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Background: Neuroinflammation and Alzheimer's disease (AD) co-pathology may contribute to disease progression and severity in dementia with Lewy bodies (DLB). This study aims to clarify whether a different pattern of neuroinflammation, such as alteration in microglial and astroglial morphology and distribution, is present in DLB cases with and without AD co-pathology. Methods: The morphology and load (% area of immunopositivity) of total (Iba1) and reactive microglia (CD68 and HLA-DR), reactive astrocytes (GFAP) and proteinopathies of alpha-synuclein (KM51/pser129), amyloid-beta (6 F/3D) and p-tau (AT8) were assessed in a cohort of mixed DLB + AD (n = 35), pure DLB (n = 15), pure AD (n = 16) and control (n = 11) donors in limbic and neocortical brain regions using immunostaining, quantitative image analysis and confocal microscopy. Regional and group differences were estimated using a linear mixed model analysis. Results: Morphologically, reactive and amoeboid microglia were common in mixed DLB + AD, while homeostatic microglia with a small soma and thin processes were observed in pure DLB cases. A higher density of swollen astrocytes was observed in pure AD cases, but not in mixed DLB + AD or pure DLB cases. Mixed DLB + AD had higher CD68-loads in the amygdala and parahippocampal gyrus than pure DLB cases, but did not differ in astrocytic loads. Pure AD showed higher Iba1-loads in the CA1 and CA2, higher CD68-loads in the CA2 and subiculum, and a higher astrocytic load in the CA1-4 and subiculum than mixed DLB + AD cases. In mixed DLB + AD cases, microglial load associated strongly with amyloid-beta (Iba1, CD68 and HLA-DR), and p-tau (CD68 and HLA-DR), and minimally with alpha-synuclein load (CD68). In addition, the highest microglial activity was found in the amygdala and CA2, and astroglial load in the CA4. Confocal microscopy demonstrated co-localization of large amoeboid microglia with neuritic and classic-cored plaques of amyloid-beta and p-tau in mixed DLB + AD cases. Conclusions: In conclusion, microglial activation in DLB was largely associated with AD co-pathology, while astrocytic response in DLB was not. In addition, microglial activity was high in limbic regions, with prevalent AD pathology. Our study provides novel insights into the molecular neuropathology of DLB, highlighting the importance of microglial activation in mixed DLB + AD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Degeneration of the locus coeruleus (LC) noradrenergic system contributes to clinical symptoms in Alzheimer's disease (AD) and Parkinson's disease (PD). Diffusion magnetic resonance imaging (MRI) has the potential to evaluate the integrity of the LC noradrenergic system. The aim of the current study was to determine whether the diffusion MRI-measured integrity of the LC and its tracts are sensitive to noradrenergic degeneration in AD and PD. Methods: Post-mortem in situ T1-weighted and multi-shell diffusion MRI was performed for 9 AD, 14 PD, and 8 control brain donors. Fractional anisotropy (FA) and mean diffusivity were derived from the LC, and from tracts between the LC and the anterior cingulate cortex, the dorsolateral prefrontal cortex (DLPFC), the primary motor cortex (M1) or the hippocampus. Brain tissue sections of the LC and cortical regions were obtained and immunostained for dopamine-beta hydroxylase (DBH) to quantify noradrenergic cell density and fiber load. Group comparisons and correlations between outcome measures were performed using linear regression and partial correlations. Results: The AD and PD cases showed loss of LC noradrenergic cells and fibers. In the cortex, the AD cases showed increased DBH + immunoreactivity in the DLPFC compared to PD cases and controls, while PD cases showed reduced DBH + immunoreactivity in the M1 compared to controls. Higher FA within the LC was found for AD, which was correlated with loss of noradrenergic cells and fibers in the LC. Increased FA of the LC-DLPFC tract was correlated with LC noradrenergic fiber loss in the combined AD and control group, whereas the increased FA of the LC-M1 tract was correlated with LC noradrenergic neuronal loss in the combined PD and control group. The tract alterations were not correlated with cortical DBH + immunoreactivity. Conclusions: In AD and PD, the diffusion MRI-detected alterations within the LC and its tracts to the DLPFC and the M1 were associated with local noradrenergic neuronal loss within the LC, rather than noradrenergic changes in the cortex. [ABSTRACT FROM AUTHOR]

Abstract: Accumulation of amyloid-beta (Aβ) in brain vessel walls and parenchyma, known as cerebral amyloid angiopathy (CAA) and senile plaques (SPs), respectively, plays a key role in Alzheimer''s disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) pathogenesis. Although the mechanisms underlying CAA and SP formation remain largely unknown, evidence is mounting that local alterations of the extracellular matrix (ECM) in the brain vessel wall and/or parenchyma play an important role. Lysyl oxidase (LOX, E.C. 1.4.3.13) is an inducible amine oxidase that modulates the ECM by catalyzing the formation of molecular covalent cross-links in ECM proteins. The aim of this study is to investigate the association of LOX with CAA and with classic and diffuse SPs in both AD and HCHWA-D cases. We observed an association of LOX with Aβ in CAA and with Aβ in both classic and diffuse SPs in AD and HCHWA-D cases. In addition, LOX staining was observed in reactive astrocytes associated with these lesions. We conclude that the ECM modulating enzyme LOX is associated with the Aβ-related pathological hallmarks of both AD and HCHWA-D, and that our findings provide additional insights into the mechanisms underlying the formation of these lesions. [Copyright &y& Elsevier]

Background: Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR). The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA). An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 gene. Also AVRPPHB SUSCEPTIBLE 3 (PBS3) plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of ICS1 and PBS3. Results: Expression studies with ICS1 promoter::β-glucuronidase (GUS) genes in Arabidopsis thaliana protoplasts cotransfected with 35S::WRKY28 showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA. Conclusions: The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of ICS1 and PBS3, respectively, and support this in silico screening as a powerful tool for identifying new components of stress signaling pathways. [ABSTRACT FROM AUTHOR]

Background: In order to comprehend the mechanisms of induced plant defense, knowledge of the biosynthesis and signaling pathways mediated by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) is essential. Potentially, many transcription factors could be involved in the regulation of these pathways, although finding them is a difficult endeavor. Here we report the use of publicly available Arabidopsis microarray datasets to generate gene co-expression networks. Results: Using 372 publicly available microarray data sets, a network was constructed in which Arabidopsis genes for known components of SA, JA and ET pathways together with the genes of over 1400 transcription factors were assayed for co-expression. After determining the Pearson Correlation Coefficient cutoff to obtain the most probable biologically relevant co-expressed genes, the resulting network confirmed the presence of many genes previously reported in literature to be relevant for stress responses and connections that fit current models of stress gene regulation, indicating the potential of our approach. In addition, the derived network suggested new candidate genes and associations that are potentially interesting for future research to further unravel their involvement in responses to stress. Conclusions: In this study large sets of stress related microarrays were used to reveal co-expression networks of transcription factors and signaling pathway components. These networks will benefit further characterization of the signal transduction pathways involved in plant defense. [ABSTRACT FROM AUTHOR]

