Radoslav Matej

A Pan-European Study of the C9orf72 Repeat Associated with FTLD: Geographic Prevalence, Genomic Instability, and Intermediate Repeats

We assessed the geographical distribution of C9orf72 G4C2 expansions in a pan‐European frontotemporal lobar degeneration (FTLD) cohort (n = 1,205), ascertained by the European Early‐Onset Dementia (EOD) consortium. Next, we performed a meta‐analysis of our data and that of other European studies, together 2,668 patients from 15 Western European countries. The frequency of the C9orf72 expansions in Western Europe was 9.98% in overall FTLD, with 18.52% in familial, and 6.26% in sporadic FTLD patients. Outliers were Finland and Sweden with overall frequencies of respectively 29.33% and 20.73%, but also Spain with 25.49%. In contrast, prevalence in Germany was limited to 4.82%. In addition, we studied the role of intermediate repeats (7–24 repeat units), which are strongly correlated with the risk haplotype, on disease and C9orf72 expression. In vitro reporter gene expression studies demonstrated significantly decreased transcriptional activity of C9orf72 with increasing number of normal repeat units, indicating that intermediate repeats might act as predisposing alleles and in favor of the loss‐of‐function disease mechanism. Further, we observed a significantly increased frequency of short indels in the GC‐rich low complexity sequence adjacent to the G4C2 repeat in C9orf72 expansion carriers (P < 0.001) with the most common indel creating one long contiguous imperfect G4C2 repeat, which is likely more prone to replication slippage and pathological expansion.

TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

We investigated the mutation spectrum of the TANK‐Binding Kinase 1 (TBK1) gene and its associated phenotypic spectrum by exonic resequencing of TBK1 in a cohort of 2,538 patients with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), or FTD plus ALS, ascertained within the European Early‐Onset Dementia Consortium. We assessed pathogenicity of predicted protein‐truncating mutations by measuring loss of RNA expression. Functional effect of in‐frame amino acid deletions and missense mutations was further explored in vivo on protein level and in vitro by an NFκB‐induced luciferase reporter assay and measuring phosphorylated TBK1. The protein‐truncating mutations led to the loss of transcript through nonsense‐mediated mRNA decay. For the in‐frame amino acid deletions, we demonstrated loss of TBK1 or phosphorylated TBK1 protein. An important fraction of the missense mutations compromised NFκB activation indicating that at least some functions of TBK1 are lost. Although missense mutations were also present in controls, over three times more mutations affecting TBK1 functioning were found in the mutation fraction observed in patients only, suggesting high‐risk alleles (P = 0.03). Total mutation frequency for confirmed TBK1 LoF mutations in the European cohort was 0.7%, with frequencies in the clinical subgroups of 0.4% in FTD, 1.3% in ALS, and 3.6% in FTD‐ALS.

Expression of proteinase-activated receptor 2 during taurocholate-induced acute pancreatic lesion development in Wistar rats.

Background: Proteinase-activated receptor 2 (PAR-2) is a G-protein coupled transmembrane receptor activated by trypsin by site-specific cleavage. Its presence on pancreatic structures was demonstrated in the past. PAR-2 physiologically involves in duct/ acinary cells secretion, arterial tonus regulation or capillary liquid turnover. During development of acute pancreatitis/ acute pancreatic lesion (APL) these mentioned structures are influenced by very high concentration of trypsin due to its increased basolateral secretion into the interstitium. The aim of our study as presented was to investigate whether PAR-2 is also involved in APL following changes of PAR-2 expression.Methods: APL was investigated in Wistar rats after injection of 0.1 mL taurocholate into the ductus choledochus. Anatomy, histology, reverse transcriptase polymerase chain reaction (RT PCR) as well as immunohistochemistry and Western-blot analysis of pancreatic tissue were performed using antibody mapping of the new NH2 terminal of PAR-2 after trypsin cleavage. Results from control rats and d 1 or d 4 rats after taurocholate injection were compared.Results: Much higher positivity on acinary/ duct cells was observed in APL induced animals than in controls. Similar findings were noticed on arterial smooth muscle cells. Surprisingly, parallel to the exocrine pancreas and vessel findings, enhanced Langerhans’ islet cell positivity was observed in experimental animals.Conclusions: Based on these results, we have demonstrated that during APL development PAR-2 expression increases. This effect is caused by conformational changes after PAR-2 activation, and the new NH2 terminal of activated receptor presentation. We suggest that PAR-2 physiological functions are enhanced during APL development.

