Altered TFEB subcellular localization in nigral neurons of subjects with incidental, sporadic and GBA-related Lewy body diseases.

Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA-related and sporadic Parkinson's disease (PD), and dementia with Lewy bodies (DLB). TFEB activation results in its translocation from the cytosol to the nucleus. Here, we investigated TFEB subcellular localization and its relation to intracellular alpha-synuclein (aSyn) accumulation in post-mortem human brain of individuals with either incidental Lewy body disease (iLBD), GBA-related PD/DLB (GBA-PD/DLB) or sporadic PD/DLB (sPD/DLB), compared to control subjects. We analyzed nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the TFEB subcellular localization patterns. We observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both in sporadic and GBA-related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn accumulation in all groups. Importantly, we observed previously-unidentified TFEB-immunopositive perinuclear clusters in human dopaminergic neurons, which localized at the Golgi apparatus. These TFEB clusters were more frequently observed and more severe in iLBD, sPD/DLB and GBA-PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons, but also in neurons lacking detectable aSyn accumulation. In aSyn-negative cells, cytoplasmic TFEB clusters were more frequently observed in GBA-PD/DLB and iLBD patients, and correlated with reduced GBA enzymatic activity as well as increased Braak LB stage. Altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected TFEB-regulated genes, indicating a possible early dysfunction of lysosomal regulation. Overall, we observed cytoplasmic TFEB retention and accumulation at the Golgi in cells without apparent pSer129 aSyn accumulation in iLBD and PD/DLB patients. This suggests potential TFEB impairment at the early stages of cellular disease and underscores TFEB as a promising therapeutic target for synucleinopathies.

Distinct tau and alpha-synuclein molecular signatures in Alzheimer's disease with and without Lewy bodies and Parkinson's disease with dementia.

Alpha-synuclein (aSyn) pathology is present in approximately 50% of Alzheimer's disease (AD) cases at autopsy and might impact the age-of-onset and disease progression in AD. Here, we aimed to determine whether tau and aSyn profiles differ between AD cases with Lewy bodies (AD-LB), pure AD and Parkinson's disease with dementia (PDD) cases using epitope-, post-translational modification- (PTM) and isoform-specific tau and aSyn antibody panels spanning from the N- to C-terminus. We included the middle temporal gyrus (MTG) and amygdala (AMY) of clinically diagnosed and pathologically confirmed cases and performed dot blotting, western blotting and immunohistochemistry combined with quantitative and morphological analyses. All investigated phospho-tau (pTau) species, except pT181, were upregulated in AD-LB and AD cases compared to PDD and control cases, but no significant differences were observed between AD-LB and AD subjects. In addition, tau antibodies targeting the proline-rich regions and C-terminus showed preferential binding to AD-LB and AD brain homogenates. Antibodies targeting C-terminal aSyn epitopes and pS129 aSyn showed stronger binding to AD-LB and PDD cases compared to AD and control cases. Two pTau species (pS198 and pS396) were specifically detected in the soluble protein fractions of AD-LB and AD subjects, indicative of early involvement of these PTMs in the multimerization process of tau. Other phospho-variants for both tau (pT212/S214, pT231 and pS422) and aSyn (pS129) were only detected in the insoluble protein fraction of AD-LB/AD and AD-LB/PDD cases, respectively. aSyn load was higher in the AMY of AD-LB cases compared to PDD cases, suggesting aggravated aSyn pathology under the presence of AD pathology, while tau load was similar between AD-LB and AD cases. Co-localization of pTau and aSyn could be observed within astrocytes of AD-LB cases within the MTG. These findings highlight a unique pathological signature for AD-LB cases compared to pure AD and PDD cases.

Distinct gene expression in demyelinated white and grey matter areas of patients with multiple sclerosis.