Abstract: Parkin is implicated in the pathogenesis of Parkinson''s disease. Furthermore, parkin targets misfolded proteins for degradation and protects cells against various forms of cellular stress, including unfolded-protein and oxidative stress. This points towards a protective role of parkin in neurological disorders in which these stressors are implicated, including Alzheimer''s disease (AD) and multiple sclerosis (MS). Here, we assessed parkin distribution in AD and MS brain tissue using immunohistochemistry. In AD brains, parkin colocalized with classic senile plaques and amyloid-laden vessels as well as astrocytes associated with both lesions. Similarly, we observed enhanced astrocytic parkin immunoreactivity in MS lesions, particularly in inflammatory lesions. Furthermore, parkin mRNA expression was increased in an astrocytoma cell line after free radical exposure. Our data indicate that parkin is upregulated in AD and MS brain tissue and might represent a defense mechanism to counteract stress-induced damage in AD and MS pathogenesis. [Copyright &y& Elsevier]

In the family Bromoviridae, a mixture of the three genomic RNAs of bromo-, cucumo-, and oleaviruses is infectious as such, whereas the RNAs of alfamo-and ilarviruses require binding of a few molecules of coat protein (CP) to the 3′ end to initiate infection. Most studies on the early function of CP have been done on the alfamovirus Alfalfa mosaic virus (AMV). The 3′ 112 nucleotides of AMV RNAs can adopt two different conformations. One conformer consists of a tRNA-like structure that, together with an upstream hairpin, is required for minus-strand promoter activity. The other conformer consists of four hairpins interspersed by AUGC-sequences and represents a strong binding site for CP. Binding of CP to this conformer enhances the translational efficiency of viral RNAs in vivo 40-fold and blocks viral minus-strand RNA synthesis in vitro. AMV CP is proposed to initiate infection by mimicking the function of the poly(A)-binding protein. [ABSTRACT FROM AUTHOR]

A pseudotriloop is formed by transloop base pairing between the first (5') and the fifth nucleotide in a hexanucleotide RNA loop ('hexaloop") to subtend a triloop of nucleotides 2-4. This structure has been found in hairpins involved in the regulation of iron metabolism in mammalian cells and in transcription of plant virus subgenomic RNA. Several hexaloop hairpins, including HIV-transactivation-responsive element and hepatitis B virus ∊, potentially adopt a pseudotriloop conformation. Here we show that an RNA plant virus whose replication depends on a conventional triloop hairpin can be used to verify the existence of pseudotriloop structures in vivo. Our data suggest that the pseudotriloop may represent a common motif in RNA-protein recognition. [ABSTRACT FROM AUTHOR]

Focuses on untranslated regions (UTR) in genomic RNAs of the alfalfa mosaic virus (AMV). Yield of infectious replicons by replacement of the UTR RNA; Sequence of a stem-loop structure required for negative strand RNA synthesis; Replication of the AMV RNA.

SUMMARY: Taxonomy: Alfalfa mosaic virus (AMV) is the type species of the genus Alfamovirus and belongs to the family Bromoviridae. In this family, the tripartite RNA genomes of bromo-, cucumo- and probably oleaviruses are infectious as such, whereas infection with the three genomic RNAs of alfamo- and ilarviruses requires addition to the inoculum of a few molecules of coat protein (CP) per RNA molecule. RNAs 1 and 2 encode the replicase proteins P1 and P2, RNA 3 encodes the movement protein and CP. CP is translated from the subgenomic RNA 4.. Physical properties: RNAs 1 (3.65 kb), 2 (2.6 kb) and 3 (2.2 kb) are separately encapsidated into bacilliform particles which are 19 nm wide and 35–56 nm long. In addition, the virus preparations contain spheroidal particles each containing two copies of RNA 4 (0.88 kb). Virus particles contain 16–17% RNA and are mainly stabilized by protein–RNA interactions. The 3′-termini of the viral RNAs contain a homologous sequence of 145 nucleotides that can adopt two alternative conformations: one represents a high-affinity binding site for CP, the other resembles a tRNA-like structure and is required for minus-strand promoter activity. Hosts: AMV mostly infects herbaceous plants, but several woody species are included in the natural host range. The experimental and natural host ranges include over 600 species in 70 families. At least 15 aphid species are known to transmit the virus in the stylet-borne or non-persistent manner. Economic importance: AMV is a significant pathogen in alfalfa and sweet clover and can spread from these forages to neighbouring crops like pepper, tobacco or soybean. The recent introduction of the soybean aphid (Aphis glycines) in the mid-west states of the USA has increased the incidence of AMV in soybean. AMV occurs world-wide in potato and is referred to as ‘calico mosaic’ because of its characteristic symptoms on the foliage. However, the economic importance of... [ABSTRACT FROM AUTHOR]

Although various neurodegenerative disorders have been associated with coeliac disease (CD), the underlying neuropathological link between these brain and gut diseases remains unclear. We postulated that the neuronal damage sporadically observed in CD patients is immune-mediated. Our aim was to determine if the loss of neurons, especially Purkinje cells, coincides with microglia activation and T- and B-cell infiltration in the cerebellum of patients with CD and a concomitant idiopathic neurological disease affecting the cerebellum (NeuroCD). Post-mortem cerebellar tissue was collected of validated NeuroCD cases. Gender- and age-matched genetic spinocerebellar ataxia (SCA) controls and non-neurological controls (NNC) were selected based on clinical reports and pathological findings. Cerebellar tissue of seventeen patients was included (6 NeuroCD, 5 SCA, 6 NNC). In SCA cases we found that the Purkinje cell layer was 58.6% reduced in comparison with NNC. In NeuroCD cases this reduction was even more prominent with a median reduction of 81.3% compared to NNC. Marked increased numbers of both CD3+ and CD8+ cells were observed in the NeuroCD but not in SCA patients. This coincided with significantly more microglial reactivity in NeuroCD patients. These findings demonstrate that the massive loss of Purkinje cells in the cerebellum of neuro CD patients is accompanied by local innate and T-cell mediated immune responses. [ABSTRACT FROM AUTHOR]

