Zdenek Rohan

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.

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.

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.

Common and rare TBK1 variants in early-onset Alzheimer disease in a European cohort

TANK-binding kinase 1 (TBK1) loss-of-function (LoF) mutations are known to cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), often combined with memory deficits early in the disease course. We performed targeted resequencing of TBK1 in 1253 early onset Alzheimers disease (EOAD) patients from 8 European countries to investigate whether pathogenic TBK1 mutations are enriched among patients with clinical diagnosis of EOAD. Variant frequencies were compared against 2117 origin-matched controls. We identified only 1 LoF mutation (p.Thr79del) in a patient clinically diagnosed with Alzheimers disease and a positive family history of ALS. We did not observe enrichment of rare variants in EOAD patients compared to controls, nor of rare variants affecting NFκB induction. Of 3 common coding variants, rs7486100 showed evidence of association (OR 1.46 [95% CI 1.13-1.9]; p-value 0.01). Homozygous carriers of the risk allele showed reduced expression of TBK1 (p-value 0.03). Our findings are not indicative of a significant role for TBK1 mutations in EOAD. The association between common variants in TBK1, disease risk and reduced TBK1 expression warrants follow-up in FTD/ALS cohorts.

The contribution of proteinase-activated receptors to intracellular signaling, transcellular transport and autophagy in Alzheimer's disease.

The etiopathogenesis of Alzheimer´s disease is characterized by beta amyloid Aβ(1-42) toxic fragment aggregation and its association with impaired autophagy. In mitochondria, chronic damage due to transport and enzymatic processes together with the production of reactive oxygen species (ROS) are followed by the subsequent accumulation of Aβ in the form of senile plaques and the accumulation of hyperphosphorylated tau protein in intracellular deposits called tangles. Proteinase-activated receptors (PARs), members of the G protein-coupled receptor (GPCR) family, facilitate and modulate the transcellular transport and distribution of a variety of subcellular molecular components to the lysosomal system and, thus, influence their degradation. A review of the data shows that the activation or inhibition of PARs leads to changes in the process of autophagy, which may influence ROS production and Aβ (1-42) degradation in lysosomes and result in AD pathogenesis.

Proteinase-activated receptor 2 and disease biomarkers in cerebrospinal fluid in cases with autopsy-confirmed prion diseases and other neurodegenerative diseases

BackgroundProteinase-activated receptor 2 (PAR-2) has been shown to promote both neurotoxic and neuroprotective effects. Similarly, other routinely used nonspecific markers of neuronal damage can be found in cerebrospinal fluid (CSF) and can be used as biomarkers for different neurodegenerative disorders.MethodsUsing enzyme-linked immunosorbent assays and western blotting we assessed PAR-2, total-tau, phospho-tau, beta-amyloid levels, and protein 14-3-3 in the CSF of former patients who had undergone a neuropathological autopsy after death and who had been definitively diagnosed with a prion or other neurodegenerative disease.ResultsWe did not find any significant correlation between levels of PAR-2 and other biomarkers, nor did we find any differences in PAR-2 levels between prion diseases and other neurodegenerative conditions. However, we confirmed that very high total-tau levels were significantly associated with definitive prion diagnoses and exhibited greater sensitivity and specificity than protein 14-3-3, which is routinely used as a marker.ConclusionsOur study showed that PAR-2, in CSF, was not specifically altered in prion diseases compared to other neurodegenerative conditions. Our results also confirmed that very high total-tau protein CSF levels were significantly associated with a definitive Creutzfeldt-Jakob disease (CJD) diagnosis and should be routinely tested as a diagnostic marker. Observed individual variability in CSF biomarkers provide invaluable feedback from neuropathological examinations even in “clinically certain” cases.