Demyelination of the central nervous system is a prominent pathological hallmark of multiple sclerosis and affects both white and grey matter. However, demyelinated white and grey matter exhibit clear pathological differences, most notably the presence or absence of inflammation and activated glial cells in white and grey matter, respectively. In order to gain more insight into the differential pathology of demyelinated white and grey matter areas, we micro-dissected neighbouring white and grey matter demyelinated areas as well as normal-appearing matter from leucocortical lesions of human post-mortem material and used these samples for RNA sequencing. Our data show that even neighbouring demyelinated white and grey matter of the same leucocortical have a distinct gene expression profile and cellular composition. We propose that, based on their distinct expression profile, pathological processes in neighbouring white and grey matter are likely different which could have implications for the efficacy of treating grey matter lesions with current anti-inflammatory-based multiple sclerosis drugs.

The Transglutaminase-2 Interactome in the APP23 Mouse Model of Alzheimer's Disease.

Amyloid-beta (Aß) deposition in the brain is closely linked with the development of Alzheimer's disease (AD). Unfortunately, therapies specifically targeting Aß deposition have failed to reach their primary clinical endpoints, emphasizing the need to broaden the search strategy for alternative targets/mechanisms. Transglutaminase-2 (TG2) catalyzes post-translational modifications, is present in AD lesions and interacts with AD-associated proteins. However, an unbiased overview of TG2 interactors is lacking in both control and AD brain. Here we aimed to identify these interactors using a crossbreed of the AD-mimicking APP23 mouse model with wild type and TG2 knock-out (TG2-/-) mice. We found that absence of TG2 had no (statistically) significant effect on Aß pathology, soluble brain levels of Aß1-40 and Aß1-42, and mRNA levels of TG family members compared to APP23 mice at 18 months of age. Quantitative proteomics and network analysis revealed a large cluster of TG2 interactors involved in synaptic transmission/assembly and cell adhesion in the APP23 brain typical of AD. Comparative proteomics of wild type and TG2-/- brains revealed a TG2-linked pathological proteome consistent with alterations in both pathways. Our data show that TG2 deletion leads to considerable network alterations consistent with a TG2 role in (dys)regulation of synaptic transmission and cell adhesion in APP23 brains.

Absence of tissue transglutaminase reduces amyloid-beta pathology in APP23 mice.

AIMS: Alzheimer's disease (AD) is characterised by amyloid-beta (Aß) aggregates in the brain. Targeting Aß aggregates is a major approach for AD therapies, although attempts have had little to no success so far. A novel treatment option is to focus on blocking the actual formation of Aß multimers. The enzyme tissue transglutaminase (TG2) is abundantly expressed in the human brain and plays a key role in post-translational modifications in Aß resulting in covalently cross-linked, stable and neurotoxic Aß oligomers. In vivo absence of TG2 in the APP23 mouse model may provide evidence that TG2 plays a key role in development and/or progression of Aß-related pathology. METHODS: Here, we compared the effects on Aß pathology in the presence or absence of TG2 using 12-month-old wild type, APP23 and a crossbreed of the TG2-/- mouse model and APP23 mice (APP23/TG2-/-). RESULTS: Using immunohistochemistry, we found that the number of Aß deposits was significantly reduced in the absence of TG2 compared with age-matched APP23 mice. To pinpoint possible TG2-associated mechanisms involved in this observation, we analysed soluble brain Aß1-40 , Aß1-42 and/or Aß40/42 ratio, and mRNA levels of human APP and TG2 family members present in brain of the various mouse models. In addition, using immunohistochemistry, both beta-pleated sheet formation in Aß deposits and the presence of reactive astrocytes associated with Aß deposits were analysed. CONCLUSIONS: We found that absence of TG2 reduces the formation of Aß pathology in the APP23 mouse model, suggesting that TG2 may be a suitable therapeutic target for reducing Aß deposition in AD.

Tissue Transglutaminase Expression Associates With Progression of Multiple Sclerosis.

