Humoral immunity response to human endogenous retroviruses K/W differentiates between amyotrophic lateral sclerosis and other neurological diseases.

BACKGROUND AND PURPOSE: Human endogenous retroviruses (HERV) K/W seem to play a role in fostering and exacerbation of some neurological diseases, including amyotrophic lateral sclerosis (ALS). Given these findings, the immunity response against HERV-K and HERV-W envelope surface (env-su) glycoprotein antigens in serum and cerebrospinal fluid (CSF) was investigated for ALS, multiple sclerosis (MS) and Alzheimer's disease patients and in healthy controls. METHODS: Four antigenic peptides derived respectively from HERV-K and HERV-W env-su proteins were studied in 21 definite or probable ALS patients, 26 possible or definite relapsing-remitting MS patients, 18 patients with Alzheimer's disease and 39 healthy controls. An indirect enzyme-linked immunosorbent assay was set up to detect specific antibodies (Abs) against env-su peptides. RESULTS: Amongst the measured levels of Abs against the four different HERV-K peptide fragments, only HERV-K env-su19-37 was significantly elevated in ALS compared to other groups, both in serum and CSF. Instead, amongst the Abs levels directed against the four different HERV-W peptide fragments, only HERV-W env-su93-108 and HERV-W env-su248-262 were significantly elevated, in the serum and CSF of the MS group compared to other groups. In ALS patients, the HERV-K env-su19-37 Abs levels were significantly correlated with clinical measures of disease severity, both in serum and CSF. CONCLUSIONS: Increased circulating levels of Abs directed against the HERV-W env-su93-108 and HERV-W env-su248-262 peptide fragments could serve as possible biomarkers in patients with MS. Similarly, increased circulating levels of Abs directed against the HERV-K env-su19-37 peptide fragment could serve as a possible early novel biomarker in patients with ALS.

Differential overexpression of SERPINA3 in human prion diseases.

Prion diseases are fatal neurodegenerative disorders with sporadic, genetic or acquired etiologies. The molecular alterations leading to the onset and the spreading of these diseases are still unknown. In a previous work we identified a five-gene signature able to distinguish intracranially BSE-infected macaques from healthy ones, with SERPINA3 showing the most prominent dysregulation. We analyzed 128 suitable frontal cortex samples, from prion-affected patients (variant Creutzfeldt-Jakob disease (vCJD) n = 20, iatrogenic CJD (iCJD) n = 11, sporadic CJD (sCJD) n = 23, familial CJD (gCJD) n = 17, fatal familial insomnia (FFI) n = 9, Gerstmann-Sträussler-Scheinker syndrome (GSS)) n = 4), patients with Alzheimer disease (AD, n = 14) and age-matched controls (n = 30). Real Time-quantitative PCR was performed for SERPINA3 transcript, and ACTB, RPL19, GAPDH and B2M were used as reference genes. We report SERPINA3 to be strongly up-regulated in the brain of all human prion diseases, with only a mild up-regulation in AD. We show that this striking up-regulation, both at the mRNA and at the protein level, is present in all types of human prion diseases analyzed, although to a different extent for each specific disorder. Our data suggest that SERPINA3 may be involved in the pathogenesis and the progression of prion diseases, representing a valid tool for distinguishing different forms of these disorders in humans.

Neurosyphilis manifesting with rapidly progressive dementia: report of three cases.

Neurosyphilis is rather an unusual cause of dementia characterized by a rapidly progressive course and psychiatric symptoms. Diagnosis of neurosyphilis should be suspected in the presence of a global cognitive impairment consisting in disorientation, amnesia and severe impairment of speech and judgement and psychiatric symptoms such as depression, mania and psychosis, with a subacute onset. More commonly, clinical manifestations of neurosyphilis include general PARESIS (involvement of Personality, Affect, Reflexes, Eye, Sensorium, Intellect and Speech). Upon clinical suspicion, diagnosis of neurosyphilis is confirmed by a reactive cerebrospinal fluid (CSF)-Venereal Disease Research Laboratory. Here we report three Human Immunodeficiency Virus (HIV)-negative male patients presenting with psychiatric symptoms and a rapidly evolving dementia. Although magnetic resonance imaging did not address to diagnosis, CSF examination was mandatory in neurosyphilis diagnosis. Other diagnostic tools such as neuropsychology and single-photon emission computed tomography resulted supportive in the diagnosis. We showed that a prompt antibiotic treatment might stop disease progression. Therefore, neurosyphilis should be always considered even in HIV-negative patients in the presence of unexpected psychiatric symptoms accompanied by a rapidly evolving cognitive decline.

