Pedro Piccardo

Host PrP Glycosylation: A Major Factor Determining the Outcome of Prion Infection

The expression of the prion protein (PrP) is essential for transmissible spongiform encephalopathy (TSE) or prion diseases to occur, but the underlying mechanism of infection remains unresolved. To address the hypothesis that glycosylation of host PrP is a major factor influencing TSE infection, we have inoculated gene-targeted transgenic mice that have restricted N-linked glycosylation of PrP with three TSE strains. We have uniquely demonstrated that mice expressing only unglycosylated PrP can sustain a TSE infection, despite altered cellular location of the host PrP. Moreover we have shown that brain material from mice infected with TSE that have only unglycosylated PrPSc is capable of transmitting infection to wild-type mice, demonstrating that glycosylation of PrP is not essential for establishing infection within a host or for transmitting TSE infectivity to a new host. We have further dissected the requirement of each glycosylation site and have shown that different TSE strains have dramatically different requirements for each of the glycosylation sites of host PrP, and moreover, we have shown that the host PrP has a major role in determining the glycosylation state of de novo generated PrPSc.

Immunohistochemical localization of prion protein in spongiform encephalopathies and normal brain tissue

We used polyclonal antibodies raised against hamster and mouse PrP27–30 as immunologic probes to study the localization of intracellular and extracellular deposits of prion protein in normal and scrapie-infected mouse and hamster brains and in Creutzfeldt-Jakob disease (CJD)-infected mouse brains. In addition, we examined normal human brain and brain tissues from patients with CJD, kuru, Alzheimers disease, and idiopathic chronic encephalitis. There was positive staining in the cytoplasm of neurons of normal and scrapie- and CJD-infected mice, and in the neurons of normal and scrapie-infected hamsters. The staining pattern suggests the localization of PrP in an intracellular membrane compartment, most likely the rough endoplasmic reticulum or Golgi apparatus. Antibodies raised against a 15-amino-acid synthetic peptide of the N-terminal of hamster PrP27–30 displayed a similar pattern of staining in mouse brain sections. We observed no intracellular staining in human brain sections obtained at autopsy. Antibodies prepared against mouse and hamster PrP27–30 reacted with amyloid plaques in scrapie-infected mouse and kuru- and CJD-infected human brain sections but not with amyloid plaques in the brain of a patient with Alzheimers disease.

Neurodegenerative Illness in Transgenic Mice Expressing a Transmembrane Form of the Prion Protein

Although PrPSc is thought to be the infectious form of the prion protein, it may not be the form that is responsible for neuronal cell death in prion diseases. CtmPrP is a transmembrane version of the prion protein that has been proposed to be a neurotoxic intermediate underlying prion-induced pathogenesis. To investigate this hypothesis, we have constructed transgenic mice that express L9R-3AV PrP, a mutant prion protein that is synthesized exclusively in the CtmPrP form in transfected cells. These mice develop a fatal neurological illness characterized by ataxia and marked neuronal loss in the cerebellum and hippocampus. CtmPrP in neurons cultured from transgenic mice is localized to the Golgi apparatus, rather than to the endoplasmic reticulum as in transfected cell lines. Surprisingly, development of the neurodegenerative phenotype is strongly dependent on coexpression of endogenous, wild-type PrP. Our results provide new insights into the cell biology of CtmPrP, the mechanism by which it induces neurodegeneration, and possible cellular activities of PrPC.

Clinical and molecular genetic study of a large German kindred with Gerstmann‐Straussler‐Scheinker syndrome

We have verified, by full open reading frame sequencing, the presence of an amino-acid-altering mutation in codon 102 of the scrapie amyloid protein gene in three affected members of a large and well-documented German family with experimentally transmitted Gerstmann-Straussler-Scheinker syndrome. In addition, we identified the mutation by partial sequencing or DNA restriction enzyme analysis in three of 12 presently healthy family members with an affected parent, and none of 12 members without an affected parent. Thus, a total of six of 15 family members at risk for the disease (including the three established cases) had the same codon 102 mutation, a proportion consistent with the autosomal dominant inheritance pattern of disease expression. It is undetermined whether the mutation influences susceptibility to infection by an exogenous agent or is itself a proximate cause of disease.