OBJECTIVE: The clinical course of multiple sclerosis (MS) is variable and largely unpredictable pointing to an urgent need for markers to monitor disease activity and progression. Recent evidence revealed that tissue transglutaminase (TG2) is altered in patient-derived monocytes. We hypothesize that blood cell-derived TG2 messenger RNA (mRNA) can potentially be used as biomarker in patients with MS. METHODS: In peripheral blood mononuclear cells (PBMCs) from 151 healthy controls and 161 patients with MS, TG2 mRNA was measured and correlated with clinical and MRI parameters of disease activity (annualized relapse rate, gadolinium-enhanced lesions, and T2 lesion volume) and disease progression (Expanded Disability Status Scale [EDSS], normalized brain volume, and hypointense T1 lesion volume). RESULTS: PBMC-derived TG2 mRNA levels were significantly associated with disease progression, i.e., worsening of the EDSS over 2 years of follow-up, normalized brain volume, and normalized gray and white matter volume in the total MS patient group at baseline. Of these, in patients with relapsing-remitting MS, TG2 expression was significantly associated with worsening of the EDSS scores over 2 years of follow-up. In the patients with primary progressive (PP) MS, TG2 mRNA levels were significantly associated with EDSS, normalized brain volume, and normalized gray and white matter volume at baseline. In addition, TG2 mRNA associated with T1 hypointense lesion volume in the patients with PP MS at baseline. CONCLUSION: PBMC-derived TG2 mRNA levels hold promise as biomarker for disease progression in patients with MS. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that in patients with MS, PBMC-derived TG2 mRNA levels are associated with disease progression.

Viscoelastic properties of white and gray matter-derived microglia differentiate upon treatment with lipopolysaccharide but not upon treatment with myelin.

BACKGROUND: The biomechanical properties of the brain have increasingly been shown to relate to brain pathology in neurological diseases, including multiple sclerosis (MS). Inflammation and demyelination in MS induce significant changes in brain stiffness which can be linked to the relative abundance of glial cells in lesions. We hypothesize that the biomechanical, in addition to biochemical, properties of white (WM) and gray matter (GM)-derived microglia may contribute to the differential microglial phenotypes as seen in MS WM and GM lesions. METHODS: Primary glial cultures from WM or GM of rat adult brains were treated with either lipopolysaccharide (LPS), myelin, or myelin+LPS for 24 h or left untreated as a control. After treatment, microglial cells were indented using dynamic indentation to determine the storage and loss moduli reflecting cell elasticity and cell viscosity, respectively, and subsequently fixed for immunocytochemical analysis. In parallel, gene expression of inflammatory-related genes were measured using semi-quantitative RT-PCR. Finally, phagocytosis of myelin was determined as well as F-actin visualized to study the cytoskeletal changes. RESULTS: WM-derived microglia were significantly more elastic and more viscous than microglia derived from GM. This heterogeneity in microglia biomechanical properties was also apparent when treated with LPS when WM-derived microglia decreased cell elasticity and viscosity, and GM-derived microglia increased elasticity and viscosity. The increase in elasticity and viscosity observed in GM-derived microglia was accompanied by an increase in Tnfα mRNA and reorganization of F-actin which was absent in WM-derived microglia. In contrast, when treated with myelin, both WM- and GM-derived microglia phagocytose myelin decrease their elasticity and viscosity. CONCLUSIONS: In demyelinating conditions, when myelin debris is phagocytized, as in MS lesions, it is likely that the observed differences in WM- versus GM-derived microglia biomechanics are mainly due to a difference in response to inflammation, rather than to the event of demyelination itself. Thus, the differential biomechanical properties of WM and GM microglia may add to their differential biochemical properties which depend on inflammation present in WM and GM lesions of MS patients.

Tissue Transglutaminase contributes to myelin phagocytosis in interleukin-4-treated human monocyte-derived macrophages.

Macrophages exert either a detrimental or beneficial role in Multiple Sclerosis (MS) pathology, depending on their inflammatory environment. Tissue Transglutaminase (TG2), a calcium-dependent cross-linking enzyme, has been described as a novel marker for anti-inflammatory, interleukin-4 (IL-4) polarized macrophages (M(IL-4)), which represent a subpopulation of macrophages with phagocytic abilities. Since TG2 is expressed in macrophages in active human MS lesions, we questioned whether TG2 drives the differentiation of M(IL-4) into an anti-inflammatory phenotype and whether it plays a role in the phagocytosis of myelin by these cells. In macrophage-differentiated THP-1 monocytes, TG2 was increased upon IL-4 treatment. Reducing TG2 expression impairs the differentiation of M(IL-4) macrophages into an anti-inflammatory phenotype and drives them into a pro-inflammatory state. In addition, reduced TG2 expression resulted in increased presence of myelin basic protein in macrophages upon myelin exposure of M(IL-4) macrophages. Moreover, the elevated presence of an early endosome marker and equal expression of a lysosome marker compared to control macrophages, suggest that TG2 plays a role in phagosome maturation in M(IL-4) macrophages These data suggest that tuning macrophages into TG2 producing anti-inflammatory cells by IL-4 treatment may benefit effective myelin phagocytosis in e.g. demyelinating MS lesions and open avenues for successful regeneration.

