The conversion of the prion protein (PrPC) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrPC interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrPC constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrPC coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.

The pathological deposition of the transactive response DNA-binding protein of 43 kDa (TDP-43) occurs in the majority (∼97%) of amyotrophic lateral sclerosis and in around 45% of frontotemporal lobar degeneration cases. Amyotrophic lateral sclerosis and frontotemporal lobar degeneration clinically overlap, presenting a continuum of phenotypes. Both amyotrophic lateral sclerosis and frontotemporal lobar degeneration lack treatments able to interfere with the underlying pathological process and early detection of TDP-43 pathology would facilitate the development of disease modifying drugs. The Real Time Quaking Induced Conversion reaction (RT-QuIC) showed the ability to detect prions in several peripheral tissues of patients with different forms of prion and prion-like diseases. Despite TDP-43 displays prion-like properties, to date the RT-QuIC technology has not yet been adapted to this protein. The aim of this study was to adapt the RT-QuIC technique for the TDP-43 substrate and to exploit the intrinsic ability of this technology to amplify minutes amount of misfolded proteins for the detection of pathological TDP-43 species in the CSF of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. We first optimized the technique with synthetic TDP-43 preformed aggregates and with autopsy-verified brain homogenate samples and subsequently analyzed CSF samples from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients and controls. TDP-43 RT-QuIC was able to detect as little as 15 picograms of TDP-43 aggregates, discriminating between a cohort of subjects affected by amyotrophic lateral sclerosis and frontotemporal lobar degeneration and age-matched controls with a total sensitivity of 94% and a specificity of 85%. Our data give a proof-of-concept that TDP-43 is a suitable substrate for the RT-QuIC. TDP-43 RT-QuIC could be an innovative and useful tool for diagnosis and drug development in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. CSF detection of TDP-43 pathological aggregates may be exploited as a disease biomarker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.

The cellular prion protein (PrP C ), encoded by the PRNP gene, is a ubiquitous glycoprotein, which is highly expressed in the brain. This protein, mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neurite outgrowth. By using a novel focal stimulation technique, we explored the potential function of PrP C , in its soluble form, as a signaling molecule. Thus, soluble recombinant prion proteins (recPrP) encapsulated in micro-vesicles were released by photolysis near the hippocampal growth cones. Local stimulation of wild-type growth cones with full-length recPrP induced neurite outgrowth and rapid growth cone turning towards the source. This effect was shown to be concentration dependent. Notably, PrP C -knockout growth cones were insensitive to recPrP stimulation, but this property was rescued in PrPknockout growth cones expressing GFP-PrP. Taken together, our findings indicate that recPrP functions as a signaling molecule, and that its homophilic interaction with membrane-anchored PrP C might promote neurite outgrowth and facilitate growth cone guidance.

The cellular prion protein (PrPC), mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neuritogenesis. By combining genomic approaches, cellular assays and focal stimulation technique, we have explored the molecular mechanism underlining PrPC function as a signaling molecule in neuritogenesis. Several recombinant prion protein (recPrP) mutants were obtained to treat primary hippocampal cultures in bulk or exposed near the hippocampal growth cones (GC) of single neurons in a local stimulation manner. While focal stimulation of GC with wild‐type recPrP induced neurite outgrowth and rapid GC turning towards the source, N‐terminal mutants fail to support this function. In particular, the copper‐binding sites mutants present at the N‐terminus of PrPC are toxic to neurons indicating this region being crucial for the function of the protein. Mutants of recPrP including a key mutation for prion conversion H95Y (Giachin, Mai et al. 2015) or a GSS‐linked mutation abolish the function on neuritogenesis. Altogether, our findings indicate the functional regions for PrPC involved in neuritogenesis, suggest a potential link between loss‐of‐function of the protein and disease initiation. Support or Funding Information Fondo per gli Investimenti della Ricerca di Base (FIRB) program project (grant number RBAP11FRE9_001 to GL) from Ministero dell'Istruzione, dell'Università e della Ricerca(MIUR), Italy This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Serpins represent the most broadly distributed superfamily of proteases inhibitors. They contribute to a variety of physiological functions and any alteration of the serpin-protease equilibrium can lead to severe consequences. SERPINA3 dysregulation has been associated with Alzheimer’s disease (AD) and prion diseases. In this study, we investigated the differential expression of serpin superfamily members in neurodegenerative diseases. SERPIN expression was analyzed in human frontal cortex samples from cases of sporadic Creutzfeldt-Jakob disease (sCJD), patients at early stages of AD–related pathology, and age-matched controls not affected by neurodegenerative disorders. In addition, we studied whether Serpin expression was dysregulated in two animal models of prion disease and AD.Our analysis revealed that, besides the already observed upregulation of SERPINA3 in patients with prion disease and AD, SERPINB1, SERPINB6, SERPING1, SERPINH1, and SERPINI1 were dysregulated in sCJD individuals compared to controls, while only SERPINB1 was upregulated in AD patients. Furthermore, we analyzed whether other serpin members were differentially expressed in prion-infected mice compared to controls and, together with SerpinA3n, SerpinF2 increased levels were observed. Interestingly, SerpinA3n transcript and protein were upregulated in a mouse model of AD. The SERPINA3/SerpinA3nincreased anti-protease activity found in post-mortem brain tissue of AD and prion disease samples suggest its involvement in the neurodegenerative processes. A SERPINA3/SerpinA3n role in neurodegenerative disease-related protein aggregation was further corroborated by in vitro SerpinA3n-dependent prion accumulation changes. Our results indicate SERPINA3/SerpinA3n is a potential therapeutic target for the treatment of prion and prion-like neurodegenerative diseases.