Multiple system atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy. [ABSTRACT FROM AUTHOR]

Aims: Although the neuroanatomical distribution of tau and amyloid‐β is well studied in Alzheimer's disease (AD) (non)‐amnestic clinical variants, that of neuroinflammation remains unexplored. We investigate the neuroanatomical distribution of activated myeloid cells, astrocytes, and complement alongside amyloid‐β and phosphorylated tau in a clinically well‐defined prospectively collected AD cohort. Methods: Clinical variants were diagnosed antemortem, and brain tissue was collected post‐mortem. Typical AD (n = 10), behavioural/dysexecutive AD (n = 6), posterior cortical atrophy (PCA) AD (n = 3), and controls (n = 10) were neuropathologically assessed for AD neuropathology, concurrent pathology including Lewy body disease, limbic‐predominant age‐related TDP‐43 encephalopathy neuropathologic change (LATE‐NC), and vascular pathology. For quantitative assessment, we analysed the corticolimbic distribution of phosphorylated tau, amyloid‐β, CD68, MHC‐II, C4b, and glial fibrillary acidic protein (GFAP) using digital pathology. Results: Phosphorylated tau was distinctly distributed in each variant. In all variants, amyloid‐β was neocortical‐dominant, with a notable increase in the middle frontal cortex of behavioural/dysexecutive AD. Typical AD and PCA AD had no concurrent Lewy body disease, whereas three out of six cases with behavioural/dysexecutive AD did. LATE‐NC stage >0 was observed in three AD cases, two typical AD (stage 1/3), and one behavioural/dysexecutive AD (stage 2/3). Vascular pathology was present in each variant. In typical AD, CD68 and MHC‐II were hippocampal‐dominant. In behavioural/dysexecutive AD, C4b was elevated in the middle frontal and inferior parietal cortex. In PCA AD, MHC‐II was increased in the fusiform gyrus, and GFAP in parietal cortices. Correlations between AD neuropathology and neuroinflammation were distinct within variants. Conclusions: Our data suggests that different involvement of neuroinflammation may add to clinical heterogeneity in AD, which has implications for neuroinflammation‐based biomarkers and future therapeutics. [ABSTRACT FROM AUTHOR]

Background: Axons, crucial for impulse transmission and cellular trafficking, are thought to be primary targets of neurodegeneration in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Axonal degeneration occurs early, preceeding and exceeding neuronal loss, and contributes to the spread of pathology, yet is poorly described outside the nigrostriatal circuitry. The insula, a cortical brain hub, was recently discovered to be highly vulnerable to pathology and plays a role in cognitive deficits in PD and DLB. The aim of this study was to evaluate morphological features as well as burden of proteinopathy and axonal degeneration in the anterior insular sub-regions in PD, PD with dementia (PDD), and DLB. Methods: α-Synuclein, phosphorylated (p-)tau, and amyloid-β pathology load were evaluated in the anterior insular (agranular and dysgranular) subregions of post-mortem human brains (n = 27). Axonal loss was evaluated using modified Bielschowsky silver staining and quantified using stereology. Cytoskeletal damage was comprehensively studied using immunofluorescent multi-labelling and 3D confocal laser-scanning microscopy. Results: Compared to PD and PDD, DLB showed significantly higher α-synuclein and p-tau pathology load, argyrophilic grains, and more severe axonal loss, particularly in the anterior agranular insula. Alternatively, the dysgranular insula showed a significantly higher load of amyloid-β pathology and its axonal density correlated with cognitive performance. p-Tau contributed most to axonal loss in the DLB group, was highest in the anterior agranular insula and significantly correlated with CDR global scores for dementia. Neurofilament and myelin showed degenerative changes including swellings, demyelination, and detachment of the axon-myelin unit. Conclusions: Our results highlight the selective vulnerability of the anterior insular sub-regions to various converging pathologies, leading to impaired axonal integrity in PD, PDD and DLB, disrupting their functional properties and potentially contributing to cognitive, emotional, and autonomic deficits. [ABSTRACT FROM AUTHOR]

