Plasma total prion protein as a potential biomarker for neurodegenerative dementia: diagnostic accuracy in the spectrum of prion diseases.

AIMS: In the search for blood-based biomarkers of neurodegenerative diseases, we characterized the concentration of total prion protein (t-PrP) in the plasma of neurodegenerative dementias. We aimed to assess its accuracy in this differential diagnostic context. METHODS: Plasma t-PrP was measured in 520 individuals including healthy controls (HC) and patients diagnosed with neurological disease control (ND), Alzheimer's disease (AD), sporadic Creutzfeldt-Jakob disease (sCJD), frontotemporal dementia (FTD), Lewy body dementia (LBD) and vascular dementia (VaD). Additionally, t-PrP was quantified in genetic prion diseases and iatrogenic CJD. The accuracy of t-PrP discriminating the diagnostic groups was evaluated and correlated with demographic, genetic and clinical data in prion diseases. Markers of blood-brain barrier impairment were investigated in sCJD brains. RESULTS: Compared to HC and ND, elevated plasma t-PrP concentrations were detected in sCJD, followed by FTD, AD, VaD and LBD. In sCJD, t-PrP was associated neither with age nor sex, but with codon 129 PRNP genotype. Plasma t-PrP concentrations correlated with cerebrospinal fluid (CSF) markers of neuro-axonal damage, but not with CSF t-PrP. In genetic prion diseases, plasma t-PrP was elevated in all type of mutations investigated. In sCJD brain tissue, extravasation of immunoglobulin G and the presence of swollen astrocytic end-feet around the vessels suggested leakage of blood-brain barrier as a potential source of increased plasma t-PrP. CONCLUSIONS: Plasma t-PrP is elevated in prion diseases regardless of aetiology. This pilot study opens the possibility to consider plasma t-PrP as a promising blood-based biomarker in the diagnostic of prion disease.

Differential diagnosis of intracranial meningiomas based on magnetic resonance spectroscopy.

BACKGROUND AND PURPOSE: To determine in vivo magnetic resonance spectroscopy (MRS) characteristics of intracranial meningiomas and to assess MRS reliability in meningioma grading and discrimination from tumours of similar radiological appearance, such as lymphomas, schwannomas and haemangiopericytomas. MATERIAL AND METHODS: Analysis of spectra of 14 patients with meningiomas, 6 with schwannomas, 2 with lymphomas, 2 with haemangiopericytomas and 17 control spectra taken from healthy hemispheres. RESULTS: All the patients with meningiomas had a high Cho signal (long TE). There were very low signals of Naa and Cr in the spectra of 10 patients. A reversed Ala doublet was seen only in 2 cases. Four patients had a negative Lac signal, whereas 3 had high Lac-Lip spectra. Twelve spectra showed high Cho signals (short TE). In one case the Cho signal was extremely low. All spectra displayed a very low Cr signal, but high Glx and Lac-Lip signals. Ala presence was found only in 3 patients. The mean Cho/Cr ratio (PRESS) was 5.97 (1.12 in normal brain, p < 0.05). Lac-Lip was present in all the meningiomas (STEAM). The Ala signal was seen only in 2 spectra with long TE and in 3 sequences of the short TE sequences. There were both ß/γ-Glx and α-Glx/glutathione signals in all 14 meningiomas. CONCLUSIONS: MRS is unable to discriminate low and high grade meningiomas. The method seems to be helpful in discriminating lymphomas (absent Glx signal), schwannomas (mI signal in the short TE sequences) and haemangiopericytomas (presence of mI band) from meningiomas.

Magnetic resonance spectroscopy in intracranial tumours of glial origin.

