Alessia Farinazzo

Two‐dimensional mapping of three phenotype‐associated isoforms of the prion protein in sporadic Creutzfeldt‐Jakob disease

Transmissible spongiform encephalopathies (TSE), or prion diseases, are mammalian neurodegenerative disorders characterized by a conformational modification of the host‐encoded prion protein (PrPC) into an isoform which is detergent‐insoluble and partially resistant to protease treatment (PrPSc). Distinct types of PrPSc, differing in conformation and variation in the relative amount of their glycoforms, have been associated with different phenotypes of TSE. In sporadic Creutzfeldt‐Jakob disease (sCJD), two major types of PrPSc, with proteinase K (PK)‐resistant fragments of 21 and 19 kDa, have been described. No consensus exists, however, on the molecular classification of PrPSc in sCJD, since further heterogeneity within PrPSc conformers has been reported. We studied 19 subjects with dementia or dementia/ataxia at onset and 12 subjects with ataxia at onset. Following two‐dimensional gel electrophoresis, we characterized PrPC and PrPSc species in normal and sCJD brains by immunoblotting with antibodies recognizing N‐terminal and C‐terminal PrP regions. Three types of PrPSc were detected in detergent‐insoluble fractions from sCJD brains, mainly consisting of full‐length PrPSc in subjects with rapidly progressive dementia, and two different sets of amino‐truncated PrPSc glycoforms in subjects with dementia/ataxia and ataxia at onset. Examination of the PrPSc core fragment, following PK treatment and deglycosylation, confirmed the existence of three distinctive patterns. These findings have immediate implications for the molecular classification of sCJD.

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

The cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)‐anchored glycoprotein abundant in neurons. Although its precise function is unknown, PrPC represents the substrate for the generation of a conformational pathogenic isoform (PrPSc) in human and animal transmissible spongiform encephalopathies, or prion diseases. By applying novel solubilization cocktails, we analyzed normal human brain and cerebrospinal fluid (CSF) PrPC by immunoblot of two‐dimensional (2‐D) gel electrophoresis preparations, using specific antibodies. Here, we show that PrPC from brain and CSF is composed of several charge isomers of differently glycosylated isoforms of the full‐length PrPC and two N‐terminally truncated fragments of 20 and 18 kDa. In the CSF, substantial amounts of the highly glycosylated PrPC isoforms and of the unglycosylated 18 kDa fragment are detected. Our study, for the first time, provides a detailed 2‐D map of human PrPC both in brain and CSF, and establishes an innovative and sensitive method that might help in detecting the CSF pathological PrPSc 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).

Allelic Origin of Protease-Sensitive and Protease- Resistant Prion Protein Isoforms in Gerstmann- Straussler-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.

Analysis of mammalian scrapie protein by novel monoclonal antibodies recognizing distinct prion protein glycoforms: an immunoblot and immunohistochemical study at the light and electron microscopic levels.

The availability of specific monoclonal antibodies (mAbs) recognizing the aberrant form (PrP(Sc)) of the cellular prion protein (PrP(C)) in different mammalian species is important for molecular diagnostics, PrP(Sc) typing and future immunotherapy. We obtained a panel of anti-PrP monoclonal antibodies in PrP(0/0) knock-out mice immunized with recombinant human PrP(23-231). Two mAbs, recognizing PrP epitopes in the alpha-helix 1 (mAb SA65) and alpha-helix 2 (mAb SA21) regions, immunoreacted with PrP(C) and PrP(Sc) and its proteolytic product, PrP27-30, from human, murine, bovine, caprine and ovine brains by Western blot. Remarkably, mAb SA21 recognized unglycosylated and monoglycosylated PrP with the second site occupied by glycan moieties, but not monoglycosylated PrP with the first consensus site occupied or highly glycosylated species. Immunoblots with mAb SA21 disclosed that PrP glycosylated at the second site accounted for the slower migrating form of the customary monoglycosylated PrP doublet. mAb SA65 immunolabelled all PrP glycoforms by Western blot and was highly efficient in detecting tissue PrP by immunohistochemistry in light microscopy and in immunoelectron microscopy. These novel anti-PrP mAbs provide tools to investigate the subcellular site of PrP deposition in mammalian prion diseases and may also contribute to assess the role of different PrP glycoforms in human and animal prion diseases.

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.