Parkinson's Disease is the second most common neurodegenerative disease and is characterized by neurodegeneration in various areas of the brain, most notably in the substantia nigra. Neuroinflammation and, more specifically, the activation of microglia in the substantia nigra is suggested to play an important role in the pathological processes of Parkinson's Disease. Upon activation, microglia may adopt a stimulus-dependent phenotype that can be pro-inflammatory or anti-inflammatory. To gain more insight into the actual inflammatory status of affected brain regions in Parkinson's Disease it is, therefore, important to identify the different microglial phenotypes present. The aim of this study was to investigate the distribution of different microglia phenotypes present in post-mortem substantia nigra tissue of control, Incidental Lewy Body Disease and Parkinson's Disease cases using semi-quantitative immunohistochemical analysis of microglia phenotype-specific markers. Our results show an increase in the number of anti-CD68 and anti-class II human leukocyte antigen antibody immunoreactive amoeboid microglia in the substantia nigra of Parkinson's Disease cases compared to Incidental Lewy Body Disease and control cases, while no differences were found in the number of anti-ionized calciumbinding adaptor molecule 1- and class II human leukocyte antigen antibody immunoreactive microglia. In addition, no differences were found in the number of anti-transmembrane protein 119- and anti-Purinergic Receptor P2Y12 antibody immunoreactive microglia, suggesting that the microglia phenotype present in the SN remains relatively constant between the control, incidental Lewy Body Disease and Parkinson's Disease cases. Together, our results suggest a subtle switch of part of the microglia population in the substantia nigra of Parkinson's Disease cases to a specific morphologic phenotype indicative of enhanced lysosomal activity. Although usually interpreted as expression of a pro-inflammatory condition, further characterization of the functional status of this subtype of amoeboid microglia may provide better insight into the role of neuroinflammation in PD pathogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Motor and cognitive impairment in Parkinson's disease (PD) is associated with dopaminergic dysfunction that stems from substantia nigra (SN) degeneration and concomitant α‐synuclein accumulation. Diffusion magnetic resonance imaging (MRI) can detect microstructural alterations of the SN and its tracts to (sub)cortical regions, but their pathological sensitivity is still poorly understood. Objective: To unravel the pathological substrate(s) underlying microstructural alterations of SN, and its tracts to the dorsal striatum and dorsolateral prefrontal cortex (DLPFC) in PD. Methods: Combining post‐mortem in situ MRI and histopathology, T1‐weighted and diffusion MRI, and neuropathological samples of nine PD, six PD with dementia (PDD), five dementia with Lewy bodies (DLB), and 10 control donors were collected. From diffusion MRI, mean diffusivity (MD) and fractional anisotropy (FA) were derived from the SN, and tracts between the SN and caudate nucleus, putamen, and DLPFC. Phosphorylated‐Ser129‐α‐synuclein and tyrosine hydroxylase immunohistochemistry was included to quantify nigral Lewy pathology and dopaminergic degeneration, respectively. Results: Compared to controls, PD and PDD/DLB showed increased MD of the SN and SN‐DLPFC tract, as well as increased FA of the SN‐caudate nucleus tract. Both PD and PDD/DLB showed nigral Lewy pathology and dopaminergic loss compared to controls. Increased MD of the SN and FA of SN‐caudate nucleus tract were associated with SN dopaminergic loss. Whereas increased MD of the SN‐DLPFC tract was associated with increased SN Lewy neurite load. Conclusions: In PD and PDD/DLB, diffusion MRI captures microstructural alterations of the SN and tracts to the dorsal striatum and DLPFC, which differentially associates with SN dopaminergic degeneration and Lewy neurite pathology. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Growing evidence suggests a crucial role of neuroinflammation in the pathophysiology of Parkinson's disease (PD). Neuroinflammation is linked to the accumulation and aggregation of a-synuclein (αSyn), the primary pathological hallmark of PD. Toll-like receptors 4 (TLR4) can have implications in the development and progression of the pathology. In this study, we analyzed the expression of TLR4 in the substantia nigra (SN) and medial temporal gyrus (GTM) of well-characterized PD patients and age-matched controls. We also assessed the co-localization of TLR4 with pSer129 αSyn. Using qPCR, we observed an upregulation of TLR4 expression in the SN and GTM in PD patients compared to controls, which was accompanied by a reduction in αSyn expression likely due to the depletion of dopaminergic (DA) cells. Additionally, using immunofluorescence and confocal microscopy, we observed TLR4-positive staining and co-localization with pSer129-αSyn in Lewy bodies of DA neurons in the SN, as well as in pyramidal neurons in the GTM of PD donors. Furthermore, we observed a co-localization of TLR4 and Iba-1 in glial cells of both SN and GTM. Our findings provide evidence for the increased expression of TLR4 in the PD brain and suggest that the interaction between TLR4 and pSer129-αSyn could play a role in mediating the neuroinflammatory response in PD. [ABSTRACT FROM AUTHOR]

Background: Increased neurofilament levels in biofluids are commonly used as a proxy for neurodegeneration in several neurodegenerative disorders. In this study, we aimed to investigate the distribution of neurofilaments in the cerebral cortex of Parkinson's disease (PD), PD with dementia (PDD) and dementia with Lewy bodies (DLB) donors, and its association with pathology load and MRI measures of atrophy and diffusivity. Methods: Using a within-subject post-mortem MRI-pathology approach, we included 9 PD, 12 PDD/DLB and 18 age-matched control donors. Cortical thickness and mean diffusivity (MD) metrics were extracted respectively from 3DT1 and DTI at 3T in-situ MRI. After autopsy, pathological hallmarks (pSer129-αSyn, p-tau and amyloid-β load) together with neurofilament light-chain (NfL) and phosphorylated-neurofilament medium- and heavy-chain (p-NfM/H) immunoreactivity were quantified in seven cortical regions, and studied in detail with confocal-laser scanning microscopy. The correlations between MRI and pathological measures were studied using linear mixed models. Results: Compared to controls, p-NfM/H immunoreactivity was increased in all cortical regions in PD and PDD/DLB, whereas NfL immunoreactivity was increased in the parahippocampal and entorhinal cortex in PDD/DLB. NfL-positive neurons showed degenerative morphological features and axonal fragmentation. The increased p-NfM/H correlated with p-tau load, and NfL correlated with pSer129-αSyn but more strongly with p-tau load in PDD/DLB. Lastly, neurofilament immunoreactivity correlated with cortical thinning in PD and with increased cortical MD in PDD/DLB. Conclusions: Taken together, increased neurofilament immunoreactivity suggests underlying axonal injury and neurofilament accumulation in morphologically altered neurons with increased pathological burden. Importantly, we demonstrate that such neurofilament markers at least partly explain MRI measures that are associated with the neurodegenerative process. [ABSTRACT FROM AUTHOR]

Background: Synaptic and neuronal loss contribute to network dysfunction and disability in multiple sclerosis (MS). However, it is unknown whether excitatory or inhibitory synapses and neurons are more vulnerable and how their losses impact network functioning. Objective: To quantify excitatory and inhibitory synapses and neurons and to investigate how synaptic loss affects network functioning through computational modeling. Methods: Using immunofluorescent staining and confocal microscopy, densities of glutamatergic and GABAergic synapses and neurons were compared between post-mortem MS and non-neurological control cases. Then, a corticothalamic biophysical model was employed to study how MS-induced excitatory and inhibitory synaptic loss affect network functioning. Results: In layer VI of normal-appearing MS cortex, excitatory and inhibitory synaptic densities were significantly lower than controls (reductions up to 14.9%), but demyelinated cortex showed larger losses of inhibitory synapses (29%). In our computational model, reducing inhibitory synapses impacted the network most, leading to a disinhibitory increase in neuronal activity and connectivity. Conclusion: In MS, excitatory and inhibitory synaptic losses were observed, predominantly for inhibitory synapses in demyelinated cortex. Inhibitory synaptic loss affected network functioning most, leading to increased neuronal activity and connectivity. As network disinhibition relates to cognitive impairment, inhibitory synaptic loss seems particularly relevant in MS. [ABSTRACT FROM AUTHOR]

Aims: Alzheimer's disease (AD) is characterised by amyloid‐beta (Aβ) aggregates in the brain. Targeting Aβ 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 Aβ multimers. The enzyme tissue transglutaminase (TG2) is abundantly expressed in the human brain and plays a key role in post‐translational modifications in Aβ resulting in covalently cross‐linked, stable and neurotoxic Aβ 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 Aβ‐related pathology. Methods: Here, we compared the effects on Aβ 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 Aβ 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 Aβ1–40, Aβ1–42 and/or Aβ40/42 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 Aβ deposits and the presence of reactive astrocytes associated with Aβ deposits were analysed. Conclusions: We found that absence of TG2 reduces the formation of Aβ pathology in the APP23 mouse model, suggesting that TG2 may be a suitable therapeutic target for reducing Aβ deposition in AD. [ABSTRACT FROM AUTHOR]