BACKGROUND AND PURPOSE: To determine in vivo magnetic resonance spectroscopy (MRS) characteristics of intracranial glial tumours and to assess MRS reliability in glioma grading and discrimination between different histopathological types of tumours. MATERIAL AND METHODS: Analysis of spectra of 26 patients with glioblastomas, 6 with fibrillary astrocytomas, 4 with anaplastic astrocytomas, 2 with pilocytic astrocytoma, 3 with oligodendrogliomas, 3 with anaplastic oligodendrogliomas and 17 control spectra taken from healthy hemispheres. RESULTS: All tumours' metabolite ratios, except for Cho/Cr in fibrillary astrocytomas (p = 0.06), were statistically significantly different from the control. The tumours showed decreased Naa and Cr contents and a high Cho signal. The Lac-Lip signal was high in grade III astrocytomas and glioblastomas. Reports that Cho/Cr ratio increases with glioma's grade whereas Naa/Cr decreases were not confirmed. Anaplastic astrocytomas compared to grade II astrocytomas had a statistically significantly greater mI/Cr ratio (p = 0.02). In pilocytic astrocytomas the Naa/Cr value (2.58 ± 0.39) was greater, whilst the Cho/Naa ratio was lower (2.14 ± 0.64) than in the other astrocytomas. The specific feature of oligodendrogliomas was the presence of glutamate/glutamine peak Glx. However, this peak was absent in two out of three anaplastic oligodendrogliomas. Characteristically, the latter tumours had a high Lac-Lip signal. CONCLUSIONS: MRS in vivo cannot be used as a reliable method for glioma grading. The method is useful in discrimination between WHO grade I and WHO grade II astrocytomas as well as oligodendrogliomas from other gliomas.

Glioma cells showing IDH1 mutation cannot be propagated in standard cell culture conditions.

BACKGROUND: It has recently been reported by several sources that original (i.e., present in vivo) glioma cell phenotypes or genotypes cannot be maintained in vitro. For example, glioblastoma cell lines presenting EGFR amplification cannot be established. METHODS AND RESULTS: IDH1 sequencing and loss of heterozygosity analysis was performed for 15 surgery samples of astrocytoma and early and late passages of cells derived from those and for 11 archival samples. We were not able to culture tumour cells presenting IDH1 mutations originating from currently proceeded 10 tumours; the same results were observed in 7 samples of archival material. CONCLUSION: The IDH1 mutation is expected to be almost mutually exclusive with EGFR amplification, so glioma cells with IDH1 mutations seem to represent a new group of tumour cells, which cannot be readily analysed in vitro because of their elimination. The reasons for this intriguing phenomenon should be investigated since its understanding can help to define a new therapeutic approach based on simulating in vivo conditions, responsible for tumour cells elimination in vitro. Moreover, a new model for culturing glioma cells in vitro should be designed since the current one does not provide conditions corresponding to in vivo growth.

Ultrastructural study of florid plaques in variant Creutzfeldt-Jakob disease: a comparison with amyloid plaques in kuru, sporadic Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease.

BACKGROUND: Although the histological features of the amyloid plaques in variant Creutzfeldt-Jakob disease (vCJD) are distinct from those in other forms of prion disease [kuru, sporadic Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Sträussler-Scheinker disease (GSS)], their ultrastructural features have only been described in a single case report. AIMS: To study vCJD plaques systematically and compare them with plaques in kuru, sCJD, GSS and Alzheimer disease (AD). METHODS: Amyloid plaques were studied by transmission electron microscopy and image analysis in five cases of vCJD, three cases of GSS, two cases of sCJD, one case of kuru and five cases of AD. Immunohistochemistry was performed on paraffin sections from one case of vCJD, two cases of GSS, one case of kuru and two cases of sCJD. RESULTS: The florid plaques in vCJD were either compact or more diffuse; in both forms, the radiating fibrils were organized into thick 'tongues', in contrast to kuru plaques. Dystrophic neurites (DNs) containing lysosomal electron-dense bodies or vesicles surrounded florid plaques. Microglial cells were found within florid plaques; occasional amyloid fibrils were identified in membrane-bound pockets of microglial cells. In vCJD, there was significant tau immunoreactivity in DNs around florid plaques while, in sCJD, GSS and kuru, minimal tau immunoreactivity was observed around plaques. CONCLUSIONS: The ultrastructure of the florid plaques and DNs in vCJD is more reminiscent of neuritic plaques in AD than kuru or multicentric plaques. These findings may reflect differences both in the strains of the transmissible agents responsible for these disorders and in host factors.

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53.

We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.

The infrequent simultaneous genetic alterations in glioblastoma multiforme (LOH 10, 17, 19q, TP53 mutation and EGFR amplification) with short clinical course.

We described the case of an unusual, complex genetic alteration in 57 year-old male patient with glioblastoma multiforme (GBM) with short survival (6 and half months). Alterations consisted of p53 mutation, LOH 10, LOH 17, LOH 19q and EGFR amplification. LOH1p, LOH 9 and LOH 13 were negative. Immunohistochemical study did not correlate with molecular results. The overexpression of TP53 protein and RB protein was detected only in small percentage of cells and interestingly the overexpression of EGFR was present only focally. Immnunostainings for PTEN, P16, PI3-K were negative. Additionally, we observed an overexpression of IGFB2 protein. This case indicates the accumulation of molecular changes in glioblastoma multiforme in patient with short survival.