Clinical and biomarker assessment of demyelinating events suggesting multiple sclerosis.

BACKGROUND: Initial demyelinating event (IDE) diagnosis and prognosis are not straightforward. OBJECTIVE: To identify potential diagnostic markers and outcome predictors of IDEs suggestive of multiple sclerosis (MS), that is, clinically isolated syndromes (CISs). METHODS: Clinically isolated syndrome cases (i.e., subjects with an IDE compatible with MS onset and no alternative explanation) with at least 1.5 years' follow-up were retrospectively identified. All cases underwent clinical, neurophysiological, MRI, and cerebrospinal fluid (CSF) assessment, including exploratory tau, 14-3-3, and cystatin C testing. CIS recovery, conversion to MS, and long-term neurological disability were used as outcome measures. Patients with neuromyelitis optica spectrum disorders, idiopathic acute transverse myelitis (IATM), Creutzfeldt-Jacob disease, and non-inflammatory/non-neurodegenerative disorders served as controls for CSF analysis. RESULTS: Forty-six CIS cases were included. Severe presentation was associated with incomplete recovery, while presence of at least 3 periventricular lesions on baseline MRI correlated with MS conversion. Initial pyramidal tract involvement, incomplete CIS recovery, and number of relapses predicted neurological disability. CSF tau, 14-3-3, and cystatin C did not correlate with any outcome measure. CIS cases had significantly lower tau and cystatin C levels compared to IATM. CONCLUSIONS: An extensive diagnostic evaluation of patients with an IDE is worthwhile to make prognostic predictions. More robust molecular biomarkers are needed.

The oldest old Creutzfeldt-Jakob disease case.

Sporadic Creutzfeldt-Jakob Disease (sCJD) is a rapidly progressive neurodegenerative disorder usually affecting people between 60 and 70 years old, with only anecdotal cases presenting at 90 years or older. The clinical phenotype of sCJD is highly variable. Diagnosis of sCJD should be considered in the differential diagnosis of rapidly evolving ataxic or dementing syndromes with or without epileptic seizures, regardless of the patient age. While the recognition of atypical phenotypes in subject 90 years or older can provide additional diagnostic challenge, it must be underlined that neuropathology is still the "gold standard" for sCJD diagnosis.

Detection of CSF 14-3-3 protein in Guillain-Barré syndrome.

OBJECTIVE: To search for biologic markers in the Guillain-Barré syndrome (GBS), we studied CSF samples from patients with GBS and neuropathy of various etiologies for the presence of 14-3-3 protein. METHODS: CSF samples from patients with GBS, chronic neuropathies, motor neuron disease (MND), definite sporadic Creutzfeldt-Jakob disease (sCJD), and normal control subjects were analyzed by standard immunoblot assay, using a polyclonal anti-14-3-3 antibody. CSF samples were also tested with antibodies recognizing specific isoforms of 14-3-3 proteins, either after one-dimensional or two-dimensional electrophoretic separation. RESULTS: A positive 14-3-3 assay was observed in 29 of 38 patients with GBS and in 4 patients with MND and other neuropathies, including 2 subjects with vasculitic neuropathy (VN). In GBS, 14-3-3 protein was detected as early as 12 to 48 hours after disease onset and showed an isoform pattern different from that encountered in patients with noninflammatory neuropathies, VN, MND, and sCJD. Immunohistochemical studies performed in archival fatal GBS cases disclosed marked 14-3-3 expression by mononuclear inflammatory infiltrates and Schwann cells. CONCLUSION: CSF 14-3-3 assay may represent a useful biologic marker in patients with Guillain-Barré syndrome.

