Neurological illness in transgenic mice expressing a prion protein with an insertional mutation.

Familial prion diseases are caused by mutations in the gene encoding the prion protein (PrP). We have produced transgenic mice that express the mouse homolog of a mutant human PrP containing a nine octapeptide insertion associated with prion dementia. These mice exhibit a slowly progressive neurological disorder characterized clinically by ataxia and neuropathologically by cerebellar atrophy and granule cell loss, gliosis, and PrP deposition that is most prominent in the cerebellum and hippocampus. Mutant PrP molecules expressed in the brains of these mice are resistant to digestion by low concentrations of proteinase K and display several other biochemical properties reminiscent of PrP(Sc), the pathogenic isoform of PrP. These results establish a new transgenic animal model of an inherited human prion disorder.

Codeposition of cystatin C with amyloid-beta protein in the brain of Alzheimer disease patients.

Immunohistochemical analysis of brains of patients with Alzheimer disease (AD) revealed that the cysteine proteinase inhibitor cystatin C colocalizes with amyloid beta-protein (Abeta) in parenchymal and vascular amyloid deposits. No evidence of cerebral hemorrhage was observed in any of the brains studied. Immunoelectron microscopy demonstrated dual staining of amyloid fibrils with anti-Abeta and anti-cystatin C antibodies. Cystatin C immunoreactivity was also observed in amyloid deposits in the brain of transgenic mice overexpressing human beta amyloid precursor protein. Massive deposition of the variant cystatin C in the cerebral vessels of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis is thought to be responsible for the pathological processes leading to stroke. Anti-cystatin C antibodies strongly labeled pyramidal neurons within cortical layers most prone to amyloid deposition in the brains of AD patients. Immunohistochemistry with antibodies against the carboxyl-terminus of Abeta(x-42) showed intracellular immunoreactivity in the same neuronal subpopulation. It remains to be established whether the association of cystatin C to Abeta plays a primary role in amyloidogenesis of AD or is a late event in which the protein is bound to the previously formed Abeta amyloid fibrils.

Gerstmann-Sträussler-Scheinker disease (PRNP P102L): amyloid deposits are best recognized by antibodies directed to epitopes in PrP region 90-165.

Gerstmann-Sträussler-Scheinker (GSS) disease is a familial neurological disorder pathologically characterized by accumulation of prion protein (PrP) in the form of fibrillary and non-fibrillary deposits within the cerebrum and cerebellum. We have studied two patients in whom the disease is caused by a leucine for proline amino acid substitution at residue 102 of PrP. In both patients, the neuropathologic findings are similar, consisting of spongiform changes, amyloid deposits, and gliosis. To investigate the antigenic profile of PrP deposits, we used antibodies raised against several peptides that correspond to segments of the N-terminus, repeat region, midregion, and C-terminus of PrP. By immunohistochemistry, PrP amyloid cores are best labeled by antibodies directed to epitopes spanning PrP residues 90-165. In GSS disease caused by a substitution of thymine to cytosine at PRNP codon 198 (Indiana kindred), the major amyloidogenic peptide spans residues 58-150; therefore, in these two genetic forms of GSS disease, amyloid may be composed of different peptides.

Proteinase-K-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease (Indiana kindred).

Gerstmann-Sträussler-Scheinker (GSS) disease is a cerebral prion protein (PrP) amyloidosis associated with mutations in the PrP gene (PRNP). A GSS disease variant with mutation at codon 198 (F198S) has been studied in a large Indiana kindred. Biochemical investigations showed that the amyloid protein consists of 11 and 7 kDa fragments of PrP. Immunohistochemical studies showed that in addition to amyloid, these patients accumulate PrP deposits which are neither fluorescent nor birefringent when stained with thioflavin S and Congo red. In the present paper, we analyzed proteinase-K (PK)-resistant PrP in 7 patients with GSS F198S disease. Immunoblots of PK-treated brain extracts show prominent bands of ca. 27-29, 18-19, and 8 kDa. Immunohistochemistry and thioflavin-S-fluorescence show that the amyloid deposits are conspicuous in the cerebellum but sparse in the caudate nucleus. However, immunoblot analysis reveals PK-resistant PrP bands of similar intensity in both regions. Treatment with PK and PNGase F generates a pattern similar to that of PK alone. Our findings suggest that brain extracts from GSS F198S disease contain 3 prominent nonglycosylated PK-resistant PrP fragments forming a pattern not previously described in other prion diseases, which may in part explain the pathology of this GSS disease variant.

