Parkinson’s disease (PD) is a slowly progressing neurodegenerative disorder that affects approximately seven million patients worldwide. Despite intensive research, the molecular mechanisms ...initiating and promoting PD are still unknown. However, it is assumed that environmental factors trigger PD. Recent research demonstrated that long noncoding RNAs (lncRNA) interfere in transcriptional and translational processes modulating gene expression reflecting environmental influences. Nevertheless, there is no systematic analysis available that investigates the impact of lncRNAs on PD. In the current study, we performed a comprehensive analysis on expression levels of 90 well-annotated lncRNAs in 30 brain specimens deriving from 20 PD patients and 10 controls as a preliminary report on the significance of lncRNAs in PD. Expression profiling of lncRNAs revealed that five lncRNAs are significantly differentially expressed in PD. While H19 upstream conserved 1 and 2 is significantly downregulated in PD, lincRNA-p21, Malat1, SNHG1, and TncRNA are significantly upregulated. An analysis on expression levels and PD stages revealed that the identified dysregulated lncRNA are altered already in early disease stage and that they precede the course of PD. In summary, this is the first comprehensive analysis on lncRNAs in PD revealing significantly altered lncRNAs. Additionally, we found that lncRNA dysregulations precede the course of the disease. Thus, the five newly identified lncRNAs may serve as potential new biomarkers appropriate even in early PD. They may be used in monitoring disease progression and they may serve as potential new targets for novel therapeutic approaches.
Accumulation of hyperphosphorylated, ubiquitinated and N-terminally truncated TAR DNA-binding protein (TDP-43) is the pathological hallmark lesion in most familial and sporadic forms of FTLD-U and ...ALS, which can be subsumed as TDP-43 proteinopathies. In order to get more insight into the role of abnormal phosphorylation in the disease process, the identification of specific phosphorylation sites and the generation of phosphorylation-specific antibodies are mandatory. Here, we developed and characterized novel rat monoclonal antibodies (1D3 and 7A9) raised against phosphorylated S409/410 of TDP-43. These antibodies were used to study the presence of S409/410 phosphorylation by immunohistochemistry and biochemical analysis in a large series of 64 FTLD-U cases with or without motor neuron disease including familial cases with mutations in progranulin (
n
= 5), valosin-containing protein (
n
= 4) and linkage to chromosome 9p (
n
= 4), 18 ALS cases as well as other neurodegenerative diseases with concomitant TDP-43 pathology (
n
= 5). Our data demonstrate that phosphorylation of S409/410 of TDP-43 is a highly consistent feature in pathologic inclusions in the whole spectrum of sporadic and familial forms of TDP-43 proteinopathies. Physiological nuclear TDP-43 was not detectable with these mAbs by immunohistochemistry and by immunoblot analyses. While the accumulation of phosphorylated C-terminal fragments was a robust finding in the cortical brain regions of FTLD-U and ALS, usually being much more abundant than the phosphorylated full-length TDP-43 band, spinal cord samples revealed a predominance of full-length TDP-43 over C-terminal fragments. This argues for a distinct TDP-43 species composition in inclusions in cortical versus spinal cord cells. Overall, these mAbs are powerful tools for the highly specific detection of disease-associated abnormal TDP-43 species and will be extremely useful for the neuropathological routine diagnostics of TDP-43 proteinopathies and for the investigation of emerging cellular and animal models for TDP-43 proteinopathies.
Neuronal intermediate filament inclusion disease (NIFID) is an uncommon neurodegenerative condition that typically presents as early-onset, sporadic frontotemporal dementia (FTD), associated with a ...pyramidal and/or extrapyramidal movement disorder. The neuropathology is characterized by frontotemporal lobar degeneration with neuronal inclusions that are immunoreactive for all class IV intermediate filaments (IF), light, medium and heavy neurofilament subunits and α-internexin. However, not all the inclusions in NIFID are IF-positive and the primary molecular defect remains uncertain. Mutations in the gene encoding the
fused in sarcoma
(FUS) protein have recently been identified as a cause of familial amyotrophic lateral sclerosis (ALS). Because of the recognized clinical, genetic and pathological overlap between FTD and ALS, we investigated the possible role of FUS in NIFID. We found abnormal intracellular accumulation of FUS to be a consistent feature of our NIFID cases (
n
= 5). More neuronal inclusions were labeled using FUS immunohistochemistry than for IF. Several types of inclusions were consistently FUS-positive but IF-negative, including neuronal intranuclear inclusions and glial cytoplasmic inclusions. Double-label immunofluorescence confirmed that many cells had only FUS-positive inclusions and that all cells with IF-positive inclusions also contained pathological FUS. No mutation in the
FUS
gene was identified in a single case with DNA available. These findings suggest that FUS may play an important role in the pathogenesis of NIFID.