Rare Variants in PLD3 Do Not Affect Risk for Early-Onset Alzheimer Disease in a European Consortium Cohort.

Rare variants in the phospholipase D3 gene (PLD3) were associated with increased risk for late‐onset Alzheimer disease (LOAD). We identified a missense mutation in PLD3 in whole‐genome sequence data of a patient with autopsy confirmed Alzheimer disease (AD) and onset age of 50 years. Subsequently, we sequenced PLD3 in a Belgian early‐onset Alzheimer disease (EOAD) patient (N = 261) and control (N = 319) cohort, as well as in European EOAD patients (N = 946) and control individuals (N = 1,209) ascertained in different European countries. Overall, we identified 22 rare variants with a minor allele frequency <1%, 20 missense and two splicing mutations. Burden analysis did not provide significant evidence for an enrichment of rare PLD3 variants in EOAD patients in any of the patient/control cohorts. Also, meta‐analysis of the PLD3 data, including a published dataset of a German EOAD cohort, was not significant (P = 0.43; OR = 1.53, 95% CI 0.60–3.31). Consequently, our data do not support a role for PLD3 rare variants in the genetic etiology of EOAD in European EOAD patients. Our data corroborate the negative replication data obtained in LOAD studies and therefore a genetic role of PLD3 in AD remains to be demonstrated.

Clinicopathological description of two cases with SQSTM1 gene mutation associated with frontotemporal dementia

There is a strong genetic influence on the clinicopathological phenotypes associated with frontotemporal lobar degeneration (FTLD) and frontotemporal dementia (FTD). Intracellular deposition of TDP‐43 is the phenotypical hallmark of a frequent subgroup of cases. Mutations in the sequestosome 1 (SQSTM1) gene have rarely been found in individuals with FTD. Here we provide a comprehensive clinicopathological description of two cases with a SQSTM1 mutation. The clinical phenotype of patient 1 (mutation p.Glu396*) was compatible with the behavioural variant (bv) of FTD. TDP‐43 pathology was consistent with the features of type B of FTLD‐TDP pathology. However, prominent neuronal granular cytoplasmic TDP‐43 immunoreactivity and abundant oligodendroglial inclusions, proven by colocalization with the oligodendroglial‐marker TPPP/p25, were also seen. The clinical phenotype of patient 2 was compatible with bvFTD associated with parkinsonism and bulbar symptoms in the later stage. Genetic testing of patient 2 identified a C9orf72 repeat expansion mutation together with a missense mutation (p.Arg212Cys) in SQSTM1. TDP‐43 pathology was characterized by neuritic profiles compatible mostly with type A. In contrast to patient 1, p62 pathology was seen to a greater extent as TDP‐43 immunoreactivity in neurons. Using an antibody that detects poly(GP) peptides produced via repeat associated non‐ATG translation associated with expanded hexanucleotide repeat in the C9orf72 gene, we confirmed the presence of pathognomonic inclusions. The present study supports previous observations on amyotrophic lateral sclerosis (ALS) that SQSTM1 mutations consistently associate with TDP‐43 pathology. The co‐presence of C9orf72 mutation may influence the phenotype, thus finding one FTLD (or ALS) related mutation does not exclude the presence of further influential genetic alterations. Oligodendroglial TDP‐43 pathology is considerable in some forms of FTLD‐TDP, thus their evaluation might be considered to be included in classification systems.