Neuroserpin mutation S52R causes neuroserpin accumulation in neurons and is associated with progressive myoclonus epilepsy.

Mutations in the Neuroserpin gene have been reported to cause familial presenile dementia. We describe a new family in which the S52R Neuroserpin mutation is associated with progressive myoclonus epilepsy in 2 siblings. The proband presented myoclonus and epilepsy at age 24, his brother and mother presented a similar disorder when they were 25. A clinical diagnosis of progressive myoclonus epilepsy was made on the proband and his brother. Skin and liver biopsies did not reveal the presence of cytological alterations in the proband. His neurological status worsened over the subsequent 19 yr during which he became demented and had uncontrollable seizures. He died at 43 yr of age from aspiration pneumonia. Neuropathologically, eosinophilic bodies, which were positive for periodic acid-Schiff and immunoreactive with antibodies against human neuroserpin, were present in the perikarya and cell processes of the neurons. They were found in large numbers in the cerebral cortex and substantia nigra and to a lesser extent, in most subcortical gray areas, spinal cord, and dorsal root ganglia. By electron microscopy, the intracytoplasmic bodies were contained within the membranes of the rough endoplasmic reticulum. Occasionally neuroserpin immunopositivity was seen throughout the cytoplasm, even without the presence of well-defined bodies. Our study characterizes for the first time the neuropathologic phenotype associated with hereditary progressive myoclonus epilepsy caused by the S52R Neuroserpin mutation.

Ectopic white matter neurons, a developmental abnormality that may be caused by the PSEN1 S169L mutation in a case of familial AD with myoclonus and seizures.

We report clinical, neuropathologic and molecular genetic data from an individual affected by a familial Alzheimer disease (AD) variant. The proband had an onset of dementia at age 29 followed by generalized seizures a year later. He died at age 40. Neuropathologically, he had severe brain atrophy and characteristic histopathologic lesions of AD. Three additional neuropathologic features need to be emphasized: 1) severe deposition of Abeta in the form of diffuse deposits in the cerebral and cerebellar cortices, 2) numerous Abeta deposits in the subcortical white matter and in the centrum semiovale, and 3) numerous ectopic neurons, often containing tau-immunopositive neurofibrillary tangles, in the white maner of the frontal and temporal lobes. A molecular genetic analysis of DNA extracted from brain tissue of the proband revealed a S169L mutation in the Presenilin 1 (PSEN1) gene. The importance of this case lies in the presence of ectopic neurons in the white matter, early-onset seizures, and a PSEN1 mutation. We hypothesize that the PSEN1 mutation may have a causal relationship with an abnormality in neuronal development.

Phenotypic variability of Gerstmann-Sträussler-Scheinker disease is associated with prion protein heterogeneity.

Gerstmann-Sträussler-Scheinker disease (GSS), a cerebello-pyramidal syndrome associated with dementia and caused by mutations in the prion protein gene (PRNP), is phenotypically heterogeneous. The molecular mechanisms responsible for such heterogeneity are unknown. Since we hypothesize that prion protein (PrP) heterogeneity may be associated with clinico-pathologic heterogeneity, the aim of this study was to analyze PrP in several GSS variants. Among the pathologic phenotypes of GSS, we recognize those without and with marked spongiform degeneration. In the latter (i.e. a subset of GSS P102L patients) we observed 3 major proteinase-K resistant PrP (PrPres) isoforms of ca. 21-30 kDa, similar to those seen in Creutzfeldt-Jakob disease. In contrast, the 21-30 kDa isoforms were not prominent in GSS variants without spongiform changes, including GSS A117V, GSS D202N, GSS Q212P, GSS Q217R, and 2 cases of GSS P102L. This suggests that spongiform changes in GSS are related to the presence of high levels of these distinct 21-30 kDa isoforms. Variable amounts of smaller, distinct PrPres isoforms of ca. 7-15 kDa were seen in all GSS variants. This suggests that GSS is characterized by the presence PrP isoforms that can be partially cleaved to low molecular weight PrPres peptides.