Abstract: Tissue transglutaminase (tTG) is a cross-linking enzyme involved in protein aggregation during Parkinson’s disease (PD) pathogenesis. Autophagy is inhibited by tTG activation via a mechanism in which cross-linking of beclin 1, an autophagy initiator at the level of the endoplasmic reticulum (ER), has been implicated. We reported increased tTG protein levels and activity at the ER in both PD brain and in a PD-mimicking cell system. Here we characterized the interaction between tTG and beclin 1 at the ER membrane and the role of tTG in reduced autophagy in an in vitro model of PD, using differentiated SH-SY5Y neurons treated with the PD-mimic MPP+. We found that under PD-mimicking conditions, beclin 1 and tTG partially colocalized at the ER, beclin 1 levels increased at the ER, and tTG readily cross-linked beclin 1 which was prevented by enzymatic blockade of tTG. Under these conditions, accumulation of beclin 1 at the ER was enhanced by inhibition of tTG activity. In line with these observations and the role of beclin 1 in autophagy, levels of the autophagy marker protein LC3II in MPP+-treated cells, were significantly increased by inhibition of tTG activity. Our data provide first evidence for a role of tTG-mediated regulation of beclin 1 and autophagy in MPP+-treated human SH-SY5Y cells. [Copyright &y& Elsevier]

Abstract: Parkinson''s disease (PD) is characterized by accumulation of α-synuclein aggregates and degeneration of melanized, catecholaminergic neurons. The tissue transglutaminase (tTG) enzyme catalyzes molecular protein cross-linking. In PD, tTG levels are increased and cross-linking has been identified as an important factor in α-synuclein aggregation. In our quest to link tTGs distribution in the human brain to the hallmarks of PD pathology, we recently reported that catecholaminergic neurons in PD disease-affected brain areas display typical endoplasmic reticulum (ER) granules showing tTG immunoreactivity. In the present study, we set out to elucidate the nature of the interaction between tTG and the ER in PD pathogenesis, using retinoic-acid differentiated SH-SY5Y cells exposed to the PD-mimetic 1-methyl-4-phenylpyridinium (MPP+). Alike our observations in PD brain, MPP+-treated cells displayed typical TG-positive granules, that were also induced by other PD mimetics and by ER-stress inducing toxins. Additional immunocytochemical and biochemical investigation revealed that tTG is indeed associated to the ER, in particular at the cytoplasmic face of the ER. Upon MPP+ exposure, additional recruitment of tTG toward the ER was found. In addition, we observed that MPP+-induced tTG activity results in transamidation of ER membrane proteins, like calnexin. Our data provide strong evidence for a, so far unrecognized, localization of tTG at the ER, at least in catecholaminergic neurons, and suggests that in PD activation of tTG may have a direct impact on ER function, in particular via post-translational modification of ER membrane proteins. [Copyright &y& Elsevier]

Abstract: Corpora amylacea (CA) are both age and neurodegeneration-related spherical bodies, consisting of polymerized proteins, often thought to be involved in sequestration of hazardous products of cellular metabolism in brain. Although CA formation is associated with cellular stress, the process underlying their formation remains obscure. Transglutaminases (TGs) are stress associated enzymes that induce molecular cross-links, leading to polymerization of substrate proteins. TG expression and activity are elevated in Alzheimer''s disease (AD) and Parkinson''s disease (PD), and TG-catalyzed cross-links are present in their lesions. Considering the nature of CA, the aim of this study was to investigate the presence of TGs and TG cross-links in CA of healthy aging brain, AD and PD brain, using immunohistochemistry. We observed TG1 and TG cross-links in CA, together with typical cytoskeletal proteins. Furthermore, the presence of proteins associated with AD or PD pathogenesis was not altered in CA of disease brain compared to controls. We propose that TG1-catalyzed cross-linking and consequent polymerization of cytoskeletal and cytoskeleton-associated proteins may underlie CA formation. [Copyright &y& Elsevier]

Multiple Sclerosis (MS) is a neuroinflammatory disease mainly affecting young adults. A major pathological hallmark of MS is the presence of demyelinated lesions in the central nervous system. In the active phase of the disease, astrocytes become activated, migrate and contribute to local tissue remodeling that ultimately can result in an astroglial scar. This process is facilitated by extracellular matrix proteins, including fibronectin. Tissue Transglutaminase (TG2) is a multifunctional enzyme with a ubiquitous tissue distribution and it has been shown that inflammatory cytokines can induce TG2 activity. In addition, TG2 is known to mediate cell adhesion and migration. We therefore hypothesized that TG2 is present in MS lesions and plays a role in cell adhesion and/or migration. Our studies showed that TG2 immunoreactivity appeared in astrocytes in active and chronic active MS lesions. These TG2 positive astrocytes partly co-localized with fibronectin. Additional in vitro studies showed that TG2 mediated astrocytoma adhesion to and migration on the extracellular matrix protein fibronectin. We therefore speculate that TG2 mediates the enhanced interaction of astrocytes with fibronectin in the extracellular matrix of MS lesions, thereby contributing to astrocyte adhesion and migration, and thus in tissue remodeling and possibly glial scarring. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by pathological lesions, in particular senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of self-aggregated proteins amyloid beta (Aβ) and tau, respectively. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular covalent cross-links. Both Aβ and tau are substrates for TG cross-linking activity, which links TGs to the aggregation process of both proteins in AD brain. The aim of this study was to investigate the association of transglutaminase 1 (TG1), transglutaminase 2 (TG2) and TG-catalyzed cross-links with the pathological lesions of AD using immunohistochemistry. We observed immunoreactivity for TG1, TG2 and TG-catalyzed cross-links in NFTs. In addition, both TG2 and TG-catalyzed cross-links colocalized with Aβ in SPs. Furthermore, both TG2 and TG-catalyzed cross-links were associated with CAA. We conclude that these TGs demonstrate cross-linking activity in AD lesions, which suggests that both TG1 and TG2 are likely involved in the protein aggregation processes underlying the formation of SPs, CAA and/or NFTs in AD brain. [ABSTRACT FROM AUTHOR]