Tissue Transglutaminase Promotes Early Differentiation of Oligodendrocyte Progenitor Cells.

Demyelinated lesions of the central nervous system are characteristic for multiple sclerosis (MS). Remyelination is not very effective, particular at later stages of the disease, which results in a chronic neurodegenerative character with worsening of symptoms. Previously, we have shown that the enzyme Tissue Transglutaminase (TG2) is downregulated upon differentiation of oligodendrocyte progenitor cells (OPCs) into myelin-forming oligodendrocytes and that TG2 knock-out mice lag behind in remyelination after cuprizone-induced demyelination. Here, we examined whether astrocytic or oligodendroglial TG2 affects OPCs in a cell-specific manner to modulate their differentiation, and therefore myelination. Our findings indicate that human TG2-expressing astrocytes did not modulate OPC differentiation and myelination. In contrast, persistent TG2 expression upon OPC maturation or exogenously added recombinant TG2 accelerated OPC differentiation and myelin membrane formation. Continuous exposure of recombinant TG2 to OPCs at different consecutive developmental stages, however, decreased OPC differentiation and myelin membrane formation, while it enhanced myelination in dorsal root ganglion neuron-OPC co-cultures. In MS lesions, TG2 is absent in OPCs, while human OPCs show TG2 immunoreactivity during brain development. Exposure to the MS-relevant pro-inflammatory cytokine IFN-γ increased TG2 expression in OPCs and prolonged expression of endogenous TG2 upon differentiation. However, despite the increased TG2 levels, OPC maturation was not accelerated, indicating that TG2-mediated OPC differentiation may be counteracted by other pathways. Together, our data show that TG2, either endogenously expressed, or exogenously supplied to OPCs, accelerates early OPC differentiation. A better understanding of the role of TG2 in the OPC differentiation process during MS is of therapeutic interest to overcome remyelination failure.

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

[This corrects the article DOI: 10.1371/journal.pone.0196433.].

In vivo assessment of neuroinflammation in progressive multiple sclerosis: a proof of concept study with [18F]DPA714 PET.

BACKGROUND: Over the past decades, positron emission tomography (PET) imaging has become an increasingly useful research modality in the field of multiple sclerosis (MS) research, as PET can visualise molecular processes, such as neuroinflammation, in vivo. The second generation PET radioligand [18F]DPA714 binds with high affinity to the 18-kDa translocator-protein (TSPO), which is mainly expressed on activated microglia. The aim of this proof of concept study was to evaluate this in vivo marker of neuroinflammation in primary and secondary progressive MS. METHODS: All subjects were genotyped for the rs6971 polymorphism within the TSPO gene, and low-affinity binders were excluded from participation in this study. Eight patients with progressive MS and seven age and genetic binding status matched healthy controls underwent a 60 min dynamic PET scan using [18F]DPA714, including both continuous on-line and manual arterial blood sampling to obtain metabolite-corrected arterial plasma input functions. RESULTS: The optimal model for quantification of [18F]DPA714 kinetics was a reversible two-tissue compartment model with additional blood volume parameter. For genetic high-affinity binders, a clear increase in binding potential was observed in patients with MS compared with age-matched controls. For both high and medium affinity binders, a further increase in binding potential was observed in T2 white matter lesions compared with non-lesional white matter. Volume of distribution, however, did not differentiate patients from healthy controls, as the large non-displaceable compartment of [18F]DPA714 masks its relatively small specific signal. CONCLUSION: The TSPO radioligand [18F]DPA714 can reliably identify increased focal and diffuse neuroinflammation in progressive MS when using plasma input-derived binding potential, but observed differences were predominantly visible in high-affinity binders.

