Biology subjects

Safar, J. G.

Publications and source records attributed to Safar, J. G..

3 recordsLinked to original sources

bioRxiv2024

Neuropathologically-directed profiling of PRNP somatic and germline variants in sporadic human prion disease

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, [~]1% were transmitted by misfolded PrP, [~]15% are inherited, and [~]85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate focal initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of >5,000X across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a focal presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

genetics↗

bioRxiv2020

Tau conformers in FTLD-MAPT undergo liquid-liquid phase separation and perturb the nuclear envelope

Recent studies show that a single MAPT gene mutation can promote alternative tau misfolding pathways engendering divergent forms of frontotemporal dementia and that under conditions of molecular crowding, the repertoire of tau forms can include liquid-liquid phase separation (LLPS). We show here that following pathogenic seeding, tau condenses on the nuclear envelope (NE) and disrupts nuclear-cytoplasmic transport (NCT). Interestingly, NE fluorescent tau signals and small fluorescent inclusions behaved as demixed liquid droplets in living cells. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 m2, indicating that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions. Our findings indicate that tau undergoing LLPS is more toxic amongst a spectrum of alternative conformers; LLPS droplets on the NE that disrupt NCT serve to trigger cell death and can act as nurseries for fibrillar structures abundantly detected in end-stage disease.

neuroscience↗

bioRxiv2019

Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have reproducibly shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. To date it has been unclear whether the reduced abundance of PrP in CSF results from its incorporation into plaques, retention in intracellular compartments, downregulation as a function of the disease process, or other factors. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to known concentrations of isotopically labeled standards. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In N=55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to individuals with non-prion diagnoses. This confirms the findings from ELISA studies, demonstrating that lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.

neuroscience↗