Abstract: Neuroinflammation may play a role in the pathogenesis of Parkinson’s disease (PD). The present study questioned whether this neuroinflammatory response differs between the olfactory bulb, as an early affected region and the nigrostriatal system. Indeed, increased microgliosis was shown in post-mortem olfactory bulb of PD patients. Also in olfactory bulb of MPTP-treated mice, microgliosis and increased expression of IL-1α, IL-1β and IL-1ra mRNA was observed early after treatment. These observations implicate that neuroinflammation is not restricted to the nigrostriatal system. MPTP-induced microgliosis in striatum and olfactory bulb was reduced in IL-1α/β knockout mice, indicating that IL-1 affects microglia activation. Importantly, MPTP induced differential regulation of IL-1 receptors. mRNA levels of IL-1RI and, to a lesser extent, IL-1RII were increased in striatum. Interestingly, in the olfactory bulb only IL-1RII mRNA was enhanced. We suggest that differential regulation of IL-1 signaling can serve as an important mechanism to modulate neuroinflammatory activity after MPTP treatment and possibly during PD. [Copyright &y& Elsevier]

Abstract: Phylloquinone (Vitamin K1) is an essential component of the photosynthetic electron transfer. As isochorismate is required for the biosynthesis of Vitamin K1, isochorismate synthase (ICS) activity is expected to be present in all green plants. In bacteria salicylic acid (SA) is synthesized via a two step pathway involving ICS and isochorismate pyruvate lyase (IPL). The effect of the introduction in tobacco plants of the bacterial ICS and IPL genes on the endogenous isochorismate pathway was investigated. Transgenic tobacco plants in which IPL was targeted to the chloroplast suffered severe growth retardation and had low Vitamin K1 content. Probably because isochorismate was channeled towards SA production, the plants were no longer able to produce normal levels of Vitamin K1. Transgenic tobacco plants in which the bacterial ICS was present in the chloroplast showed higher Vitamin K1 contents than wild type plants. [Copyright &y& Elsevier]

Abstract: Rotenone has been reported to induce various degrees of Parkinsonism in rats. We tested whether advancing age alters the sensitivity of dopaminergic neurons to rotenone. A low, systemic dose of rotenone had no effect on young rats, but led to a 20–30% reduction of tyrosine hydroxylase-positive neurons in the substantia nigra of older rats. The effect was specific to nigral dopaminergic neurons and may be associated with the increase of glial cell activation in older rats. These data suggest that age enhances the sensitivity of dopaminergic neurons to rotenone and should be considered when assessing models of Parkinson''s disease. [Copyright &y& Elsevier]

Abstract: The in vivo analysis of dopaminergic degeneration in animal models of Parkinson''s disease (PD), using pinhole single photon emission computed tomography (SPECT), ideally should afford a serial study design, enabling the analysis of the degenerative process as well as the potential neuroprotective and/or restorative properties of drugs over time in living animals. Previously, we demonstrated that striatal dopamine transporter (DAT) levels in rats could be analyzed reproducibly, using pinhole SPECT with the DAT probe [123I]N-ω-fluoropropyl-2β-carbomethoxy-3β-{4-iodophenyl}nortropane (FP-CIT). However, the capacity of this approach to accurately detect a range of striatal DAT levels in the most widely used animal model of PD, i.e., the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse, remains to be determined. For this purpose, various levels of DAT were induced by treating c57BL/6J mice for 1, 3, or 5 days with MPTP (25 mg/kg ip), respectively. [123I]FP-CIT SPECT scans were performed 5 days after the last MPTP injection. Mice were perfused 6 days after the last MPTP injection, and the SPECT data were compared to ex vivo striatal and nigral DAT levels as measured by immunohistochemistry within the same animals. The analysis of striatal DAT levels using SPECT and DAT immunohistochemistry yielded highly comparable results on the percentage of DAT reduction in each MPTP group. The in vivo data showed a decrease of specific striatal to non-specific binding ratios by 59%, 82%, and 76% in mice treated for 1, 3, and 5 days, respectively. Moreover, a strong, positive correlation was observed between the in vivo and ex vivo parameters. The present study provides the first evidence that [123I]FP-CIT pinhole SPECT allows the accurate detection of a range of striatal DAT (i.e., losses of approximately 60–80%) levels in mice. Since such large dopaminergic lesions could be detected, this SPECT method may at least be useful for analyzing neuroprotective treatment with a clear-cut positive (i.e., complete protection) or negative (i.e., not any protection) effect. Whether this method is also useful for analyzing more subtle effects of neuroprotective treatment (partial protection) remains to be established, by studying mice with small dopaminergic lesions. [Copyright &y& Elsevier]

Promoter regions required for minus-strand and subgenomic RNA synthesis have been mapped for several plus-strand RNA viruses. In general, the two types of promoters do not share structural features even though they are recognized by the same viral polymerase. The minus-strand promoter of Alfalfa mosaic virus (AMV), a plant virus of the family Bromoviridae, consists of a triloop hairpin (hpE) which is attached to a 3′ tRNA-like structure (TLS). In contrast, the AMV subgenomic promoter consists of a single triloop hairpin that bears no sequence homology with hpE. Here we show that hpE, when detached from its TLS, can function as a subgenomic promoter in vitro and can replace the authentic subgenomic promoter in the live virus. Thus, the AMV subgenomic and minus-strand promoters are basically the same, but the minus-strand promoter is linked to a 3′ TLS to force the polymerase to initiate at the very 3′ end. [ABSTRACT FROM AUTHOR]

Reports on the study of cis- and trans-acting functions of the helicase-like domain replicase proteins (RP) by agroinfiltration plant leaves. Synthesis of minus-strand RNAs in mutations of conserved motifs; Inability of transient RP to complement the defect in RNA replication; Existence of a mechanism which coordinates the RNA replication.

Adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mediate leukocyte infiltration into the CNS, in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). Because exogenous interleukin-10 (IL-10) inhibits ICAM-1 and VCAM-1 expression and clinical EAE, we hypothesize that endogenous IL-10 signaling may suppress expression of adhesion molecules. In a rat model of chronic relapsing EAE, expression levels of IL-10 and its receptor (IL-10R1), ICAM-1 and VCAM-1 mRNA in the spinal cord are markedly increased, whereas levels of IL-10 mRNA remain relatively low. The temporal pattern of mRNA and protein expression showed marked differences between spinal cord levels. During relapse, IL-10, IL-10R1, ICAM-1, VCAM-1 mRNA levels and neurological scores show positive correlations. We conclude that endogenous IL-10 is not a crucial factor inhibiting adhesion molecule expression in this model. [Copyright &y& Elsevier]

