genes that showed increases in the TG2 astrocytes had predicted cJun/AP-1 binding motifs in their promoters. Furthermore, phospho-cJun levels were robustly elevated in TG2 astrocytes, a finding which was consistent with the increase in expression of AP-1 responsive genes. These in vitro data were subsequently extended into an in vivo model to determine whether the absence of astrocytic TG2 improves outcomes after CNS injury. Our results show that, following a spinal cord injury, scar formation is significantly attenuated in mice with astrocyte-specific TG2 deletion compared to mice expressing normal TG2 levels. Taken together, these data indicate that TG2 plays a pivotal role in mediating reactive astrocyte properties following CNS injury. Further, the data suggest that limiting the AP-1 mediated pro-survival injury response may be a contributing factor to that the detrimental effects of astrocytic TG2.

Deletion or inhibition of astrocytic transglutaminase 2 promotes functional recovery after spinal cord injury

“…Immunohistochemical analyses of the spinal cords from the TG2-A-cKO and TG2fl/fl mice at 3 and 7 dpi suggests a modulation of the glial scar in animals with astrocytic deletion of TG2. In agreement with what we had observed previously [39], GFAP immunoreactivity as a general indicator of astrogliosis was significantly reduced in the TG2-A-cKO mice. Likewise, we find a significant reduction in the expression of the neurite growth inhibitory CSPG NG2 and the upstream regulator of inhibitory CSPGs, Sox9.…”

“…Previously we demonstrated that deletion of TG2 from astrocytes or treatment with the selective TG2 inhibitor VA4, significantly increases their survival in OGD conditions [6, 13]. Deletion of TG2 from astrocytes also significantly increases their ability to protect neurons from ischemic-induced death [6, 39]. Given the fact that TG2 regulates transcriptional processes [40, 41], we carried out RNA-seq on wild type and TG2-/-astrocytes and found that pro-survival/pro-regenerative genes were upregulated in the TG2-/-astrocytes [39].…”

“…Deletion of TG2 from astrocytes also significantly increases their ability to protect neurons from ischemic-induced death [6, 39]. Given the fact that TG2 regulates transcriptional processes [40, 41], we carried out RNA-seq on wild type and TG2-/-astrocytes and found that pro-survival/pro-regenerative genes were upregulated in the TG2-/-astrocytes [39]. Although these findings indicate that deletion of TG2 from astrocytes is beneficial in the context of CNS injury, this needs to be tested in an in vivo model.…”

“…Although these findings indicate that deletion of TG2 from astrocytes is beneficial in the context of CNS injury, this needs to be tested in an in vivo model. Therefore, we used TG2-A-cKO and TG2fl/fl mice [39] in a contusion SCI model followed by analysis of motor function over a 42 day period. Remarkably, motor function in the mice with astrocytic TG2 deleted was significantly better starting at 7 dpi and continuing through 42 dpi.…”

Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2

“…amyloid-β and α-synuclein), thus favouring formation of protein aggregates (Andringa, Lam et al, 2004;Benilova, Karran et al, 2012;Grosso, Woo et al, 2014;Hartley, Zhao et al, 2008;Junn, Ronchetti et al, 2003). However a number of recent observations, such as the potentiation of Ca 2+ -induced hippocampal damage by TG2 in mice brain and higher sensitivity to kainic acid-induced seizures (Tucholski, Roth et al, 2006), as well as the neurotoxic role of astrocytic TG2 following acute brain injury (Feola, Barton et al, 2017;Monteagudo, Feola et al, 2018), have hinted to a possible role of TG2 in excitotoxicity-induced neuronal cell death, which may be either consequent to [Ca 2+ ]i increases or, as a new hypothesis, triggered by TG2-mediated changes in [Ca 2+ ]i. Astrocytes are an abundant source of TG2, which is externalised and accumulates in the extracellular matrix (ECM) in response to inflammatory stimuli (Pinzon, Breve et al, 2017). Importantly, astrocytes are key mediators of brain immune responses (Colombo & Farina, 2016) and TG2 has been shown to play a role in neuroinflammation (Ientile, Curro et al, 2015).…”