Transglutaminase 2: Friend or foe? The discordant role in neurons and astrocytes

Abstract

Members of the transglutaminase family catalyze the formation of isopeptide bonds between a polypeptide-bound glutamine and a low molecular weight amine (e.g., spermidine) or the ɛ-amino group of a polypeptide-bound lysine. Transglutaminase 2 (TG2), a prominent member of this family, is unique because in addition to being a transamidating enzyme, it exhibits numerous other activities. As a result, TG2 plays a role in many physiological processes, and its function is highly cell type specific and relies upon a number of factors, including conformation, cellular compartment location, and local concentrations of Ca²⁺ and guanine nucleotides. TG2 is the most abundant transglutaminase in the central nervous system (CNS) and plays a pivotal role in the CNS injury response. How TG2 affects the cell in response to an insult is strikingly different in astrocytes and neurons. In neurons, TG2 supports survival. Overexpression of TG2 in primary neurons protects against oxygen and glucose deprivation (OGD)-induced cell death and in vivo results in a reduction in infarct volume subsequent to a stroke. Knockdown of TG2 in primary neurons results in a loss of viability. In contrast, deletion of TG2 from astrocytes results in increased survival following OGD and improved ability to protect neurons from injury. Here, a brief overview of TG2 is provided, followed by a discussion of the role of TG2 in transcriptional regulation, cellular dynamics, and cell death. The differing roles TG2 plays in neurons and astrocytes are highlighted and compared to how TG2 functions in other cell types.

Keywords: astrocytes; cell death; cytoskeleton; injury; neurites; neurons; transcription regulation.

Figure 1

Domains (A), closed (B) and open (C) structures of human transglutaminase 2 (TG2) (A)The catalytic triad is mediates the transamidating activity in a Ca2+ dependent manner. Six Ca2+ binding sites have been identified in the catalytic core. W241 is essential for the stabilization of the intermediate state of the transamidation. R580 is required for efficient GTP/GDP binding. C230, C370 and C371 act as redox sensors maintaining TG2 in an inactive state in oxidizing conditions. (B) Closed, guanine nucleotide bound form of TG2 as a dimer (Liu et al. 2002). Bound GDP is shown as a ball stick model between the first and second β-barrels (PDB ID code 1KV3). (C) Open, transamidating active form of TG2 (Pinkas et al. 2007) (PDB ID code 2Q3Z).

Figure 2

Depletion of TG2 in neurons attenuates Cre luciferase reporter activity. Primary rat cortical neurons were transduced with TG2 shRNA as indicated. Five days after transduction all neurons were transfected with a Cre luciferase reporter and a promoterless null Renilla construct. Two days later the neurons were treated with IBMX or vehicle overnight followed by measurement of luciferase and renilla activity. The luciferase measurements were normalized to its Renilla measurement. Results are shown as fold increase relative to vehicle-treated measurements and expressed as mean ± SEM. *p < 0.05, n= 3 (Y. Nuzbrokh and S. Gundemir, unpublished data).

Figure 3

The depletion of TG2 increases NF-κB luciferase reporter activity. Primary rat cortical neurons were transduced with TG2 shRNA or scrambled control. Five days after transduction the neurons were transfected with an NF-κB luciferase reporter and a promoterless null Renilla construct. Two days later the neurons were exposed to hypoxia (0.1% O₂) or normoxia for 14 hours. After hypoxia the luciferase and Renilla expression were measured. The luciferase measurements were normalized to its Renilla measurement. Final results were normalized to control measurements. Results are shown as mean ± SEM. **p < 0.01, ****p < 0.0001. n= 5 biological replicates with 5–6 separate wells for each condition.

Figure 4

Deletion of transglutaminase 2 (TG2) in astrocytes significant increases the levels of lactate, malate and α-ketoglutarate after ischemic stress. Primary astrocytes from wild type (closed symbols) and TG2−/− (open symbols) mice were exposed to 90 minutes of oxygen and glucose deprivation (OGD, 0.1% O₂) followed by 24 hrs of reperfusion. Cells were snap frozen and metabolites were extracted with 80% methanol. Extracted metabolites were analyzed using reverse phase chromatography with an ion-pairing reagent in a Shimadzu HPLC coupled to a Thermo Quantum triple-quadrupole mass spectrometer (Nadtochiy et al. 2015). Data are expressed relative to wild type astrocytes. Results are shown as mean ± SEM. *p < 0.05, n= 8. (A. Monteagudo, unpublished data).

Figure 5

Hypothetical model illustrating how TG2 mediated suppression of NF-κB signaling in astrocytes could compromise the ability of astrocytes to protect against neurons against excitotoxicity. In pathological conditions where there is excessive excitation of glutamatergic neurons and release of glutamate the primary protective response is uptake of glutamate by astrocytes and GLT-1 plays a major role in this process. GLT-1 expression in astrocytes is mediated by NF-κB signaling (Ghosh et al. 2011). Glutamate treatment of astrocytes results in increased nuclear localization of NF-κB subunits, as well as increased binding of these subunits to the promoter of TG2 (Caccamo et al. 2005) which can increase TG2 expression (Mirza et al. 1997). Given that in astrocytes TG2 represses NF-κB activity (Feola et al. 2017) this could lead to decreased GLT-1 expression which would result in decreased glutamate uptake.