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Self-interaction, chaperone binding and posttranslational modification of amyloid-beta (Aβ) is essential in the initiation and propagation of Aβ aggregation. Aggregation results in insoluble Aβ deposits characteristic of Alzheimer's disease (AD) brain lesions, i.e. senile plaques and cerebral amyloid angiopathy. Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes posttranslational modifications including the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds (molecular crosslinks), and colocalizes with Aβ deposits in AD. Two independent groups recently found that apart from the induction of Aβ oligomerization, the blood-derived transglutaminase member FXIIIa forms stable protein-protein complexes with Aβ independent of the transamidation reaction. Here, we investigated whether also tTG forms rigid protein complexes with Aβ in the absence of catalytic activation. We found that both Aβ 1-40 and Aβ 1-42 are substrates for tTG-catalyzed crosslinking. In addition, in the absence of calcium or the presence of a peptidergic inhibitor of tTG, stable tTG-Aβ 1-40 complexes were found. Interestingly, the stable complexes between tTG and Aβ 1-40 , were only found at 'physiological' concentrations of Aβ 1-40. Together, our data suggest that depending on the Aβ species at hand, and on the concentration of Aβ, rigid protein-complexes are formed between tTG and Aβ 1-40 without the involvement of the crosslinking reaction. Image 1 • Both Aβ 1-40 and Aβ 1-42 are substrates for tTG-catalyzed crosslinking. • Endogenous tTG is not reactive in senile plaques using an in situ assay. • Absence of calcium or the presence of an inhibitor of tTG, stable tTG-Aβ 1-40 complexes are formed. • Stable tTG/Aβ 1-40 complexes are only found at 'physiological' concentrations of Aβ 1-40. [ABSTRACT FROM AUTHOR]

Leukocyte infiltration into the central nervous system (CNS) is a key pathological feature in multiple sclerosis (MS) and the MS animal model experimental autoimmune encephalomyelitis (EAE). Recently, preventing leukocyte influx into the CNS of MS patients is the main target of MS therapies and insight into cell behaviour in the circulation is needed for further elucidation of such therapies. In this study, we aimed at in vivo visualization of monocytes in a time-dependent manner during EAE. Using intravital two-photon microscopy (IVM), we imaged CX3CR1 mice during EAE, visualizing CX3CR1-GFP monocytes and their dynamics in the spinal cord vasculature. Our observations showed that intraluminal crawling of CX3CR1-GFP monocytes increased even before the clinical onset of EAE due to immunization of the animals. Furthermore, intraluminal crawling remained elevated during ongoing clinical disease. Besides, the displacement of these cells was larger during the peak of EAE compared to the control animals. In addition, we showed that the enzyme tissue transglutaminase (TG2), which is present in CNS-infiltrated cells in MS patients, is likewise found in CX3CR1-GFP monocytes in the spinal cord lesions and at the luminal side of the vasculature during EAE. It might thereby contribute to adhesion and crawling of monocytes, facilitating extravasation into the CNS. Thus, we put forward that interference with monocyte adhesion, by e.g. inhibition of TG2, should be applied at a very early stage of EAE and possibly MS, to effectively combat subsequent pathology. [ABSTRACT FROM AUTHOR]

Introduction Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme capable of forming metabolically and mechanically stable crosslinks between the γ-carboxamide of a glutamine acyl-acceptor substrate and the ε-amino functionality of a lysine acyl-donor substrate resulting in protein oligomers. High TG2 crosslinking activity has been implicated in the pathogenesis of various diseases including celiac disease, cancer and fibrotic and neurodegenerative diseases. Development of a PET tracer specific for active TG2 provides a novel tool to further investigate TG2 biology in vivo in disease states. Recently, potent irreversible active site TG2 inhibitors carrying an acrylamide warhead were synthesized and pharmacologically characterized. Methods Three of these inhibitors, compound 1 , 2 and 3 , were successfully radiolabeled with carbon-11 on the acrylamide carbonyl position using a palladium mediated [ 11 C]CO aminocarbonylation reaction. Ex vivo biodistribution and plasma stability were evaluated in healthy Wistar rats. Autoradiography was performed on MDA-MB-231 tumor sections. Results [ 11 C] 1 , - 2 and - 3 were obtained in decay corrected radiochemical yields of 38–55%. Biodistribution showed low uptake in peripheral tissues, with the exception of liver and kidney. Low brain uptake of < 0.05% ID/g was observed. Blood plasma analysis demonstrated that [ 11 C] 1 and [ 11 C] 2 were rapidly metabolized, whereas [ 11 C] 3 was metabolized at a more moderate rate (63.2 ± 6.8 and 28.7 ± 10.8% intact tracer after 15 and 45 min, respectively). Autoradiography with [ 11 C] 3 on MDA-MB-231 tumor sections showed selective and specific binding of the radiotracer to the active state of TG2. Conclusions Taken together, these results identify [ 11 C] 3 as the most promising of the three compounds tested for development as PET radiotracer for the in vivo investigation of TG2 activity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cerebral amyloid angiopathy ( CAA) is a pathological hallmark of Alzheimer's disease ( AD) and characterized by deposition of amyloid-β (Aβ) protein and smooth muscle cell ( SMC) death in cerebral vessel walls. Apolipoprotein E (ApoE) is of importance in both Aβ accumulation and Aβ-mediated toxicity towards SMCs in the cerebral vessel wall, although its exact role in CAA pathogenesis remains unclear. Tissue transglutaminase ( tTG) is an enzyme capable of inducing both protein complexes and altered protein bioactivity via post-translational cross-linking. In CAA, tTG and its catalytic activity are associated with deposited Aβ. Furthermore, several apolipoproteins are known substrates of tTG. We therefore investigated whether ApoE is a substrate for tTG and if this affects ApoE's bioactivity. We found strong binding of different ApoE isoforms with tTG and demonstrated tTG-catalysed ApoE multimers. In post-mortem human AD cases, ApoE colocalized with in situ active tTG in CAA. Moreover, human brain SMCs treated with Aβ demonstrated enhanced secretion of both ApoE and tTG, and of TG cross-links in the extracellular matrix. Interestingly, tTG-catalysed cross-linked ApoE failed to protect SMCs against Aβ-mediated cytotoxicity. Together, our data demonstrate a novel tTG-driven post-translational modification of ApoE that might play an important role in CAA. [ABSTRACT FROM AUTHOR]