Screening for α-synuclein immunoreactive neuronal inclusions in the hippocampus allows identification of atypical MSA (FTLD-synuclein).

brainstem, and cerebellum. According to Aoki et al. [2], we used a four-tiered scoring system (none—0, mild—1, moderate—2, severe—3) for the evaluation of α-synuclein NCIs regardless of their morphology (ring, NFT, or Pick body like). The selection was blinded to the clinical diagnosis, age, gender, and macroscopic observations. We observed α-synuclein immunoreactive NCIs in the granule cells of the dentate gyrus in seven cases (38 %). Five out of seven cases (online supplemental file 1) showed only relatively few NCIs in the dentate gyrus and CA1/Subiculum (score 1) (Fig. 1a, b). There was a lack of α-synuclein immunoreactive thin neurites and eosinophilic Pick body-like spherical inclusions in the hematoxylin and eosin (H&E) staining. Three of these five cases did not show clinical symptoms of dementia. Gait disturbance, parkinsonism, cerebellar symptoms, and dementia (not compatible with FTD) were reported in the two additional cases (57and 71-year-old women) during the final 24 months of illness (total duration of illness was 120 and 75 months, respectively). Both brains showed Aβ plaques (both Thal phase 3) [9] and neurofibrillary degeneration (both Braak stage II) [1, 3]. In the Atypical multiple system atrophy (aMSA) is a term recently introduced by Aoki et al. to describe cases that show hallmark neuropathological changes of glial cytoplasmic inclusions (Papp–Lantos bodies) characteristic of MSA, while clinically presenting with frontotemporal dementia (FTD) syndromes associated with frontotemporal lobar degeneration (FTLD) and severe limbic and cortical α-synuclein neuronal pathology [2]. The authors evaluated FTD syndrome cases and showed that the evaluation of α-synuclein immunoreactive neuronal cytoplasmic inclusions (NCIs) in the hippocampus (dentate gyrus and CA1/Subiculum) seems to be of great importance [2]. We aimed to determine if these morphological features in the hippocampus are reliable to identify similar cases in our archives. We evaluated α-synuclein immunostaining in the hippocampus from a cohort of 18 neuropathologically confirmed MSA cases [10]: all cases contained characteristic Papp–Lantos bodies and NCIs in the basal ganglia,

Shared and Distinct Patterns of Oligodendroglial Response in α-Synucleinopathies and Tauopathies

Pathological protein deposits in oligodendroglia are common but variable features of various neurodegenerative conditions. To evaluate oligodendrocyte response in neurodegenerative diseases (NDDs) with different extents of oligodendroglial protein deposition we performed immunostaining for tubulin polymerization-promoting protein p25&agr; (TPPP/p25&agr;), &agr;-synuclein (&agr;-syn), phospho-tau, ubiquitin, myelin basic protein, and the microglial marker HLA-DR. We investigated cases of multiple system atrophy ([MSA] n = 10), Lewy body disease ([LBD] n = 10), globular glial tauopathy ([GGT] n = 7) and progressive supranuclear palsy ([PSP] n = 10). Loss of nuclear TPPP/p25&agr; immunoreactivity correlated significantly with the degree of microglial reaction and loss of myelin basic prtein density as a marker of tract degeneration. This was more prominent in MSA and GGT, which, together with enlarged cytoplasmic TPPP/p25&agr; immunoreactivity and inclusion burden allowed these disorders to be grouped as predominant oligodendroglial proteinopathies. However, distinct features, ie more colocalization of &agr;-syn than tau with TPPP/p25&agr;, more obvious loss of oligodendrocyte density in MSA, but more prominent association of tau protein inclusions in GGT to loss of nuclear TPPP/p25&agr; immunoreactivity, were also recognized. In addition, we observed previously underappreciated oligodendroglial &agr;-synuclein pathology in the pallidothalamic tract in LBD. Our study demonstrates common and distinct aspects of oligodendroglial involvement in the pathogenesis of diverse NDDs.