Differential Expression of Tissue Transglutaminase Splice Variants in Peripheral Blood Mononuclear Cells of Primary Progressive Multiple Sclerosis Patients.

Multiple Sclerosis (MS) is an inflammatory and neurodegenerative disorder of the central nervous system (CNS) characterized by inflammation and immune cell infiltration in the brain parenchyma. Tissue transglutaminase (TG2), a calcium-dependent cross-linking enzyme, has been shown to be present in infiltrating MHC-II positive cells in lesions of patients suffering from MS. Moreover, TG2 mRNA levels in peripheral blood mononuclear cells (PBMC)-derived from primary progressive (PP)-MS patients correlated with clinical parameters, thus highlighting the importance of TG2 in MS pathology. In the present study, we further characterized TG2 expression by measuring the mRNA levels of full-length TG2 and four TG2 alternative splice variants in PBMCs derived from PP-MS patients and healthy control (HC) subjects. In PP-MS-derived PBMCs, TG2 variant V4b was significantly higher expressed, and both V4a and V4b variants were relatively more expressed in relation to full-length TG2. These observations open new avenues to unravel the importance of TG2 alternative splicing in the pathophysiology of PP-MS.

In vivo evaluation of two tissue transglutaminase PET tracers in an orthotopic tumour xenograft model.

BACKGROUND: The protein cross-linking enzyme tissue transglutaminase (TG2; EC 2.3.2.13) is associated with the pathogenesis of various diseases, including cancer. Recently, the synthesis and initial evaluation of two high-potential radiolabelled irreversible TG2 inhibitors were reported by us. In the present study, these two compounds were evaluated further in a breast cancer (MDA-MB-231) tumour xenograft model for imaging active tissue transglutaminase in vivo. RESULTS: The metabolic stability of [11C]1 and [18F]2 in SCID mice was comparable to the previously reported stability in Wistar rats. Quantitative real-time polymerase chain reaction analysis on MDA-MB-231 cells and isolated tumours showed a high level of TG2 expression with very low expression of other transglutaminases. PET imaging showed low tumour uptake of [11C]1 (approx. 0.5 percentage of the injected dose per gram (%ID/g) at 40-60 min p.i.) and with relatively fast washout. Tumour uptake for [18F]2 was steadily increasing over time (approx. 1.7 %ID/g at 40-60 min p.i.). Pretreatment of the animals with the TG2 inhibitor ERW1041E resulted in lower tumour activity concentrations, and this inhibitory effect was enhanced using unlabelled 2. CONCLUSIONS: Whereas the TG2 targeting potential of [11C]1 in this model seems inadequate, targeting of TG2 using [18F]2 was achieved. As such, [18F]2 could be used in future studies to clarify the role of active tissue transglutaminase in disease.

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

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.

Monocyte-derived tissue transglutaminase in multiple sclerosis patients: reflecting an anti-inflammatory status and function of the cells?

BACKGROUND: Leukocyte infiltration into the central nervous system is an important feature of multiple sclerosis (MS) pathology. Among the infiltrating cells, monocytes comprise the largest population and are considered to play a dual role in the course of the disease. The enzyme tissue transglutaminase (TG2), produced by monocytes, plays a central role in monocyte adhesion/migration in animal models of MS. In the present study, we questioned whether TG2 expression is altered in monocytes from MS patients compared to healthy control (HC) subjects. Moreover, we determined the inflammatory status of these TG2-expressing monocytes, what inflammatory factor regulates TG2 expression, and whether TG2 can functionally contribute to their adhesion/migration processes. METHODS: Primary human monocytes from MS patients and HC subjects were collected, RNA isolated and subjected to qPCR analysis. Human THP-1 monocytes were lentivirally transduced with TG2 siRNA or control and treated with various cytokines. Subsequently, mRNA levels of inflammatory factors, adhesion properties, and activity of RhoA were analyzed in interleukin (IL)-4-treated monocytes. RESULTS: TG2 mRNA levels are significantly increased in monocytes derived from MS patients compared to HC subjects. In addition, correlation analyses indicated that TG2-expressing cells display a more anti-inflammatory, migratory profile in MS patients. Using THP-1 monocytes, we observed that IL-4 is a major trigger of TG2 expression in these cells. Furthermore, knockdown of TG2 expression leads to a pro-inflammatory profile and reduced adhesion/migration properties of IL-4-treated monocytes. CONCLUSIONS: TG2-expressing monocytes in MS patients have a more anti-inflammatory profile. Furthermore, TG2 mediates IL-4-induced anti-inflammatory status in THP-1 monocytes, adhesion, and cytoskeletal rearrangement in vitro. We thus propose that IL-4 upregulates TG2 expression in monocytes of MS patients, driving them into an anti-inflammatory status.