Insomnia associated with thalamic involvement in E200K Creutzfeldt–Jakob disease

BackgroundInsomnia with predominant thalamic involvement and minor cortical and cerebellar pathologic changes is not characteristic of familial Creutzfeldt–Jakob disease (CJD) but is a hallmark of fatal familial insomnia. ObjectiveTo report a 53-year-old woman with intractable insomnia as her initial symptom of disease. MethodsThe authors characterized clinical, pathologic, and molecular features of the disease using EEG, polysomnography, neurohistology, Western blotting, protein sequencing, and prion protein (PrP) gene (PRNP) analysis. ResultsThe patient developed dysgraphia, dysarthria, bulimia, myoclonus, memory loss, visual hallucinations, and opisthotonos, as well as pyramidal, extrapyramidal, and cerebellar signs. Polysomnographic studies showed an absence of stages 3 and 4, and REM. She died 8 months after onset. On neuropathologic examination, there was major thalamic involvement characterized by neuronal loss, spongiform changes, and prominent gliosis. The inferior olivary nuclei exhibited chromatolysis, neuronal loss, and gliosis. Spongiform changes were mild in the neocortex and not evident in the cerebellum. PrP immunopositivity was present in these areas as well as in the thalamus. PRNP analysis showed the haplotype E200K-129M. Western blot analysis showed the presence of proteinase K (PK)–resistant PrP (PrPsc) with the nonglycosylated isoform of approximately 21 kd, corresponding in size to that of type 1 PrPsc. N-terminal protein sequencing demonstrated PK cleavage sites at glycine (G) 82 and G78, as previously reported in CJD with the E200K-129 M haplotype. ConclusionsInsomnia may be a prominent early symptom in cases of CJD linked to the E200K-129M haplotype in which the thalamus is severely affected.

Prion Protein Interacts with BACE1 Protein and Differentially Regulates Its Activity toward Wild Type and Swedish Mutant Amyloid Precursor Protein

In Alzheimer disease amyloid-β (Aβ) peptides derived from the amyloid precursor protein (APP) accumulate in the brain. Cleavage of APP by the β-secretase BACE1 is the rate-limiting step in the production of Aβ. We have reported previously that the cellular prion protein (PrPC) inhibited the action of BACE1 toward human wild type APP (APPWT) in cellular models and that the levels of endogenous murine Aβ were significantly increased in PrPC-null mouse brain. Here we investigated the molecular and cellular mechanisms underlying this observation. PrPC interacted directly with the prodomain of the immature Golgi-localized form of BACE1. This interaction decreased BACE1 at the cell surface and in endosomes where it preferentially cleaves APPWT but increased it in the Golgi where it preferentially cleaves APP with the Swedish mutation (APPSwe). In transgenic mice expressing human APP with the Swedish and Indiana familial mutations (APPSwe,Ind), PrPC deletion had no influence on APP proteolytic processing, Aβ plaque deposition, or levels of soluble Aβ or Aβ oligomers. In cells, although PrPC inhibited the action of BACE1 on APPWT, it did not inhibit BACE1 activity toward APPSwe. The differential subcellular location of the BACE1 cleavage of APPSwe relative to APPWT provides an explanation for the failure of PrPC deletion to affect Aβ accumulation in APPSwe,Ind mice. Thus, although PrPC exerts no control on cleavage of APPSwe by BACE1, it has a profound influence on the cleavage of APPWT, suggesting that PrPC may be a key protective player against sporadic Alzheimer disease.

Creutzfeldt-Jakob disease and kuru patients lack a mutation consistently found in the Gerstmann-Sträussler-Scheinker syndrome

We and others have recently reported that patients with the Gerstmann-Sträussler-Scheinker syndrome have a mutation at codon 102 of the gene coding for amyloid protein that accumulates in this disease. We report here that this mutation was not found in 5 familial and 27 sporadic cases of Creutzfeldt-Jakob disease or in 3 patients with kuru, so that although this mutation may be responsible for amyloidogenesis and transmissibility in Gerstmann-Sträussler-Scheinker syndrome, it cannot be the only cause of human spongiform encephalopathy.

