Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantification of ARID1A in Tissue Lysates.

ARID1A is a subunit of the mammalian SWI/SNF complex, which is thought to regulate gene expression through restructuring chromatin structures. Its gene ARID1A is frequently mutated and ARID1A levels are lowered in several human cancers, especially gynecologic ones. A functional ARID1A loss may have prognostic or predictive value in terms of therapeutic strategies but has not been proposed based on a quantitative method. Hardly any literature is available on ARID1A levels in tumor samples. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for ARID1A based on the current EMA and FDA criteria. We demonstrated that our ELISA provides the objective, accurate, and precise quantification of ARID1A concentrations in recombinant protein solutions, cell culture standards, and tissue lysates of tumors. A standard curve analysis yielded a 'goodness of fit' of R2 = 0.99. Standards measured on several plates and days achieved an inter-assay accuracy of 90.26% and an inter-assay precision with a coefficient of variation of 4.53%. When tumor lysates were prepared and measured multiple times, our method had an inter-assay precision with a coefficient of variation of 11.78%. We believe that our suggested method ensures a high reproducibility and can be used for a high sample throughput to determine the ARID1A concentration in different tumor entities. The application of our ELISA on various tumor and control tissues will allow us to explore whether quantitative ARID1A measurements in tumor samples are of predictive value.

Bisulfite profiling of the MGMT promoter and comparison with routine testing in glioblastoma diagnostics.

BACKGROUND: Promoter methylation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Patients with methylated CpGs in the MGMT promoter benefit from treatment with alkylating agents, such as temozolomide, and show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methylation is of importance for diagnostic decisions. We experienced that different methods show partially divergent results in a daily routine. For an integrated neuropathological diagnosis of malignant gliomas, we therefore currently apply a combination of methylation-specific PCR assays and pyrosequencing. RESULTS: To better rationalize the variation across assays, we compared these standard techniques and assays to deep bisulfite sequencing results in a cohort of 80 malignant astrocytomas. Our deep analysis covers 49 CpG sites of the expanded MGMT promoter, including exon 1, parts of intron 1 and a region upstream of the transcription start site (TSS). We observed that deep sequencing data are in general in agreement with CpG-specific pyrosequencing, while the most widely used MSP assays published by Esteller et al. (N Engl J Med 343(19):1350-1354, 2000. https://doi.org/10.1056/NEJM200011093431901 ) and Felsberg et al. (Clin Cancer Res 15(21):6683-6693, 2009. https://doi.org/10.1158/1078-0432.CCR-08-2801 ) resulted in partially discordant results in 22 tumors (27.5%). Local deep bisulfite sequencing (LDBS) revealed that CpGs located in exon 1 are suited best to discriminate methylated from unmethylated samples. Based on LDBS data, we propose an optimized MSP primer pair with 83% and 85% concordance to pyrosequencing and LDBS data. A hitherto neglected region upstream of the TSS, with an overall higher methylation compared to exon 1 and intron 1 of MGMT, is also able to discriminate the methylation status. CONCLUSION: Our integrated analysis allows to evaluate and redefine co-methylation domains within the MGMT promoter and to rationalize the practical impact on assays used in daily routine diagnostics.

Neurodegeneration and neuroinflammation are linked, but independent of alpha-synuclein inclusions, in a seeding/spreading mouse model of Parkinson's disease.

A key pathological process in Parkinson's disease (PD) is the transneuronal spreading of α-synuclein. Alpha-synuclein (α-syn) is a presynaptic protein that, in PD, forms pathological inclusions. Other hallmarks of PD include neurodegeneration and microgliosis in susceptible brain regions. Whether it is primarily transneuronal spreading of α-syn particles, inclusion formation, or other mechanisms, such as inflammation, that cause neurodegeneration in PD is unclear. We used a model of spreading of α-syn induced by striatal injection of α-syn preformed fibrils into the mouse striatum to address this question. We performed quantitative analysis for α-syn inclusions, neurodegeneration, and microgliosis in different brain regions, and generated gene expression profiles of the ventral midbrain, at two different timepoints after disease induction. We observed significant neurodegeneration and microgliosis in brain regions not only with, but also without α-syn inclusions. We also observed prominent microgliosis in injured brain regions that did not correlate with neurodegeneration nor with inclusion load. Using longitudinal gene expression profiling, we observed early gene expression changes, linked to neuroinflammation, that preceded neurodegeneration, indicating an active role of microglia in this process. Altered gene pathways overlapped with those typical of PD. Our observations indicate that α-syn inclusion formation is not the major driver in the early phases of PD-like neurodegeneration, but that microglia, activated by diffusible, oligomeric α-syn, may play a key role in this process. Our findings uncover new features of α-syn induced pathologies, in particular microgliosis, and point to the necessity for a broader view of the process of α-syn spreading.

Prion type 2 selection in sporadic Creutzfeldt-Jakob disease affecting peripheral ganglia.

In sporadic Creutzfeldt-Jakob disease (sCJD), the pathological changes appear to be restricted to the central nervous system. Only involvement of the trigeminal ganglion is widely accepted. The present study systematically examined the involvement of peripheral ganglia in sCJD utilizing the currently most sensitive technique for detecting prions in tissue morphologically. The trigeminal, nodose, stellate, and celiac ganglia, as well as ganglia of the cervical, thoracic and lumbar sympathetic trunk of 40 patients were analyzed with the paraffin-embedded tissue (PET)-blot method. Apart from the trigeminal ganglion, which contained protein aggregates in five of 19 prion type 1 patients, evidence of prion protein aggregation was only found in patients associated with type 2 prions. With the PET-blot, aggregates of prion protein type 2 were found in all trigeminal (17/17), in some nodose (5 of 7) and thoracic (3 of 6) ganglia, as well as in a few celiac (4 of 19) and lumbar (1 of 5) ganglia of sCJD patients. Whereas aggregates of both prion types may spread to dorsal root ganglia, more CNS-distant ganglia seem to be only involved in patients accumulating prion type 2. Whether the prion type association is due to selection by prion type-dependent replication, or due to a prion type-dependent property of axonal spread remains to be resolved in further studies.

Defining the Prion Type of Fatal Familial Insomnia.

Fatal familial insomnia (FFI) belongs to the genetic human transmissible spongiform encephalopathies (TSE), such as genetic Creutzfeldt-Jakob disease (CJD) or Gerstmann-Straeussler-Scheinker syndrome (GSS). Here, we analyzed the properties of the pathological prion protein in six FFI cases by Western blot analysis, a protein aggregate stability assay, and aggregate deposition characteristics visualized with the paraffin-embedded tissue blot. While in all cases the unglycosylated fragment in Western blot analysis shared the same size with sporadic CJD prion type 2, the reticular/synaptic deposition pattern of the prion aggregates resembled the ones found in sporadic CJD type 1 (CJD types according to the Parchi classification from 1999). Regarding the conformational stability against denaturation with GdnHCl, FFI prion aggregates resembled CJD type 1 more than type 2. Our results suggest that the size of the proteinase-K-resistant fragments is not a valid criterion on its own. Additional criteria supplying information about conformational differences or similarities need to be taken into account. FFI may resemble a prion type with its own conformation sharing properties partly with type 1 and type 2 prions.