The transcription factor Sox2 has been shown to play essential roles during embryonic development as well as in cancer. To more precisely understand tumor biology and to identify potential ...therapeutical targets, we thoroughly investigated the expression and function of Sox2 in medulloblastoma, a malignant embryonic brain tumor that initiates in the posterior fossa and eventually spreads throughout the entire cerebrospinal axis. We examined a large series of tumor samples (n = 188) to show that SOX2 is specifically expressed in Sonic hedgehog (SHH)-associated medulloblastoma with an interesting preponderance in adolescent and adult cases. We further show that cerebellar granule neuron precursors (CGNP), which are believed to serve as the cell of origin for this medulloblastoma subgroup, express Sox2 in early stages. Also, Shh-associated medulloblastoma can be initiated from such Sox2-positive CGNPs in mice. Independent of their endogenous Sox2 expression, constitutive activation of Shh signaling in CGNPs resulted in significantly enhanced proliferation and ectopic expression of Sox2 in vitro and Sox2-positive medulloblastoma in vivo. Genetic ablation of Sox2 from murine medulloblastoma did not affect survival, most likely due to a compensatory overexpression of Sox3. However, acute deletion of Sox2 from primary cultures of CGNPs with constitutive Shh signaling significantly decreased proliferation, whereas overexpression of Sox2 enhanced proliferation of murine medulloblastoma cells. We conclude that Sox2 is a marker for Shh-dependent medulloblastomas where it is required and sufficient to drive tumor cell proliferation.
Accumulation of the DNA/RNA binding protein fused in sarcoma as cytoplasmic inclusions in neurons and glial cells is the pathological hallmark of all patients with amyotrophic lateral sclerosis with ...mutations in FUS as well as in several subtypes of frontotemporal lobar degeneration, which are not associated with FUS mutations. The mechanisms leading to inclusion formation and fused in sarcoma-associated neurodegeneration are only poorly understood. Because fused in sarcoma belongs to a family of proteins known as FET, which also includes Ewing's sarcoma and TATA-binding protein-associated factor 15, we investigated the potential involvement of these other FET protein family members in the pathogenesis of fused in sarcoma proteinopathies. Immunohistochemical analysis of FET proteins revealed a striking difference among the various conditions, with pathology in amyotrophic lateral sclerosis with FUS mutations being labelled exclusively for fused in sarcoma, whereas fused in sarcoma-positive inclusions in subtypes of frontotemporal lobar degeneration also consistently immunostained for TATA-binding protein-associated factor 15 and variably for Ewing's sarcoma. Immunoblot analysis of proteins extracted from post-mortem tissue of frontotemporal lobar degeneration with fused in sarcoma pathology demonstrated a relative shift of all FET proteins towards insoluble protein fractions, while genetic analysis of the TATA-binding protein-associated factor 15 and Ewing's sarcoma gene did not identify any pathogenic variants. Cell culture experiments replicated the findings of amyotrophic lateral sclerosis with FUS mutations by confirming the absence of TATA-binding protein-associated factor 15 and Ewing's sarcoma alterations upon expression of mutant fused in sarcoma. In contrast, all endogenous FET proteins were recruited into cytoplasmic stress granules upon general inhibition of Transportin-mediated nuclear import, mimicking the findings in frontotemporal lobar degeneration with fused in sarcoma pathology. These results allow a separation of fused in sarcoma proteinopathies caused by FUS mutations from those without a known genetic cause based on neuropathological features. More importantly, our data imply different pathological processes underlying inclusion formation and cell death between both conditions; the pathogenesis in amyotrophic lateral sclerosis with FUS mutations appears to be more restricted to dysfunction of fused in sarcoma, while a more global and complex dysregulation of all FET proteins is involved in the subtypes of frontotemporal lobar degeneration with fused in sarcoma pathology.
