Characterization of Transglutaminase 2 activity inhibitors in monocytes in vitro and their effect in a mouse model for multiple sclerosis

Abstract

The neurodegenerative disease multiple sclerosis (MS) is pathologically characterized by the massive influx of immune cells into the central nervous system. This contributes to demyelination and axonal damage which causes symptoms such as motor and cognitive dysfunctions. The migration of leukocytes from the blood vessel is orchestrated by a multitude of factors whose determination is essential in reducing cellular influx in MS patients and the experimental autoimmune encephalomyelitis (EAE) animal model. The here studied enzyme tissue Transglutaminase (TG2) is present intracellularly, on the cell surface and extracellularly. There it contributes to cellular adhesion and migration via its transamidation activity and possibly by facilitating cellular interaction with the extracellular matrix. Previous data from our group showed reduced motor symptoms and cellular infiltration after using a pharmacological TG2 transamidation activity inhibitor in a rat EAE model. However, it remained elusive if the cross-linking activity of the enzyme resulted in the observed effects. To follow-up, we now characterized two new small molecule TG2 activity inhibitors, BJJF078 and ERW1041E. Both compounds are potent inhibitor of recombinant human and mouse Transglutaminase enzyme activity, mainly TG2 and the close related enzyme TG1. In addition they did not affect the binding of TG2 to the extracellular matrix substrate fibronectin, a process via which TG2 promotes cellular adhesion and migration. We found, that ERW1041E but not BJJF078 resulted in reduced EAE disease motor-symptoms while neither caused apparent changes in pathology (cellular influx), Transglutaminase activity or expression of inflammation related markers in the spinal cord, compared to vehicle treated controls. Although we cannot exclude issues on bioavailability and in vivo efficacy of the used compounds, we hypothesize that extracellular TG1/TG2 activity is of greater importance than (intra-)cellular activity in mouse EAE pathology.

Fig 1. ERW1041E and BJJF078: Name and chemical structure of the TG2 inhibitors.

Fig 2. Inhibition of TG activity and TG2-fibronectin interaction by BJJF078 and ERW1041E in solution and in vitro.

(a) BJFF078 and ERW1041E dose-dependently inhibited human (hTG2) and mouse (mTG2) recombinant TG2 activity. BJJF078 and ERW1041E inhibited also (b) human recombinant TG1 and (c) human recombinant FXIII. (d) Cellular activity of human TG2 in THP1 cells was dose-dependently inhibited by BJJF078 and Z006 but not by ERW1041E. (e,f) Effect of BJJF078 and ERW1041E on TG2 binding to fibronectin without (e) or with added calcium (f). n = 1–3 independent experiments with triplicates each.

Fig 3. Disease scores and bodyweight of EAE animals treated with BJJF078 or ERW1041E.

(a) BJJF078 did not affect motor symptoms or (b) bodyweight of EAE affected mice compared to vehicle treated animals. (c) ERW1041E reduced motor symptoms significantly without (d) affecting the loss of bodyweight of EAE affected mice. n = 11–12 animals per treatment group. Statistics: generalized linear model, *P<0.05.

Fig 4. Transglutaminases mRNA, and TG1 and TG2 immunoreactivity in post-mortem spinal cord tissue of EAE animals.

(a) EAE spinal cord mRNA levels for Tgm1, Tgm2, Tgm3, Tgm6 (n.d. = not detectable) and F13A1, (b) TG2 immunoreactivity (green) associates with CD45 (red) stained EAE lesions whereas TG1 immunoreactivity (green) is found in some lesions (d) but not in others (d). Scale bar: 25 μm. n = 9 animals/group.

Fig 5. TG activity in spinal cord post-mortem tissue derived from EAE animals treated with vehicle, BJJF078 or ERW1041E.

(a-f) In situ TG activity was detected in spinal cord sections derived from (a,d) vehicle, (b,e) BJJF078 and (c,f) ERW1041E treated mice. (d-f) Co-incubation of the sections with the TG2 inhibitor Z006 diminished TG activity. Scale bar: 50 μm. n = 11 or 12 animals/group.

Fig 6. Cell infiltrates and TG2 expression in the spinal cord of BJJF078 or ERW1041E treated EAE mice.

(a-d) BJJF078 and ERW1041E did not affect the infiltration of CD45 positive leukocytes into the spinal cord. Also (e-h), CD68 positive phagocytic cells and (i-l) CD3 positive T cells were observed in similar amount in vehicle and TG2 inhibitor treated animals. (b, f, j) show co-labelling of TG2 (green) with the immune cells makers (red) (b) CD45, (f) CD68 and (j) CD3. Scale bar: 100 μm, except for b,f,j: 50 μm. n = 9–12 animals/group.

Fig 7. qPCR analysis of gene expression associated with inflammation in the spinal cord of ERW1041E and vehicle only treated EAE mice.

mRNA levels of inflammatory markers (a) IL-1ra, (b) IL-1β, (c) IFNγ, (d) TNFα and (e) iNOS were not significantly changed in spinal cord of ERW1041E animals compared to the vehicle treated group. n = 9–12 animals/group. Statistical analysis: student T-test or, if not normally distributed: Mann-Whitney U test.