Tissue transglutaminase in astrocytes is enhanced by inflammatory mediators and is involved in the formation of fibronectin fibril-like structures.

BACKGROUND: During multiple sclerosis (MS) lesion formation, inflammatory mediators are produced by microglial cells and invading leukocytes. Subsequently, hypertrophic astrocytes fill the lesion and produce extracellular matrix (ECM) proteins that together form the astroglial scar. This is beneficial because it seals off the site of central nervous system (CNS) damage. However, astroglial scarring also forms an obstacle that inhibits remyelination of brain lesions. This is possibly an important cause for incomplete remyelination of the CNS in early stage MS patients and for failure of remyelination when the disease progresses. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme that can cross-link proteins, appears in astrocytes in inflammatory MS lesions and may contribute to the rearrangement of ECM protein deposition and aggregation. METHODS: The effect of different inflammatory mediators on TG2 and fibronectin, an ECM protein, protein levels was examined in primary rat microglia and astrocytes by western blotting. Also, TG2 activity was analyzed in primary rat astrocytes by a TG activity assay. To determine the role of TG2 in the deposition and cross-linking of fibronectin, a TG2 inhibitor and TG2 knockdown astrocytes were used. RESULTS: Our data show that under inflammatory conditions in vitro, TG2 production is enhanced in astrocytes and microglia. We observed that in particular, astrocytes produce fibronectin that can be cross-linked and aggregated by exogenous TG2. Moreover, inflammatory stimulus-induced endogenously produced TG2 is involved in the appearance of morphological fibril-like fibronectin deposits but does not lead to cross-linked fibronectin aggregates. CONCLUSIONS: Our in vitro observations suggest that during MS lesion formation, when inflammatory mediators are produced, astrocyte-derived TG2 may contribute to ECM rearrangement, and subsequent astroglial scarring.

Astrocyte-derived tissue Transglutaminase affects fibronectin deposition, but not aggregation, during cuprizone-induced demyelination.

Astrogliosis as seen in Multiple Sclerosis (MS) develops into astroglial scarring, which is beneficial because it seals off the site of central nervous system (CNS) damage. However, astroglial scarring also forms an obstacle that inhibits axon outgrowth and (re)myelination in brain lesions. This is possibly an important cause for incomplete remyelination in the CNS of early stage MS patients and for failure in remyelination when the disease progresses. In this study we address whether under demyelinating conditions in vivo, tissue Transglutaminase (TG2), a Ca2+ -dependent enzyme that catalyses posttranslational modification of proteins, contributes to extracellular matrix (ECM) deposition and/or aggregation. We used the cuprizone model for de- and remyelination. TG2 immunoreactivity and enzymatic activity time-dependently appeared in astrocytes and ECM, respectively, in the corpus callosum of cuprizone-treated mice. Enhanced presence of soluble monomeric and multimeric fibronectin was detected during demyelination, and fibronectin immunoreactivity was slightly decreased in cuprizone-treated TG2-/- mice. In vitro TG2 overexpression in astrocytes coincided with more, while knock-down of TG2 with less fibronectin production. TG2 contributes, at least partly, to fibronectin production, and may play a role in fibronectin deposition during cuprizone-induced demyelination. Our observations are of interest in understanding the functional implications of TG2 during astrogliosis.

Tissue Transglutaminase contributes to experimental multiple sclerosis pathogenesis and clinical outcome by promoting macrophage migration.

