Applications of the real-time quaking-induced conversion assay in diagnosis, prion strain-typing, drug pre-screening and other amyloidopathies.

INTRODUCTION: The development of in vitro protein misfolding amplification assays for the detection and analysis of abnormally folded proteins, such as proteinase K resistant prion protein (PrPres) was a major innovation in the prion field. In prion diseases, these types of assays imitate the pathological conversion of the cellular PrP (PrPC) into a proteinase resistant associated conformer or amyloid, called PrPres. Areas covered: The most prominent protein misfolding amplification assays are the protein misfolding cyclic amplification (PMCA), which is based on sonication and the real-time quaking-induced conversion (RT-QuIC) technique based on shaking. The more recently established RT-QuIC is fully automatic and enables the monitoring of misfolded protein aggregates in real-time by using a fluorescent dye. Expert commentary: RT-QuIC is a very robust and highly reproducible test system which is applicable in diagnosis, prion strain-typing, drug pre-screening and other amyloidopathies.

Identification of new molecular alterations in fatal familial insomnia.

Fatal familial insomnia is a rare disease caused by a D178N mutation in combination with methionine (Met) at codon 129 in the mutated allele of PRNP (D178N-129M haplotype). FFI is manifested by sleep disturbances with insomnia, autonomic disorders and spontaneous and evoked myoclonus, among other symptoms. This study describes new neuropathological and biochemical observations in a series of eight patients with FFI. The mediodorsal and anterior nuclei of the thalamus have severe neuronal loss and marked astrocytic gliosis in every case, whereas the entorhinal cortex is variably affected. Spongiform degeneration only occurs in the entorhinal cortex. Synaptic and fine granular proteinase K digestion (PrPres) immunoreactivity is found in the entorhinal cortex but not in the thalamus. Interleukin 6, interleukin 10 receptor alpha subunit, colony stimulating factor 3 receptor and toll-like receptor 7 mRNA expression increases in the thalamus in FFI. PrPc levels are significantly decreased in the thalamus, entorhinal cortex and cerebellum in FFI. This is accompanied by a particular PrPc and PrPres band profile. Altered PrP solubility consistent with significantly reduced PrP levels in the cytoplasmic fraction and increased PrP levels in the insoluble fraction are identified in FFI cases. Amyloid-like deposits are only seen in the entorhinal cortex. The RT-QuIC assay reveals that all the FFI samples of the entorhinal cortex are positive, whereas the thalamus is positive only in three cases and the cerebellum in two cases. The present findings unveil particular neuropathological and neuroinflammatory profiles in FFI and novel characteristics of natural prion protein in FFI, altered PrPres and Scrapie PrP (abnormal and pathogenic PrP) patterns and region-dependent putative capacity of PrP seeding.