Clinical spectrum and IgG subclass analysis of anti-myelin oligodendrocyte glycoprotein antibody-associated syndromes: a multicenter study

Anti-myelin oligodendrocyte glycoprotein antibodies (MOG-Ab) recently emerged as a potential biomarker in patients with inflammatory demyelinating diseases of the central nervous system. We here compare the clinical and laboratory findings observed in a cohort of MOG-Ab seropositive and seronegative cases and describe IgG subclass analysis results. Consecutive serum samples referred to Verona University Neuropathology Laboratory for aquaporin-4 (AQP4)-Ab and/or MOG-Ab testing were analysed between March 2014 and May 2017. The presence of AQP4-Ab was determined using a cell-based assay. A live cell immunofluorescence assay was used for the detection of MOG-IgG and IgG subclass analysis. Among 454 analysed samples, 29 were excluded due to AQP4-Ab positivity or to the final demonstration of a disorder not compatible with MOG-Ab. We obtained clinical data in 154 out of 425 cases. Of these, 22 subjects resulted MOG-Ab positive. MOG-Ab positive patients were mainly characterised by the involvement of the optic nerve and/or spinal cord. Half of the cases presented relapses and the recovery was usually partial. Brain MRI was heterogeneous while short lesions were the prevalent observation on spinal cord MRI. MOG-Ab titre usually decreased in non-relapsing cases. In all MOG-IgG positive cases, we observed IgG1 antibodies, which were predominant in most subjects. IgG2 (5/22), IgG3 (9/22) and IgG4 (3/22) antibodies were also detectable. We confirm that MOG-Ab-related syndromes have distinct features in the spectrum of demyelinating conditions, and we describe the possible role of the different IgG subclasses in this condition.

Mycobacterium avium subspecies paratuberculosis and myelin basic protein specific epitopes are highly recognized by sera from patients with Neuromyelitis optica spectrum disorder

Epstein-Barr virus (EBV) is the main environmental agent associated to neuromyelitis optica spectrum disorder (NMOSD). Following to studies reporting an increased prevalence of antibodies against peptides derived from Mycobacterium avium subsp. paratuberculosis (MAP) homologous to EBV and human epitopes (MBP85-98, IRF5424-434) in multiple sclerosis (MS), we investigated whether seroreactivity to these antigens display a NMOSD-specific pattern. The sera of 34 NMOSD patients showed elevated levels of antibodies against MAP and MBP compared to healthy controls (44% vs. 5%, p < 0.0002 and 50% vs. 2%, p < 0.0001, respectively), while, unlike in MS, responsiveness to EBV was similar.

2D immunomic approach for the study of IgG autoantibodies in the experimental model of multiple sclerosis.

2D-immunomics may be useful in the identification of autoantigens in neurological autoimmune diseases, but its application may be limited by denaturation of target proteins. Here we compared the capacity of a single or multiple antigens to elicit autoantibodies targeting multiple neural autoantigens by ELISA and 2D-immunomics. We induced experimental autoimmune encephalomyelitis (EAE) with MBP peptide(89-104), total MBP or spinal cord homogenate. Both techniques showed anti-MBP IgG only after immunization with total MBP. In addition, 2D-immunomics revealed the presence in EAE mice of autoantibodies targeting other neural proteins, some displaying partial sequence homology with MBP. The present finding by 2D-immunomics of multiple neural proteins targeted by autoantibodies generated by a single antigen may help to explain the complex autoimmune response observed in multiple sclerosis.

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

Background 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). Objective The objective of this paper is to investigate whether antibody (Ab) response against HERV-W env-su antigenic peptides differs between NMOSD and MS. Methods Serum samples were collected from 36 patients with NMOSD, 36 patients with MS and 36 healthy control individuals (HCs). An indirect ELISA was set up to detect specific Abs against HERV-W env-su peptides. Results Our data showed that two antigenic peptides, particularly HERV-Wenv93–108 and HERV-Wenv248–262, were statistically significantly present only in serum of MS compared to NMOSD and HCs. Thus, the specific humoral immune response against HERV-W env-su glycoprotein antigens found in MS is widely missing in NMOSD. Conclusion Increased circulating serum levels of these HERV-W Abs may be suitable as additional biomarkers to better differentiate MS from NMOSD.

Nanovesicles from adipose-derived mesenchymal stem cells inhibit T lymphocyte trafficking and ameliorate chronic experimental autoimmune encephalomyelitis

Cell based-therapies represent promising strategies for the treatment of neurological diseases. We have previously shown that adipose stem cells (ASC) ameliorate chronic experimental autoimmune encephalomyelitis (EAE). Recent evidence indicates that most ASC paracrine effects are mediated by extracellular vesicles, i.e. micro- and nanovesicles (MVs and NVs). We show that preventive intravenous administration of NVs isolated from ASC (ASC-NVs) before disease onset significantly reduces the severity of EAE and decreases spinal cord inflammation and demyelination, whereas therapeutic treatment with ASC-NVs does not ameliorate established EAE. This treatment marginally inhibits antigen-specific T cell activation, while reducing microglial activation and demyelination in the spinal cord. Importantly, ASC-NVs inhibited integrin-dependent adhesion of encephalitogenic T cells in vitro, with no effect on adhesion molecule expression. In addition, intravital microscopy showed that encephalitogenic T cells treated with ASC NVs display a significantly reduced rolling and firm adhesion in inflamed spinal cord vessels compared to untreated cells. Our results show that ASC-NVs ameliorate EAE pathogenesis mainly by inhibiting T cell extravasation in the inflamed CNS, suggesting that NVs may represent a novel therapeutic approach in neuro-inflammatory diseases, enabling the safe administration of ASC effector factors.