To describe the first symptom/sign and first diagnosis in patients with sporadic Creutzfeldt-Jakob disease (sCJD) in Germany with respect to M129V polymorphism of the prion protein gene and prion ...protein type. Data on the first symptom/sign and first diagnosis were studied in 492 sCJD patients with probable and definite sCJD and known M129V polymorphism. Unspecific prodromal symptoms such as headache, fatigue, sleep disturbances, “peculiar feeling in the head”, photophobia or weight loss were found in about 10 % of the patients. No prodromal symptoms were found in MV2 and VV1 patients. Dementia was the most common first symptom (37 %) followed by cerebellar (34 %), visual (15 %), and psychiatric disturbances (14 %). The CJD diagnosis was the first diagnosis in only 35 % of the patients (in 42 % of MM, 28 % of MV, and 24.5 % of VV patients). We provide a detailed analysis on clinical presentation and first diagnosis in a large group of patients with sCJD with respect to M129V genotype and prion protein type. These data emphasize the importance of knowledge about CJD and especially rare CJD types among physicians of different specializations. Our findings may improve early recognition of atypical CJD forms.
Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is an autosomal-dominant central nervous system white-matter disease with variable clinical presentations, including personality and ...behavioral changes, dementia, depression, parkinsonism, seizures and other phenotypes. We combined genome-wide linkage analysis with exome sequencing and identified 14 different mutations affecting the tyrosine kinase domain of the colony stimulating factor 1 receptor (encoded by CSF1R) in 14 families with HDLS. In one kindred, we confirmed the de novo occurrence of the mutation. Follow-up sequencing identified an additional CSF1R mutation in an individual diagnosed with corticobasal syndrome. In vitro, CSF-1 stimulation resulted in rapid autophosphorylation of selected tyrosine residues in the kinase domain of wild-type but not mutant CSF1R, suggesting that HDLS may result from partial loss of CSF1R function. As CSF1R is a crucial mediator of microglial proliferation and differentiation in the brain, our findings suggest an important role for microglial dysfunction in HDLS pathogenesis.
We analyzed prospectively whether MGMT (O(6)-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma ...patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression.
ADULT PATIENTS WITH A HISTOLOGICALLY PROVEN MALIGNANT ASTROCYTOMA (GLIOBLASTOMA: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression.
MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined.
The current classification of human sporadic prion diseases recognizes six major phenotypic subtypes with distinctive clinicopathological features, which largely correlate at the molecular level with ...the genotype at the polymorphic codon 129 (methionine, M, or valine, V) in the prion protein gene and with the size of the protease-resistant core of the abnormal prion protein, PrP
Sc
(i.e. type 1 migrating at 21 kDa and type 2 at 19 kDa). We previously demonstrated that PrP
Sc
typing by Western blotting is a reliable means of strain typing and disease classification. Limitations of this approach, however, particularly in the interlaboratory setting, are the association of PrP
Sc
types 1 or 2 with more than one clinicopathological phenotype, which precludes definitive case classification if not supported by further analysis, and the difficulty of fully recognizing cases with mixed phenotypic features. In this study, we tested the inter-rater reliability of disease classification based only on histopathological criteria. Slides from 21 cases covering the whole phenotypic spectrum of human sporadic prion diseases, and also including two cases of variant Creutzfeldt–Jakob disease (CJD), were distributed blindly to 13 assessors for classification according to given instructions. The results showed good-to-excellent agreement between assessors in the classification of cases. In particular, there was full agreement (100 %) for the two most common sporadic CJD subtypes and variant CJD, and very high concordance in general for all pure phenotypes and the most common subtype with mixed phenotypic features. The present data fully support the basis for the current classification of sporadic human prion diseases and indicate that, besides molecular PrP
Sc
typing, histopathological analysis permits reliable disease classification with high interlaboratory accuracy.
Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence ...indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease‐associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrPSc), a β‐sheet‐rich isoform of the cellular PrP (PrPC), are dependent on neuronal expression of PrPC. In this study, we demonstrate that PrPC has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various β‐sheet‐rich (β) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid β‐peptide, (iii) yeast prion proteins or (iv) designed β‐peptides. Toxic signalling via PrPC requires the intrinsically disordered N‐terminal domain (N‐PrP) and the GPI anchor of PrP. We found that the N‐terminal domain is important for mediating the interaction of PrPC with β‐conformers. Interestingly, a secreted version of N‐PrP associated with β‐conformers and antagonized their toxic signalling via PrPC. Moreover, PrPC‐mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer‐specific antibody. Our study indicates that PrPC can mediate toxic signalling of various β‐sheet‐rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.
PrPC can interact with and mediate toxic signalling of misfolded β‐sheet‐rich oligomers of different origin.
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