Identification of distinct N-terminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes.

In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules

Antibody response against HERV-W env surface peptides differentiates multiple sclerosis and neuromyelitis optica spectrum disorder

A specific humoral immune response against HERV-W envelope surface (env-su) glycoprotein antigens has been reported in serum of patients with multiple sclerosis (MS). However, it has not been evaluated to date in patients with neuromyelitis optica spectrum disorder (NMOSD)

In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF

Allelic Origin of Protease-Sensitive and Protease-Resistant Prion Protein Isoforms in Gerstmann-Sträussler-Scheinker Disease with the P102L Mutation

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrPSc, consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrPSc isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrPSc has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrPSc allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrPSc molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrPSc quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases

Intraspecies Transmission of BASE Induces Clinical Dullness and Amyotrophic Changes

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrPTSE), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrPTSE type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle

A proteomic approach to identify autoantigens in Hashimoto's encephalopathy [Un approccio proteomico per l'identificazione di autoantigeni nell'encefalopatia di Hashimoto]

Hashimoto's encephalopathy (HE) is a syndrome involving the central nervous system (CNS), characterized by an heterogeneous clinical presentation with neurological and/or neuropsychiatric symptoms associated with high titers of anti-thyroid antibodies. Although the pathogenesis of HE is still unclear, the response to steroid treatment suggests that autoimmune mechanisms may be involved. To date, the role of anti-thyroid IgG as well as the identification of the antigen(s) targeted by IgG remain unknown. We performed a proteomic study on 19 patients with HE and 15 controls based on bidimensional electrophoresis (2D) of human CNS proteins followed by immunoblotting with cerebrospinal fluid (CSF) of patients. The comparative analysis of 2D maps showed that CSF IgG from HE patients specifically recognized 3 spots in a range of pH from 5 to 7 and of MW from 31 to 37 kDa amongst a wide autoreactivity to neural antigens. After mass spectrometry analysis and immunoblotting with specific antibodies, these proteins were identified as dimethylargininase-I (DDAHI) and aldehyde reductase-I (AKRIAI). Almost 90% of HE patients targeted at least one of two isoforms of DDAHI, while few cases recognized AKRIAI. The present findings suggest DDAHI and AKRIAI as biomarkers of HE although further experimental evidence is needed to understand their pathogenetic and diagnostic role, if any. Our 2D proteomic approach appears to be a sensitive and reliable method to assess the autoimmune repertoire in HE, helping to identify potential autoantigens in CSF and sera of HE patients

Murine adipose-derived mesenchymal stromal cell vesicles: in vitro clues for neuroprotective and neuroregenerative approaches

BACKGROUND AIMS: Adipose-derived mesenchymal stromal cells (ASC) are known to promote neuroprotection and neuroregeneration in vitro and in vivo. These biological effects are probably mediated by paracrine mechanisms. In recent years, nanovesicles (NV) and microvesicles (MV) have been shown to play a major role in cell-to-cell communication. We tested the efficacy of NV and MV obtained from ASC in mediating neuroprotection and neuroregeneration in vitro.METHODS: We exposed neuronal cells (both cell line and primary cultures) to oxidative stress in the presence or not of NV or MV.RESULTS: In this experimental setting, we found that low doses of NV or MV protected neurons from apoptotic cell death. We then assessed the neuroregenerative effect of NV/MV in cerebellar slice cultures demyelinated with lysophosphatidylcholine. We observed that low but not higher doses of NV and MV increased the process of remyelination and activated nestin-positive oligodendroglial precursors.CONCLUSIONS: Taken together, our data in vitro support the relevance of ASC vesicles as a source of protecting and regenerating factors that might modulate the microenvironment in neuro-inflammatory as well as in neurodegenerative disorders. The present findings may suggest that stromal cell-derived vesicles might represent a potential therapeutic tool, enabling the safe administration of stromal cell effector factors, avoiding the cellular counterpart

Comparative two-dimensional mapping of prion protein isoforms in human cerebrospinal fluid and central nervous system

The cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein abundant in neurons. Although its precise function is unknown, PrP(C) represents the substrate for the generation of a conformational pathogenic isoform (PrP(Sc)) in human and animal transmissible spongiform encephalopathies, or prion diseases. By applying novel solubilization cocktails, we analyzed normal human brain and cerebrospinal fluid (CSF) PrP(C) by immunoblot of two-dimensional (2-D) gel electrophoresis preparations, using specific antibodies. Here, we show that PrP(C) from brain and CSF is composed of several charge isomers of differently glycosylated isoforms of the full-length PrP(C) and two N-terminally truncated fragments of 20 and 18 kDa. In the CSF, substantial amounts of the highly glycosylated PrP(C) isoforms and of the unglycosylated 18 kDa fragment are detected. Our study, for the first time, provides a detailed 2-D map of human PrP(C) both in brain and CSF, and establishes an innovative and sensitive method that might help in detecting the CSF pathological PrP(Sc) isoform in vivo. It also shows the incredible microheterogeneity of such isoforms (ca. 60 spots!), as revealed in 2-D mapping, as opposed to 3-4 main zones by mono-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Serum and cerebrospinal neurofilament light chain levels in patients with acquired peripheral neuropathies

Neurofilament light chain (NFL) levels reflect axonal damage in different inflammatory and neurodegenerative central nervous system conditions, in correlation with disease severity. Our aim was to determine the possible diagnostic and prognostic value of serum and cerebrospinal fluid (CSF) NFL levels in subjects with different forms of acquired peripheral neuropathies (PN). Paired serum and CSF samples of 25 patients with acquired PN were analysed for NFL using an ultrasensitive technique (Quanterix, Simoa, Lexington, MA, USA) and compared with a group of 25 age-matched healthy subjects. Demographic, clinical, CSF and neurophysiological data were reviewed. Cases with Guillain-Barr\ue9 syndrome (N\u2009=\u20095), multifocal motor neuropathy (N\u2009=\u20093), chronic inflammatory demyelinating polyneuropathy (CIDP) and variants (N\u2009=\u200912), anti-myelin-associated glycoprotein (MAG) neuropathy (N\u2009=\u20093), both CIDP and anti-MAG neuropathy (N\u2009=\u20091), and non-systemic vasculitic neuropathy (N\u2009=\u20091) were studied. NFL levels were significantly (P\u2009<\u20090.001) increased in patients with PN and were higher in the CSF (median: 1407\u2009pg/mL, range: 140.2-12\u2009661) than in serum (median: 31.52\u2009pg/mL, range: 4.33-1178). A statistically significant correlation was observed between serum and CSF levels in cases with blood-nerve-barrier damage (r\u2009=\u20090.71, P\u2009<\u20090.01), and between serum NFL levels and disease activity at sampling (r\u2009=\u20090.52, P\u2009<\u20090.01) and at last follow-up (r\u2009=\u20090.53, P\u2009<\u20090.01) in all subjects. The increase of NFL values in both serum and CSF of patients with acquired PN and the significant correlation between serum NFL levels, disease severity and final outcome support the possible role of NFL as disease activity and prognostic biomarker also in peripheral nervous system disorders