Molecular analysis of chromosome 1, 10 and 19 abnormalities in human oligodendroglial tumors: relationship between frequency of LOH grade, age and gender.

BACKGROUND: Loss of heterozygosity (LOH) on 1p and 19q is observed in most oligodendroglial tumors. LOH on 10q appears to be less common in these tumors as compared to other gliomas. PATIENTS AND METHODS: We reviewed 14 patients with oligodendroglial tumors (10 low-grade and 4 anaplastic oligodendroglioma) to evaluate the frequency of LOH on 1p, 10q and 19q and correlate it with tumor grade and patients' age and gender; 5 loci on 1p and 5 on 19q as well as 4 on 10q were analyzed for LOH using PCR techniques. RESULTS: LOH on 1p together with 19q was detected in 6 tumors, 1 tumor showed deletion of 19q accompanied with deletion on 10q. Deletion on 1p was associated with deletion of 19q (p < 0.005) and mutual associations among deletions at loci on 19q (p < 0.05) were found. Patients with LOH on 1p were younger on average than patients with retained heterozygosity (p = 0.05). Grade II oligodendrogliomas predominated among younger patients (p < 0.01) while grade III oligodendrogliomas predominated among women (p < 0.005). No association between LOH on 1p nor 19q and tumor grade or patients' gender was found. CONCLUSION: Our study provides several clinically interesting findings and further supports the hypothesis of chromosome 1p and 19q involvement in the oligodendroglial cancerogenesis.

January 2000: 12 year old boy with recent onset seizures.

A 12-year old boy presented with new onset of seizures and a CT scan showed a left frontal lobe tumor which was removed completely. Neuropathological examination showed a pleomorphic ganglion cell tumor with necrosi, and endothelial proliferation. The diagnosis was extraventricular atypical neurocytic neoplasm ("cystic ganglioneurocytoma").

Severe, early and selective loss of a subpopulation of GABAergic inhibitory neurons in experimental transmissible spongiform encephalopathies.

Little is known about the pathogenetic basis of characteristic symptoms in transmissible spongiform encephalopathies (TSEs) such as myoclonus and characteristic EEG hyperactivity. We investigated the GABAergic system and its subpopulations in mice inoculated with experimental scrapie (ME7, RML, 22A strains) and Creutzfeldt-Jakob disease (CJD; Fujisaki strain), to study damage to inhibitory neurons. Since recent studies have shown electrophysiological changes in prion protein (PrP) knockout mice, we also studied mice lacking or overexpressing the PrP gene. Antibodies against glutamic acid decarboxylase (GAD), parvalbumin (PV), calbindin (CB), and calretinin (CR) were used to stain GABAergic neurons, and isolectin-B4 to stain perineuronal nets around PV+ neurons. In scrapie infected mice, cortical PV+ neurons were severely reduced while CB+ and CR+ neurons were well preserved. In CJD inoculated mice, loss of PV+ neurons was severe and occurred very early after inoculation. PrP-/- and tg20 mice showed normal appearance of PV, CB, CR, GAD+ neurons and their neuropil, and of isolectin-B4+ perineuronal nets. The early, severe and selective loss of cortical PV+ neurons in experimental scrapie and CJD suggest selective loss of PV+ GABAergic neurons as important event during disease development, possibly as one basis of excitatory symptoms in TSEs.

The original Gerstmann-Sträussler-Scheinker family of Austria: divergent clinicopathological phenotypes but constant PrP genotype.

We present new data on the original Austrian kindred with Gerstmann-Sträussler-Scheinker disease (GSS) which encompasses currently 221 members in 9 generations. The mode of inheritance is autosomal dominant. Predominant clinical features are slowly progressive ataxia and late impairment of higher cerebral functions. In contrast, a recent case with proven P102L mutation of the PRNP gene had rapidly developing dementia and severe cortical damage indistinguishable from the clinicopathological phenotype of Creutzfeldt-Jakob disease (CJD). PRNP codon 129 was homozygous for methionine in both the historic and recent cases. Neuropathology confirms spongiosis of variable degree and numerous protease resistant/prion protein (PrP) amyloid plaques scattered throughout most of the brain as constant features in this family. Some amyloid deposits are surrounded by dystrophic neurites with accumulation of phosphorylated neurofilaments and abnormal organelles, reminiscent of Alzheimer-type plaques. Severe telencephalic damage and a synaptic-type fine granular immunoreactivity in laminar distribution in the cortex with anti-PrP after hydrated autoclaving of sections were seen only in the recent patient. In conclusion, factors in addition to the PRNP genotype at codons 102 and 129 must play a role in determining clinicopathological characteristics of this inherited brain amyloidosis.