Phosphorylated 14-3-3zeta protein in the CSF of neuroleptic-treated patients.

The authors describe 12 neuroleptic-treated patients with dementia of various etiologies who showed CSF elevation of phosphorylated 14-3-3zeta and normal tau protein levels. This contrasted with elevated amounts of 14-3-3 gamma, epsilon, and unphosphorylated zeta coupled to high tau protein levels in Creutzfeldt-Jakob disease and negative 14-3-3 assay in drug-free patients with dementia. Characterization of CSF 14-3-3 isoforms and determination of tau protein level can help to distinguish different etiologies of dementia.

pH-dependent prion protein conformation in classical Creutzfeldt-Jakob disease.

In transmissible spongiform encephalopathies, the cellular prion protein (PrP(C)) undergoes a conformational change from a prevailing alpha-helical structure to a beta-sheet-rich, protease-resistant isoform, termed PrP(Sc). PrP(C) has two characteristics: a high affinity for Cu(2+) and a strong pH-dependent conformation. Lines of evidence indicate that PrP(Sc) conformation is dependent on copper and that acidic conditions facilitate the conversion of PrP(C) --> PrP(Sc). In each species, PrP(Sc) exists in multiple conformations, which are associated with different prion strains. In sporadic Creutzfeldt-Jakob disease (sCJD), different biochemical types of PrP(Sc) have been identified according to the size of the protease-resistant fragments, patterns of glycosylation, and the metal-ion occupancy. Based on the site of cleavage produced by proteinase K, we investigated the conformational stability of PrP(Sc) under acidic, neutral, and basic conditions in 42 sCJD subjects. Our study shows that only one type of sCJD PrP(Sc), associated with the classical form, shows a pH-dependent conformation, whereas two other biochemical PrP(Sc) types, detected in distinct sCJD phenotypes, are unaffected by pH variations. This novel approach demonstrates the presence of three types of PrP(Sc) in sCJD.

Increased expression of the normal cellular isoform of prion protein in inclusion-body myositis, inflammatory myopathies and denervation atrophy.

The cellular isoform of the prion protein (PrPc) is a glycosylphosphatidylinositol-anchored glycoprotein, normally expressed in neural and non-neural tissues, including skeletal muscle. In transmissible spongiform encephalopathies, or prion diseases, PrPc, which is soluble in nondenaturing detergent and sensitive to proteinase K (PK)-treatment, represents the molecular substrate for the production of a detergent-insoluble and PK-resistant isoform, termed PrP(Sc). In human prion diseases, PrP(Sc) accumulation occurs only in brain tissues, with the exception of new variant Creutzfeldt-Jakob disease, where PrP(Sc) is also detected in lymphoid tissues. Increased amounts of prion protein expression and deposition have been described in pathological muscle fibers of two human muscle disorders, called sporadic inclusion-body myositis (s-IBM) and hereditary inclusion-body myopathy, but it is unknown whether accumulated prion protein reflects normal PrPc or PrP(Sc). We investigated the biochemical characteristics of prion protein in normal human muscle, s-IBM, other inflammatory myopathies and denervation atrophy. We report that 1) both the glycoform profile and size of the normal muscle PrPc are different from those of human brain PrPc; 2) in addition to s-IBM, increased PrPc expression is seen in polymyositis, dermatomyositis and neurogenic muscle atrophy, but PrPc glycoforms are unchanged; 3) only the normal PrPc isoform, and not PrP(Sc), is detected in s-IBM. The present results exclude that s-IBM is a prion disease.

The expression and potential function of cellular prion protein in human lymphocytes.