Multisite Assessment of Aging-Related Tau Astrogliopathy (ARTAG)

Aging-related tau astrogliopathy (ARTAG) is a recently introduced terminology. To facilitate the consistent identification of ARTAG and to distinguish it from astroglial tau pathologies observed in the primary frontotemporal lobar degeneration tauopathies we evaluated how consistently neuropathologists recognize (1) different astroglial tau immunoreactivities, including those of ARTAG and those associated with primary tauopathies (Study 1); (2) ARTAG types (Study 2A); and (3) ARTAG severity (Study 2B). Microphotographs and scanned sections immunostained for phosphorylated tau (AT8) were made available for download and preview. Percentage of agreement and kappa values with 95% confidence interval (CI) were calculated for each evaluation. The overall agreement for Study 1 was >60% with a kappa value of 0.55 (95% CI 0.433-0.645). Moderate agreement (>90%, kappa 0.48, 95% CI 0.457-0.900) was reached in Study 2A for the identification of ARTAG pathology for each ARTAG subtype (kappa 0.37-0.72), whereas fair agreement (kappa 0.40, 95% CI 0.341-0.445) was reached for the evaluation of ARTAG severity. The overall assessment of ARTAG showed moderate agreement (kappa 0.60, 95% CI 0.534-0.653) among raters. Our study supports the application of the current harmonized evaluation strategy for ARTAG with a slight modification of the evaluation of its severity.

IL-4 polymorphisms, HRCT score and lung tissue markers in idiopathic pulmonary fibrosis.

AIMS We studied the influence of IL-4 gene polymorphisms on the IPF phenotype, i.e., extent of radiological changes (HRCT interstitial (IS) and alveolar (AS) score) and histopathological markers from lung biopsies. PATIENTS AND METHODS 46 IPF patients underwent genotyping, 43 of them had HRCT and 14 patients had a surgical lung biopsy. The HRCT scans were evaluated for AS and IS. The histopathological evaluation comprised myofibroblast foci (MF), intensity of inflammation and fibrosis (Ashcroft score) and numbers of eosinophils and granulomas. For immunohistochemical evaluation primary antibodies against PAR-2, CD124, TGF beta, YY-1 and TSLP were used. The IL-4 and IL-4 R alpha gene polymorphisms were characterized. RESULTS We found a correlation between eosinophils in lung biopsies and AS. The Ashcroft score was higher in IL-4 HA 2 GCC and MF were more frequent in IL-4 HA 2 TCC carriers. A relationship was found between IL-4 (-1098) A2 T and PAR-2 expression and IL-4 (-590) A1 T, IL-4 HA1TTT and CD124 expression. AS was lower in IL-4 (-590) A1 C, in IL-4 HA1 TCC and in IL-4RA (+1902) A1 A carriers. CONCLUSIONS We suggest that the polymorphisms of IL-4 genes might influence the phenotype of IPF reflected by histopathological changes in lung biopsies and HRCT score.

Oligodendroglial Response in the Spinal Cord in TDP-43 Proteinopathy with Motor Neuron Involvement

Background: TDP-43 proteinopathies represent a spectrum of neurodegenerative disorders. Variable clinical presentations including frontotemporal dementia, amyotrophic lateral sclerosis (ALS) and mixed forms are associated with the spatial heterogeneity of the TDP-43 pathology. Recent studies have emphasized the role of oligodendrocytes in the pathogenesis of ALS. Objective: To evaluate whether TDP-43 proteinopathies are associated with an oligodendroglial response. Methods: We performed a study on 7 controls and 10 diseased patients with spinal cord involvement. Using the oligodendroglia-specific antibody TPPP/p25, we assessed oligodendrocyte density in the lateral corticospinal tracts (LCSs) along with the presence of perineuronal oligodendrocytes (PNOGs) in the anterior horns. We performed a densitometry of myelin basic protein (MBP) immunoreactivity. The numbers of TDP-43 and p62 immunoreactive inclusions were counted in both the LCSs and the anterior horns. Results: Double immunolabeling confirmed that oligodendrocytes harbor TDP-43 inclusions. In the LCSs, MBP density, but not the number of oligodendrocytes, was decreased in the diseased group. However, oligodendrocyte counts in the LCS correlated positively, and the density of MBP inversely, with the number of neuronal inclusions in the anterior horn, suggestive of a compensatory response of oligodendrocytes. The number of neurons with PNOGs correlated with the amount of inclusions. Conclusion: Our study further emphasizes the importance of oligodendroglia in the pathogenesis of TDP-43 proteinopathies with spinal cord involvement.