Prion protein amyloidosis.

The prion protein (PrP) plays an essential role in the pathogenesis of a group of sporadic, genetically determined and infectious fatal degenerative diseases, referred to as "prion diseases", affecting the central nervous system of humans and other mammals. The cellular PrP is encoded by a single copy gene, highly conserved across mammalian species. In prion diseases, PrP undergoes conformational changes involving a shift from alpha-helix to beta-sheet structure. This conversion is important for PrP amyloidogenesis, which occurs to the highest degree in the genetically determined Gerstmann-Sträussler-Scheinker disease (GSS) and prion protein cerebral amyloid angiopathy (PrP-CAA), while it is less frequently seen in other prion diseases. GSS and PrP-CAA are associated with point mutations of the prion protein gene (PRNP); these conditions show a broad spectrum of clinical presentation, the main signs being ataxia, spastic paraparesis, extrapyramidal signs and dementia. In GSS, parenchymal amyloid may be associated with spongiform changes or neurofibrillary lesions; in PrP-CAA, vascular amyloid is associated with neurofibrillary lesions. A major component of the amyloid fibrils in the two diseases is a 7 kDa peptide, spanning residues 81-150 of PrP.

A new PRNP mutation (G131V) associated with Gerstmann-Sträussler-Scheinker disease.

BACKGROUND: Gerstmann-Sträussler-Scheinker disease is a rare form of prion disease. OBJECTIVE: To determine the prion mutation in a 51-year-old man without a family history of neurologic disease who died from Gerstmann-Sträussler-Scheinker disease. PATIENT AND METHODS: The patient was a 51-year-old man who died after a 9-year illness characterized by dementia and eventually ataxia. Neuropathologic studies were performed, the results of which revealed abundant prion protein-immunopositive amyloid plaques in the cerebellum without spongiform degeneration. RESULTS: Genetic analysis of the prion protein gene showed a novel mutation at codon 131 that caused a valine-for-glycine substitution (G131V) and homozygosity at codon 129 (129M). Proteinase K-resistant prion protein was detected by Western blot analysis. CONCLUSIONS: This is the first mutation described in the short, antiparallel beta-sheet domain of the prion protein. This report highlights the importance of genetic analysis of patients with atypical dementia even in the absence of a family history.

Microencapsulation and the grafting of genetically transformed cells as therapeutic strategies to rescue degenerating neurons of the CNS.

A number of approaches have been developed to deliver growth factors within the central nervous system of adult mammals. Initially a variety of neurotrophic factors were administered either by single intracerebroventricular or local injections directly into brain tissues or via permanently installed cannulae for chronic administrations. More recently delivery systems including conjugates, biodegradable and non-biodegradable implants and microspheres as well as genetically engineered cells have been introduced in order to provide a prolonged supply of neurotrophic factors and to prevent their enzymatic degradation. In this review we examine a variety of means of delivering neurotrophic factors (mainly nerve growth factor) with the primary focus upon the use of microencapsulated neurotrophins and cells genetically modified to produce them. In addition, this review highlights some difficulties and future trends in the development of novel delivery systems hopefully more suitable for investigations in different areas of neuroscience.

Scrapie infectivity and prion protein are distributed in the same pH range in agarose isoelectric focusing.

We separated lysed synaptosomal-microsomal membrane fraction from scrapie-infected hamster brain in preparative agarose isoelectric focusing. We also studied the distribution of PrP27-30 and scrapie infectivity in 13 regions of the gel in the range of pH 3.5 to 9.3. Most of the infectivity remained in the trough, where it had been placed at the beginning of the electrophoresis, along with PrP27-30. Scrapie infectious particles that encountered the gel demonstrated charge heterogeneity and were distributed in the range of pH 5.4 to 9.3. Analysis of charge heterogeneity of PrP27-30 after sodium dodecyl sulfate solubilization showed an isoelectric pattern in the same pH range as that for scrapie infectious particles. The similarity in charge heterogeneity between infectivity and PrP27-30, together with copurification, support the idea that PrP27-30 is an essential component of the scrapie infectious agent.