We examined expression of the normal cellular prion protein (PrP(C)) in human peripheral blood mononuclear cells (PBMC) and in transfected neuroblastoma cells with a panel of six monoclonal antibodies (Mabs). While all six of the Mabs reacted strongly with the neuroblastoma cells, only four of the Mabs reacted with PrP(C) expressed by human PBMC. PrP(C) is expressed at high levels in human T cells, B cells, monocytes, and dendritic cells, but not in red blood cells. Immunoblotting studies revealed that the PrP(C) glycoforms and the composition of the N-linked glycans on PrP(C) in human PBMC are different from those of the brain or the neuroblastoma cells. In human PBMC and the neuroblastoma cell lines the N-terminal portion of the PrP(C) is hypersensitive to proteolytic digestion, suggesting that the N-terminus of the PrP(C) on the surface of a living cell lacks secondary structure. We found that the level of PrP(C) expressed on the surface of human T lymphocytes was up-regulated as a consequence of cellular activation. Accordingly, memory T cells express more PrP(C) than naïve T cells. In addition, the proliferation of human T lymphocytes stimulated with an anti-CD3 Mab was inhibited by anti-PrP(C) Mabs. Collectively, these results suggest that PrP(C) can participate in signal transduction in human T lymphocytes.

The chaperone protein BiP binds to a mutant prion protein and mediates its degradation by the proteasome.

Familial prion diseases are thought to result from a change in structure of the mutant prion protein (PrP), which takes a pathogenic conformation. We have examined the role of molecular chaperones in the folding of normal and mutant PrP Q217R (PrP(217)) in transfected neuroblastoma cells. In a previous report we showed that, although most of the PrP(217) forms escape the endoplasmic reticulum quality control system and aggregate in post-Golgi compartments, a significant proportion of PrP(217) retains the C-terminal glycosylphosphatidyl inositol signal peptide (PrP32), and does not exit the endoplasmic reticulum (Singh, N., Zanusso, G., Chen, S. G., Fujioka, H., Richardson, S., Gambetti, P., and Petersen, R. B. (1997) J. Biol. Chem. 272, 28461-28470). We have now studied the folding and turnover of PrP32 to understand the mechanism by which abnormal PrP forms cause cellular toxicity in our cell culture model and in the human brain carrying the Gerstmann-Sträussler-Scheinker disease Q217R mutation. In this report, we show that PrP32 remains associated with the chaperone BiP for an abnormally prolonged period of time and is degraded by the proteasomal pathway. This study is the first demonstration that BiP is chaperoning the folding of PrP and plays a role in maintaining the quality control in the PrP maturation pathway. Our data provide new insight into the diverse pathways of mutant PrP metabolism and neurotoxicity.

Tumor necrosis factor alpha and human Schwann cells: signalling and phenotype modulation without cell death.

The aim of the study was to evaluate the biological response of human Schwann cells (SC) to tumor necrosis factor alpha (TNFalpha) in vitro and to the inflammatory milieu of chronic inflammatory demyelinating polyradiculoneuritis (CIDP). By immunocytochemical and functional assays, we found that SC expressed TNF receptors and that TNFalpha promoted in SC cultures transient activation of transcription factors NFkappaB and c-jun in the absence of apoptosis. In addition, TNFalpha significantly increased the proportion of non-myelin-forming SC expressing the p75 nerve growth factor receptor. Such phenotypic effect was dose-dependent and partially mediated by NFkappaB, as assessed by functional blockage with acetylsalicylic acid. We then extended our study to a human disease in which SC are exposed to TNFalpha. Increased signals for NFkappaB, but not c-jun, molecules were observed by immunohistochemistry on SC nuclei in nerve biopsies from patients with CIDP, as compared with controls. Irrespective of the presence of nerve inflammation, SC showed no evidence of apoptosis. Taken together, our results suggested that SC are potential targets of TNFalpha and that this cytokine exerted no cytotoxic effects either in vivo or in vitro. Rather, TNFalpha may influence the fate of SC by activating transcriptional pathways and modulating their phenotype.

Corticobasal degeneration shares a common genetic background with progressive supranuclear palsy.