Infiltration of leukocytes is a major pathological event in white matter lesion formation in the brain of multiple sclerosis (MS) patients. In grey matter lesions, less infiltration of these cells occur, but microglial activation is present. Thus far, the interaction of β-integrins with extracellular matrix proteins, e.g. fibronectin, is considered to be of importance for the influx of immune cells. Recent in vitro studies indicate a possible role for the enzyme tissue Transglutaminase (TG2) in mediating cell adhesion and migration. In the present study we questioned whether TG2 is present in white and grey matter lesions observed in the marmoset model for MS. To this end, immunohistochemical studies were performed. We observed that TG2, expressed by infiltrating monocytes in white matter lesions co-expressed β1-integrin and is located in close apposition to deposited fibronectin. These data suggest an important role for TG2 in the adhesion and migration of infiltrating monocytes during white matter lesion formation. Moreover, in grey matter lesions, TG2 is mainly present in microglial cells together with some β1-integrin, whereas fibronectin is absent in these lesions. These data imply an alternative role for microglial-derived TG2 in grey matter lesions, e.g. cell proliferation. Further research should clarify the functional role of TG2 in monocytes or microglial cells in MS lesion formation. [ABSTRACT FROM AUTHOR]

The olfactory bulb ( OB) is affected early in both Parkinson's ( PD) and Alzheimer's disease ( AD), evidenced by the presence of disease-specific protein aggregates and an early loss of olfaction. Whereas previous studies showed amoeboid microglia in the classically affected brain regions of PD and AD patients, little was known about such changes in the OB. Using a morphometric approach, a significant increase in amoeboid microglia density within the anterior olfactory nucleus ( AON) of AD and PD patients was observed. These amoeboid microglia cells were in close apposition to β-amyloid, hyperphosphorylated tau or α-synuclein deposits, but no uptake of pathological proteins by microglia could be visualized. Subsequent analysis showed (i) no correlation between microglia and α-synuclein ( PD), (ii) a positive correlation with β-amyloid ( AD), and (iii) a negative correlation with hyperphosphorylated tau ( AD). Furthermore, despite the observed pathological alterations in neurite morphology, neuronal loss was not apparent in the AON of both patient groups. Thus, we hypothesize that, in contrast to the classically affected brain regions of AD and PD patients, within the AON rather than neuronal loss, the increased density in amoeboid microglial cells, possibly in combination with neurite pathology, may contribute to functional deficits. [ABSTRACT FROM AUTHOR]

Background: Multiple sclerosis (MS) has been mainly attributed to white matter (WM) pathology. However, recent evidence indicated the presence of grey matter (GM) lesions. One of the principal mediators of inflammatory processes is interleukin-1β (IL-1β), which is known to play a role in MS pathogenesis. It is unknown whether IL-1β is solely present in WM or also in GM lesions. Using an experimental MS model, we questioned whether IL-1β and the IL-1 receptor antagonist (IL-1ra) are present in GM in addition to affected WM regions. Methods: The expression of IL-1β and IL-1ra in chronic-relapsing EAE (cr-EAE) rats was examined using in situ hybridization, immunohistochemistry and real-time PCR. Rats were sacrificed at the peak of the first disease phase, the trough of the remission phase, and at the peak of the relapse. Histopathological characteristics of CNS lesions were studied using immunohistochemistry for PLP, CD68 and CD3 and Oil-Red O histochemistry. Results: IL-1β and IL-ra expression appears to a similar extent in affected GM and WM regions in the brain and spinal cord of cr-EAE rats, particularly in perivascular and periventricular locations. IL-1β and IL-1ra expression was dedicated to macrophages and/or activated microglial cells, at sites of starting demyelination. The time-dependent expression of IL-1β and IL-1ra revealed that within the spinal cord IL-1β and IL-1ra mRNA remained present throughout the disease, whereas in the brain their expression disappeared during the relapse. Conclusions: The appearance of IL-1β expressing cells in GM within the CNS during cr-EAE may explain the occurrence of several clinical deficits present in EAE and MS which cannot be attributed solely to the presence of IL-1β in WM. Endogenously produced IL-1ra seems not capable to counteract IL-1β-induced effects. We put forward that IL-1β may behold promise as a target to address GM, in addition to WM, related pathology in MS. [ABSTRACT FROM AUTHOR]

Abstract: Cerebral amyloid angiopathy (CAA) is a key histopathological hallmark of Alzheimer''s disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). CAA is characterized by amyloid-beta (Aβ) depositions and remodeling of the extracellular matrix (ECM) in brain vessels and plays an important role in the development and progression of both AD and HCHWA-D. Tissue transglutaminase (tTG) modulates the ECM by molecular cross-linking of ECM proteins. Here, we investigated the distribution pattern, cellular source, and activity of tTG in CAA in control, AD, and HCHWA-D cases. We observed increased tTG immunoreactivity and colocalization with Aβ in the vessel wall in early stage CAA, whereas in later CAA stages, tTG and its cross-links were present in halos enclosing the Aβ deposition. In CAA, tTG and its cross-links at the abluminal side of the vessel were demonstrated to be either of astrocytic origin in parenchymal vessels, of fibroblastic origin in leptomeningeal vessels, and of endothelial origin at the luminal side of the deposited Aβ. Furthermore, the ECM proteins fibronectin and laminin colocalized with the tTG-positive halos surrounding the deposited Aβ in CAA. However, we observed that in situ tTG activity was present throughout the vessel wall in late stage CAA. Together, our data suggest that tTG and its activity might play a differential role in the development and progression of CAA, possibly evolving from direct modulation of Aβ aggregation to cross-linking of ECM proteins resulting in ECM restructuring. [Copyright &y& Elsevier]

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is characterized by the accumulation of α-synuclein aggregates and degeneration of melanized neurons. The tissue transglutaminase (tTG) enzyme catalyzes molecular protein cross-linking. In PD brain, tTG-induced cross-links have been identified in α-synuclein monomers, oligomers and α-synuclein aggregates. However, whether tTG and α-synuclein occur together in PD affected neurons remains to be established. Interestingly, using immunohistochemistry, we observed a granular distribution pattern of tTG, characteristic of melanized neurons in PD brain. Apart from tTG, these granules were also positive for typical endoplasmic reticulum (ER)-resident chaperones, that is, protein disulphide isomerase, ERp57 and calreticulin, suggesting a direct link to the ER. Additionally, we observed the presence of phosphorylated pancreatic ER kinase (pPERK), a classical ER stress marker, in tTG granule positive neurons in PD brain, although no subcellular colocalization of tTG and pPERK was found. Our data therefore suggest that tTG localization to granular ER compartments is specific for stressed melanized neurons in PD brain. Moreover, as also α-synuclein aggregates were observed in tTG granule positive neurons, these results provide a clue to the cellular site of interaction between α-synuclein and tTG. [ABSTRACT FROM AUTHOR]