Pathology and immunocytochemistry of a kuru brain.

We report here results of modern staining techniques including anti-prion protein (PrP) immunocytochemistry to a set of archival brain specimens of a 16 year-old male who died from kuru in 1967. Brain suspensions transmitted disease to chimpanzees and New World monkeys. The PrP gene is homozygous for valine at the polymorphic codon 129. Histology shows neuronal loss, spongiform change, and astrogliosis. Lesions are maximal in parasagittal and interhemispheric areas of frontal, central and parietal cortex, cingulate cortex, striatum, and thalamus, and are accentuated in middle and deep cerebral cortical layers. PrP accumulates as diffuse synaptic type deposits and mostly unicentric plaques. PrP deposition is maximal in parasagittal and interhemispheric areas of frontal, central and parietal cortex, cingulate cortex, basal ganglia, and cerebellar cortex. Plaques are prominent in the striatum, thalamus, and granular layer of cerebellar cortex. Meticulous examination reveals only rare "florid" plaques with surrounding vacuolation. We conclude that 1) pathology including immunomorphology of PrP deposition in this kuru brain is within the lesion spectrum of Creutzfeldt-Jakob disease although plaques are unusually prominent and widespread; 2) kuru does not share the neuropathological hallmarks of the new Creutzfeldt-Jakob disease variant recently reported in the UK and France; 3) topographic prominence of PrP deposition parallels that of spongiform change and/or astrogliosis.

Transmissible cerebral amyloidoses as a model for Alzheimer's disease. An ultrastructural perspective.

Alzheimer's disease, a prototypic nontransmissible cerebral amyloidosis, has no adequate experimental model. Several pathogenetic events, however, may be modeled and accurately studied in the transmissible cerebral amyloidoses of kuru, Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, and scrapie. The common neuropathological denominator in both types of cerebral amyloidoses is the presence of stellate kuru plaques, senile plaques, and pure neuritic plaques. These amyloid plaques consist of amyloid fibers, dystrophic neurites, and reactive astrocytes in different proportions. Microglial cells, which are regarded as amyloid producer/processor cells in Alzheimer's disease, may play the same function in the transmissible cerebral amyloidoses. In both transmissible and nontransmissible amyloidoses, the impairment of axonal transport leads to accumulation of abnormally phosphorylated cytoskeleton proteins (such as neurofilament proteins and microtubule-associated protein tau), which eventually produce dystrophic neurites observed as parts of plaque or as isolated pathological structures.

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.

Tubulovesicular structures: what are they really?

The tubulovesicular structures (TVS) are the only structures unique at the level of thin-section electron microscopy for all TSEs so far examined. They were first described in NIH Swiss mice infected intracerebrally with the "Chandler" strain of scrapie by David-Ferreira et al. in 1968 [Proc. Soc. Exp. Biol. Med. 127:313-320]. TVS were described as "particles and rods ranging in diameter from 320 to 360 A(o)." The exact topology of TVS is not entirely clear. In most published electron micrographs, TVS appeared as spheres measuring between 20 and 40 nm in diameter. The number of neuritic processes containing TVS increases through the incubation period and has been shown to correlate with the incubation period and titre of infectivity in three longitudinal disease studies of scrapie and CJD. These studies, therefore, suggest that TVS may represent a primary pathogenetic event rather than a pathological product of disease. The predominant theory of the scrapie agent is now the "prion hypothesis" and its derivatives, which implies that a conformationally altered abnormal isoform (PrP(Sc) or PrP*) of a normal cellular membrane glycoprotein (PrP(c)) is the agent and its accumulation merely mimicks replication. If an abnormal fraction of PrP is indeed the infectious agent, (although it is no longer suggested in some quarters that protease resistant fraction of PrP(Sc) is the agent). The absence of stainable PrP in TVS, however, would indicate that they are not the ultrastructural correlate of the agent. However, TVS appear to be specific and unique to the TSEs, appearing before the earliest pathological changes and increasing in line with incubation period or titre. The very existence of TVS and their correlation with infectivity, therefore, urgently needs an explanation.