Corticobasal degeneration is a sporadic form of tauopathy, involving the cerebral cortex and extrapyramidal motor system. A series of affected subjects was genotyped for a set of genetic markers along the tau protein gene. A specific haplotype is significantly overrepresented in patients versus controls. This haplotype is the same already reported in association with progressive supranuclear palsy. These data show that corticobasal degeneration and progressive supranuclear palsy, in addition to several clinical, pathological, and molecular features, may have the same genetic background.

Proteasomal degradation and N-terminal protease resistance of the codon 145 mutant prion protein.

An amber mutation at codon 145 (Y145stop) of the prion protein gene results in a variant of an inherited human prion disease named Gerstmann-Sträussler-Scheinker syndrome. The characteristic features of this disorder include amyloid deposits of prion protein in cerebral parenchyma and vessels. We have studied the biosynthesis and processing of the prion protein containing the Y145stop mutation (PrP(145)) in transfected human neuroblastoma cells in an attempt to clarify the effect of the mutation on the metabolism of PrP(145) and to gain insight into the underlying pathogenetic mechanism. Our results demonstrate that 1) a significant proportion of PrP(145) is not processed post-translationally and retains the N-terminal signal peptide, 2) most PrP(145) is degraded very rapidly by the proteasome-mediated pathway, 3) blockage of proteasomal degradation results in intracellular accumulation of PrP(145), 4) most of the accumulated PrP(145) is detergent-insoluble, and both the detergent-soluble and -insoluble fractions are resistant to mild proteinase K (PK) treatment, suggesting that PK resistance is not simply because of aggregation. The present study demonstrates for the first time that a mutant prion protein is degraded through the proteasomal pathway and acquires PK-resistance if degradation is impaired.

Prion protein expression in different species: analysis with a panel of new mAbs.

By immunizing prion knockout mice (Prnp-/-) with recombinant murine prion protein (PrPc), we obtained a panel of mAbs specific for murine PrPc. These mAbs can be applied to immunoblotting, cell surface immunofluorescent staining, and immunohistochemistry at light and electron microscopy. These mAbs recognize both the normal (PrPc) and protease-resistant (PrPres) isoforms of PrP. Some mAbs are species restricted, while others react with PrP from a broad range of mammals including mice, humans, monkeys, cows, sheep, squirrels, and hamsters. Moreover, some of the mAbs selectively recognize different PrP glycoforms as well as the metabolic fragments of PrPc. These newly generated PrPc antibodies will help to explore the biology of PrPc and to establish the diagnosis of prion diseases in both humans and animals.

HIV-associated PML presenting as epilepsia partialis continua.

Two patients with human immunodeficiency virus (HIV) type 1 infection presented new-onset epilepsia partialis continua (EPC) as an early manifestation of progressive multifocal leukoencephalopathy (PML). EPC occurred with no other seizures and was associated with negative radiographic and electrophysiological findings for several weeks. PML represents an increasingly recognized cause of new-onset seizures in both seropositive and seronegative patients, with no report of EPC as a presenting complaint.

Prion protein aggregation reverted by low temperature in transfected cells carrying a prion protein gene mutation.

Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC), a glycoprotein that is anchored to the cell membrane by a glycosylphosphatidylinositol moiety, into an isoform that is protease-resistant (PrPres) and pathogenic. In inherited prion diseases, mutations in the prion protein (PrPM) engender the conversion of PrPM into PrPres. We developed a cell model of Gerstmann-Sträussler-Scheinker disease, a neurodegenerative condition characterized by PrPM-containing amyloid deposits and neuronal loss, by expressing the Gerstmann-Sträussler-Scheinker haplotype Q217R-129V in human neuroblastoma cells. By comparison to PrPC, this genotype results in the following alterations of PrPM: 1) expression of an aberrant form lacking the glycosylphosphatidylinositol anchor, 2) increased aggregation and protease resistance, and 3) impaired transport to the cell surface. Most of these alterations are temperature-sensitive, indicating that they are due to misfolding of PrPM.