Subcellular distribution and physicochemical properties of scrapie‐associated precursor protein and relationship with scrapie agent

We studied the biologic properties of hamster-adapted scrapie (strain 263K) and its relationship to the precursor protein of scrapie (PrP33–35Sc). The highest titer of infectious material and the greatest concentration of PrP33-35sc were in the fractions containing microsomal and synaptosomal membranes. We found traces of infectivity in the absence of PrP33–35Sc associated with matrix protein. Partitioning of membranes with neutral chloroform-methanol resulted in concentration of PrP33–35Sc and infectivity within the interphase layer. Recombination of membrane glycoproteins (interphase) with lipids extracted from homologous brains decreased infectivity 24 logs. Temperature-dependent phase separation of infected synaptosomal and microsomal membranes with Triton X-114 yielded a phospholipid-rich phase containing a high concentration of PrP33–35S and greatest infectivity titers. This material spontaneously formed liposomes, indicating that PrP33–35Sc and PrP33–35C precursor proteins are highly hydrophobic intrinsic membrane components integrated with phospholipids. Homologous membrane phospholipids appear to prevent aggregation of the scrapie isoform of PrP and maintain high levels of infectivity.

Scrapie-associated precursor proteins: Antigenic relationship between species and immunocytochemical localization in normal, scrapie, and creutzfeldt- jakob disease brains

We describe the antigenic properties and detection of a normal isoform of scrapie-associated precursor protein (PrP33–35C) in normal, and both normal and scrapie isoforms in scrapie- or Creutzfeldt-Jakob disease (CJD)-infected mouse, hamster, and human brains, using a variety of specific antibodies. Polyclonal antibodies raised against mouse and hamster PrP27–30 and against a synthetic peptide of the N-terminal sequence of this protein were used as immunologic probes. PrP27–30 purified as a primary immunogen corresponded to the lower molecular mass peptide, with M, between 9.3 and 13.5 kd as estimated by size-exclusion high-pressure liquid chromatography. ELISA and immunoblot techniques demonstrated that antibodies recognized homologous antigens as well as precursor proteins from normal brains (PrP33–35C) and the scrapie isoform of scrapie-associated proteins (PrP33–35Sc/CJD and PrP27–30) from scrapie- and CJD-infected brains. The normal, scrapie, and CJD isoforms of scrapie-associated proteins share common epitopes with varying degrees of interspecies homology. Specific antigen detected in neurons indicated that these proteins are synthesized primarily in these cells. In infected brains, extracellular amyloid deposits formed by the scrapie isoform of PrP protein also strongly reacted with anti-PrP antibodies.

Histochemical and X-ray microanalytical localization of aluminum in amyotrophic lateral sclerosis and parkinsonism-dementia of Guam

SummaryHistochemical staining for aluminum, using Solochrome azurine or Morin, provided a rapid, simple and reliable means of identifying areas and structures of the brain of interest for closer scrutiny by X-ray microanalysis in patients with amyotrophic lateral sclerosis and parkinsonism-dementia of Guam. Neuronal perikarya, dendritic processes, and the walls of some cerebral vessels were aluminum positive by Solochrome azurine staining. In some cases, the deposition of aluminum was rather diffuse, particularly in the white matter. Fluorescent localization of aluminum using Morin was equally sensitive and specific, but provided less morphological detail than Solochrome azurine. Confirmation of histochemical detection of aluminum was achieved by examining adjacent tissue sections using wavelength-dispersive spectrometry coupled to a computer-controlled electron beam X-ray microprobe. Although the minimum detectable limits for aluminum by these histochemical procedures are unknown, the lower detection limit of our X-ray microanalytical technique is 10–100 ppm dry weight. Solochrome and Morin staining, as verified by X-ray microanalysis, afford a useful and reliable means of surveying multiple anatomical regions for aluminum deposition in naturally occurring and experimentally induced neurodegenerative disorders.