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 Mr 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 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.

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, Alzheimer's 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 Alzheimer's disease.

Creutzfeldt-Jakob disease in a husband and wife.

A 53-year-old man died of sporadic Creutzfeldt-Jakob disease (CJD) after a 1.5-year clinical course. Four and a half years later, his then 55-year-old widow died from CJD after a 1-month illness. Both patients had typical clinical and neuropathologic features of the disease, and pathognomonic proteinase-resistant amyloid protein ("prion" protein, or PrP) was present in both brains. Neither patient had a family history of neurologic disease, and molecular genetic analysis of their PrP genes was normal. No medical, surgical, or dietary antecedent of CJD was identified; therefore, we are left with the unanswerable alternatives of human-to-human transmission or the chance occurrence of sporadic CJD in a husband and wife.

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 greater than or equal to 4 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 PrP33-35Sc 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.

Gerstmann-Sträussler-Scheinker disease with mutation at codon 102 and methionine at codon 129 of PRNP in previously unreported patients.

We present two patients with Gerstmann-Sträussler-Scheinker disease (GSS), one from a previously undescribed kindred and one from the Canadian branch of a previously reported British kindred. In both patients, GSS is caused by a substitution of thymine for cytosine at codon 102 of the prion protein gene (PRNP). In each patient, we confirmed the clinical diagnosis by neuropathologic examination. The mutation, causing a substitution of leucine for proline at residue 102 (P102L) of the prion protein, has been previously reported in at least 30 other families. In the patients described here, the mutation was in coupling with methionine at PRNP codon 129.

Clinical and molecular genetic study of a large German kindred with Gerstmann-Sträussler-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-Sträussler-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.

BACKGROUND: Insomnia 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. OBJECTIVE: To report a 53-year-old woman with intractable insomnia as her initial symptom of disease. METHODS: The 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. RESULTS: The 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 (PrP(sc)) with the nonglycosylated isoform of approximately 21 kd, corresponding in size to that of type 1 PrP(sc). 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. CONCLUSIONS: Insomnia may be a prominent early symptom in cases of CJD linked to the E200K-129M haplotype in which the thalamus is severely affected.

Proteasomal inhibition induced by manganese ethylene-bis-dithiocarbamate: relevance to Parkinson's disease.

Maneb, a widely used fungicide, has been associated with Parkinsonism in humans. In experimental models, maneb and its major active element, manganese ethylene-bis-dithiocarbamate (Mn-EBDC) cause selective nigrostriatal neurodegeneration in mice and in rats, respectively. To investigate the mechanisms underlying this neurodegeneration, we studied the effects of Mn-EBDC on proteasomal function, which is decreased in patients with Parkinson's disease (PD), in a dopaminergic neuronal cell line (MES 23.5 or MES). The results demonstrated that exposure of MES cells to 6 microM Mn-EBDC for 7 days produced not only significant neurotoxicity but also inhibition of proteasomal chymotrypsin-like and postglutamyl peptidase activities. Proteasomal dysfunction was accompanied by formation of cytoplasmic inclusions that were positive for alpha-synuclein immunostaining and significantly increased sodium dodecyl sulfate-insoluble alpha-synuclein aggregation seen by Western blot analysis. In addition, there was a significant increase in oxidative stress, evidenced by elevated total protein carbonyl content, in cells treated with Mn-EBDC. Manipulation of intracellular reduced glutathione levels with N-acetyl-L-cysteine or L-buthionine sulfoximine pretreatment to modulate Mn-EBDC-mediated oxidative stress altered Mn-EBDC-mediated neurotoxicity, proteasomal dysfunction, and alpha-synuclein aggregation in these cells. These data suggest that neurotoxicity-induced by Mn-EBDC is at least partially attributable to Mn-EBDC-mediated proteasomal inhibition, and that the proteasome may be an important target by which environmental exposure modifies the risk for developing PD in vulnerable populations.