Multiple sclerosis is a serious neurological disorder, resulting in e.g., sensory, motor and cognitive deficits. A critical pathological aspect of multiple sclerosis (MS) is the influx of immunomodulatory cells into the central nervous system (CNS). Identification of key players that regulate cellular trafficking into the CNS may lead to the development of more selective treatment to halt this process. The multifunctional enzyme tissue Transglutaminase (TG2) can participate in various inflammation-related processes, and is known to be expressed in the CNS. In the present study, we question whether TG2 activity contributes to the pathogenesis of experimental MS, and could be a novel therapeutic target. In human post-mortem material, we showed the appearance of TG2 immunoreactivity in leukocytes in MS lesions, and particular in macrophages in rat chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE), an experimental MS model. Clinical deficits as observed in mouse EAE were reduced in TG2 knock-out mice compared to littermate wild-type mice, supporting a role of TG2 in EAE pathogenesis. To establish if the enzyme TG2 represents an attractive therapeutic target, cr-EAE rats were treated with TG2 activity inhibitors during ongoing disease. Reduction of TG2 activity in cr-EAE animals dramatically attenuated clinical deficits and demyelination. The mechanism underlying these beneficial effects pointed toward a reduction in macrophage migration into the CNS due to attenuated cytoskeletal flexibility and RhoA GTPase activity. Moreover, iNOS and TNFα levels were selectively reduced in the CNS of cr-EAE rats treated with a TG2 activity inhibitor, whereas other relevant inflammatory mediators were not affected in CNS or spleen by reducing TG2 activity. We conclude that modulating TG2 activity opens new avenues for therapeutic intervention in MS which does not affect peripheral levels of inflammatory mediators.

Brain region-specific gene expression profiles in freshly isolated rat microglia.

Microglia are important cells in the brain that can acquire different morphological and functional phenotypes dependent on the local situation they encounter. Knowledge on the region-specific gene signature of microglia may hold valuable clues for microglial functioning in health and disease, e.g., Parkinson's disease (PD) in which microglial phenotypes differ between affected brain regions. Therefore, we here investigated whether regional differences exist in gene expression profiles of microglia that are isolated from healthy rat brain regions relevant for PD. We used an optimized isolation protocol based on a rapid isolation of microglia from discrete rat gray matter regions using density gradients and fluorescent-activated cell sorting. Application of the present protocol followed by gene expression analysis enabled us to identify subtle differences in region-specific microglial expression profiles and show that the genetic profile of microglia already differs between different brain regions when studied under control conditions. As such, these novel findings imply that brain region-specific microglial gene expression profiles exist that may contribute to the region-specific differences in microglia responsivity during disease conditions, such as seen in, e.g., PD.

Discrepancy in CCL2 and CCR2 expression in white versus grey matter hippocampal lesions of Multiple Sclerosis patients.

A remarkable pathological difference between grey matter lesions (GML) and white matter lesions (WML) in Multiple Sclerosis (MS) patients is the paucity of infiltrating leukocytes in GML. To better understand these pathological differences, we hypothesize that the chemokine monocyte chemotactic protein-1 (MCP-1 or CCL2), of importance for leukocyte migration, and its receptor CCR2 are more abundantly expressed in WML than in GML of MS patients. To this end, we analyzed CCL2 and CCR2 expression in the hippocampus, comprising WML and GML,of post-mortem MS patients, and of control subjects. CCL2 and CCR2 mRNA were significantly increased in demyelinated MS hippocampus. Semi-quantification of CCL2 and CCR2 immunoreactivity showed that CCL2 is present in astrocytes only in active WML. CCR2 is upregulated in monocytes/macrophages or amoeboid microglia in active WML, and in ramified microglia in active GML, although to a lesser extent. As a follow-up, we observed a significantly increased CCL2 production by WM-, but not GM-derived astrocytes upon stimulation with bz-ATP in vitro. Finally, upon CCL2 stimulation, GM-derived microglia significantly increased their proliferation rate. We conclude that within hippocampal lesions, CCL2 expression is mainly restricted to WML, whereas the receptor CCR2 is upregulated in both WML and GML. The relative absence of CCL2 in GML may explain the lack of infiltrating immune cells in this type of lesions. We propose that the divergent expression of CCL2 and CCR2 in WML and GML explains or contributes to the differences in WML and GML formation in MS.