Re: Shi et al. Protease-activated receptor 2 suppresses lymphangiogenesis and subsequent lymph node metastasis in a murine pancreatic cancer model. J Pathol 2014;234: 398-409

We read with great interest the article by Shi et al [1] in the November 2014 issue of The Journal of Pathology, which discussed the role of proteinase-activated receptor (PAR)-2 in the growth and metastasis of pancreatic cancer in a murine model. In addition to the well-known role of PAR-2 in the primary proliferation of various cancer cells [2,3], including pancreatic cancer [4], the results presented highlighted convincingly the importance of stromal host receptors relative to local progression in the orthotopic pancreatic cancer model. The authors recorded an increase in the average volume of primary tumours in wild-type animals compared with PAR-2−/−knockouts. However, they found that metastatic spread to abdominal organs and lymph node metastasis was more extensive in PAR-2−/− knockout animals than in wild-type animals. The authors noted the importance of the different roles of PAR-2 in metastasis, which vary depending on the model of metastasis (ie the important role of the host receptor). However, while a similar receptor (PAR-1), which is activated by thrombin, is (1) involved in cancer cell proliferation and invasion, (2) generally considered pro-carcinogenic, and (3) unambiguously considered to be a pro-metastatic factor [5], the same level of certainty regarding these events cannot be extended to PAR-2. Nevertheless, even though the data set describing this receptor in different models and/or experimental settings is small, the available results provide a good indication of it role. No differences in the size and number of lung metastases in the murine melanoma model (using injections into the tail vein) were described by Camerer et al [6]. In contrast, our team recently published data that showed larger primary B16 melanomas growing subcutaneously in PAR-2−/− knockouts, while distant metastases (mainly to the lungs) were significantly more frequently recorded in wild-type controls [7]. Additionally, survival was prolonged in PAR-2−/− knockouts. The available data make it clear that the absence of host PAR-2 leads to different outcomes depending on (1) primary tumour type (melanoma versus pancreatic cancer), (2) primary tumour location (skin versus pancreas), and (3) the model of metastatic induction (primary tumour growth versus intravenous application). Based on the three variations mentioned above, a variety of mechanisms for PAR-2 action have been discussed, including (a) antigen presentation in dendritic cells (DCs) [8]; (b) the role of PAR-2 in DC maturation [9]; and (c) the role of tissue factor/factor VIIa PAR-2 in tumour proliferation [10] and in capillary endothelia in distant locations [6,11]. Recently, Shi et al suggested an additional role for PAR-2 with regard to lymphatic vessel maturation [1]. To evaluate the influence of PAR-2 on lymphatic vessel growth, Shi et al utilized both artificial [agonist peptide (AP)] and more natural, in vivo-generated cell culture extracts to activate PAR-2. It is worth pointing out that ligand pleiotropy and receptor redundancy are characteristic features of PARs that can adversely affect virtually all PAR-based experiments and, as the authors noted in their conclusion, often lead to ambiguous results and further complicate therapeutic applications of PAR-based research. Together with PAR-2 heteroand homo-dimerization [12], and probably also transactivation [13], these features lead to the extreme variability of effects observed under different physiological, pathological, and research conditions. In vitro or ex vivo research in this field allows only limited use of well-defined proteases or APs. Nonetheless, these can help to assign a specific role to the receptor after activation by a given ligand. In an organism where complex systems of interacting proteases and anti-proteases affect virtually all receptors, PARs (namely PAR-2 in the study by Shi et al) are exposed to the influence of various proteases, including both activating and inactivating proteases, as well as several different types of protease inhibitors. Moreover, serum-activating and -inactivating proteases can also significantly affect tissue phenotype from the perspective of PAR-2 activation status. Using complex mixtures of tissues or cell culture extracts can more reliably simulate in vivo conditions; however, this makes assessing the real composition and individual protease concentrations, and their receptor affinities, problematic under physiological conditions. When combined with complex steric interactions between ligands (both natural, ie proteases, and artificial, APs) and membrane receptors, there is a potential for ‘biased’ signalling of the PAR-2 or modified downstream signalling interactions in general. Surrogate markers for PAR-2 (eg ERK1, 2 activity, used by Shi et al) and other markers of PAR activation usually reflect only one (often the dominant) signalling axis, but do not reflect other potentially co-activated cascades that may potentially modify, or even oppose, the effect of the original stimulus.