Cytogenetic changes in two cases of subependymal giant-cell astrocytoma.

Cytogenetic analysis of subependymal giant-cell astrocytomas (SEGAs) from two patients presenting the clinical symptoms of tuberous sclerosis complex (TSC) revealed clonal chromosomal changes, resulting in the partial loss of chromosome 22q in both tumors. Immunohistochemically, tumors exhibited features of glial differentiation, while ultrastructural studies identified the characteristic paracrystalline inclusions within the tumor cells. To our knowledge, it is the first cytogenetic description of SEGAs associated with TSC.

Aberrations of chromosome 22 and polysomy of chromosome 8 as non-random changes in clear cell sarcoma.

Cytogenetic analysis was performed on a primary tumor and a metastatic lesion of a clear cell sarcoma of tendons and aponeuroses (CCS), a rare soft tissue neoplasm of uncertain histopathologic origin. Clonal chromosomal abnormalities resulting in two related clones were found in both tumors. The karyotype was near-triploid with several structural and numerical changes, comprising a der(15;22) (q10;q10). Including the present case, 14 of 15 cases of CCS have had structural or numerical aberrations of chromosome 22 and nine of them (65%) displayed a similar or identical t(12;22)(q13-14;q12-13). Our findings suggest that in the absence of specific t(12;22), other abnormalities of chromosome 22 may be significant. In addition, increased doses of chromosome 8 found in 70% of the tumors strongly suggest a significant role for this chromosome in the development of clear cell sarcoma.

Accumulation of chromosomal changes in human glioma progression. A cytogenetic study of 50 cases.

Cytogenetic studies of 50 human gliomas, including three oligodendrogliomas, 16 grade I-III astrocytomas, and 31 glioblastomas multiforme, were performed using the short-term tissue culture method. The most common numerical chromosome aberrations were +7, -9, -10, -14, and loss of a sex chromosome. Structural changes involved predominantly the following chromosome arms: 1q, 2q, 6q, 7q, 9p, 14q, 17p, and 18p. Losses of chromosomes 9, 10, and 14, often occurring simultaneously and in polyploid clones, were observed almost exclusively in high-grade gliomas, and appear to constitute important events during glioma progression.

Amyloid beta-protein in cerebral amyloid angiopathy, senile plaques, and preamyloidotic lesions in subcortical arteriosclerotic encephalopathy (Binswanger disease).

Cerebral vascular amyloid deposits, senile plaques and neurofibrillary tangles have been found in subcortical arteriosclerotic encephalopathy (Binswanger disease). A mouse antiserum, prepared against a 43-amino acid synthetic peptide homologous to the amyloid beta-protein of Alzheimer disease (anti-SP43), revealed immunoreactive amyloid deposits in meningeal and intracortical blood vessels, senile plaques, intraneuronal amyloid and preamyloid in a neuropathologically confirmed case of Binswanger disease previously reported to have cerebral vascular amyloid deposits. These lesions contained sulfated glycosaminoglycans as determined by the Alcian blue/critical electrolyte concentration method. Similar findings were not observed in a case of Binswanger encephalopathy without cerebral amyloid deposits. Our study indicates that amyloidotic lesions in Binswanger encephalopathy with cerebral amyloid deposits contain amyloid beta-protein and sulfated glycosaminoglycans.

Tubulovesicular structures (TVS): virus-like particles specific for all subacute spongiform virus encephalopathies--what are they really?

Tubulovesicular structures (TVS) are virus-like particles specific for all the subacute spongiform virus encephalopathies (SSVE). I report here the presence of TVS in the highest range of naturally occurring and experimentally induced SSVE studied so far: natural and experimental Creutzfeld-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, natural bovine spongiform encephalopathy (BSE) and BSE transmitted to pigs and four models of experimental scrapie in hamsters. TVS are spherical particles, approximately 30 nm in diameter. They are observed at both sides of synaptic cleft (i.e. in dendrites and axonal preterminals and terminals). By the ultrastructural criteria, TVS are easily differentiated from other spherical or tubular elements of the nervous system: microtubules, synaptic vesicles, multivesicular bodies and glycogen granules. The number of affected processes roughly correlates with infectivity titre. Their significance is unknown!