Spectrum of Novel Hereditary Hemorrhagic Telangiectasia Variants in an Austrian Patient Cohort.

OBJECTIVES: Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant genetic disorder characterized by pathogenic blood vessel development and maintenance. HHT type 1 (HHT1) and type 2 (HHT2) are caused by variants in endoglin (ENG) and activin receptor-like kinase-1 (ACVRL1), respectively. The aim of this study was to identify the spectrum of pathogenic variants in ENG and ACVRL1 in Austrian HHT families. METHODS: In this prospective study, eight Austrian HHT families were screened for variants in ENG and ACVRL1 by polymerase chain reaction amplification and sequencing of DNA isolated from peripheral blood. RESULTS: Heterozygous variants were identified in all families under study. HHT1 was caused by a novel c.816+1G>A splice donor variant, a novel c.1479C>A nonsense (p.Cys493X) variant and a published c.1306C>T nonsense (p.Gln436X) variant in ENG. Variants found in ACVRL1 were novel c.200G>C (p.Arg67Pro) and known c.772G>A (p.Gly258Ser) missense variants in highly conserved residues, a known heterozygous c.100dupT frameshift (p.Cys34Leufs*4) and the known c.1204G>A missense (p.Gly402Ser) and c.1435C>T nonsense (p.Arg479X) variants as causes of HHT2. CONCLUSION: Novel and published variants in ENG (37.5%) and ACVRL1 (62.5%) were exclusively identified as the cause of HHT in an Austrian patient cohort. Identification of novel causative genetics variants should facilitate the development of tailored therapeutical applications in the future treatment of autosomal dominant HHT.

Antibodies against complement-regulatory proteins on platelets in immune thrombocytopenia.

In immune thrombocytopenia (ITP), antibodies reacting with platelet membrane glycoproteins (GP) mediate premature platelet cleavage, resulting in thrombocytopenia and therefore a risk of bleeding. These antibodies may induce complement activation, thus mediating complement-induced platelet destruction. In this study, we investigated the possibility of an additional complement-related pathogenic mechanism, where antibodies against the complement-regulatory factors CD55 and CD59 may directly interfere with normal complement function. CD55 downregulates both the classic and the alternative activation pathways, while CD59 blocks the formation of the membrane attack complex; both proteins are present on platelets and may therefore be targets of autoantibodies. Using the simultaneous analysis of specific platelet antibodies (SASPA) assay, we found that in some cases of immune-mediated thrombocytopenia, anti-CD55 and -CD59 antibodies are detectable in patients' sera and/or on their autologous platelets in combination with antibodies against platelet-specific GP. Although antibodies against CD55 and CD59 seem to be a rare phenomenon, this finding may have clinical relevance due to the availability of highly effective therapeutics targeting the complement system.

Atypical sporadic CJD-MM phenotype with white matter kuru plaques associated with intranuclear inclusion body and argyrophilic grain disease.

We describe an atypical neuropathological phenotype of sporadic Creutzfeldt-Jakob disease in a 76-year-old man. The clinical symptoms were characterized by progressive dementia, gait ataxia, rigidity and urinary incontinence. The disease duration was 6 weeks. MRI did not show prominent atrophy or hyperintensities in cortical areas, striatum or thalamus. Biomarker examination of the cerebrospinal fluid deviated from that seen in pure Alzheimer's disease. Triphasic waves in the EEG were detected only later in the disease course, while 14-3-3 assay was positive. PRNP genotyping revealed methionine homozygosity (MM) at codon 129. Neuropathology showed classical CJD changes corresponding to the MM type 1 cases. However, a striking feature was the presence of abundant kuru-type plaques in the white matter. This rare morphology was associated with neuropathological signs of intranuclear inclusion body disease and advanced stage of argyrophilic grain disease. These alterations did not show correlation with each other, thus seemed to develop independently. This case further highlights the complexity of neuropathological alterations in the ageing brain.

Detection of disease-associated α-synuclein in the cerebrospinal fluid: a feasibility study.

With the aim to evaluate the significance and reliability of detecting disease-specific α-synuclein in the cerebrospinal fluid (CSF) we developed an ELISA and bead-assay. We used a commercial antibody (5G4) that does not bind to the physiological monomeric form of α-synuclein, but is highly specific for the disease-associated forms, including high molecular weight fraction of ß-sheet rich oligomers. We applied both tests in CSF from a series of neuropathologically confirmed α-synucleinopathy cases, including Parkinson' disease dementia (PDD) and dementia with Lewy bodies (DLB) (n = 7), as well as Alzheimer' disease (n = 6), and control patients without neurodegenerative pathologies (n = 9). Disease-specific α-synuclein was detectable in the CSF in a subset of patients with α-synuclein pathology in the brain. When combined with the analysis of total α-synuclein, the bead-assay for disease-specific α-synuclein was highly specific for PDD/DLB. Detection of disease-associated αsynuclein combined with the total levels of α-synuclein is a promising tool for the in-vivo diagnosis of α-synucleinopathies, including PDD and LBD.

A mutant prion protein sensitizes neurons to glutamate-induced excitotoxicity.

Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer's diseases. However, how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders attributable to toxic, ß-rich oligomers that bind to PrP(C).

The N-terminal, polybasic region of PrP(C) dictates the efficiency of prion propagation by binding to PrP(Sc).

Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrP(Sc)) binds to the cellular form (PrP(C)), generating additional molecules of PrP(Sc). While several regions of the PrP(C) molecule have been suggested to play a role in PrP(Sc) formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23-31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrP(Sc). These results, in combination with biochemical data, demonstrate that residues 23-31 represent a critical site on PrP(C) that binds to PrP(Sc) and is essential for efficient prion propagation. It may be possible to specifically target this region for treatment of prion diseases as well as other neurodegenerative disorders due to ß-sheet-rich oligomers that bind to PrP(C).

Prion protein at the crossroads of physiology and disease.

The presence of the cellular prion protein (PrP(C)) on the cell surface is critical for the neurotoxicity of prions. Although several biological activities have been attributed to PrP(C), a definitive demonstration of its physiological function remains elusive. In this review, we discuss some of the proposed functions of PrP(C), focusing on recently suggested roles in cell adhesion, regulation of ionic currents at the cell membrane and neuroprotection. We also discuss recent evidence supporting the idea that PrP(C) may function as a receptor for soluble oligomers of the amyloid ß peptide and possibly other toxic protein aggregates. These data suggest surprising new connections between the physiological function of PrP(C) and its role in neurodegenerative diseases beyond those caused by prions.

The N-terminal, polybasic region is critical for prion protein neuroprotective activity.

Several lines of evidence suggest that the normal form of the prion protein, PrP(C), exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrP(C) to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (Δ32-134, called F35). To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (Δ23-31, Δ23-111, and Δ23-134) to rescue the phenotype of Tg(F35) mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although Δ23-31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrP(C) neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases.

Peroxisomal localization of the proopiomelanocortin-derived peptides beta-lipotropin and beta-endorphin.

The peptide hormones ACTH, MSHs, ß-lipotropin (ß-LPH), and ß-endorphin are all derived from the precursor molecule proopiomelanocortin (POMC). Using confocal laser microscopy and immunoelectron microscopy in human pituitary gland, we demonstrate a peroxisomal localization of ß-endorphin and ß-LPH in cells expressing the peroxisomal ATP-binding cassette-transporter adrenoleukodystrophy protein (ALDP). The peroxisomal localization of ß-LPH and ß-endorphin was not restricted to the pituitary gland but was additionally found in other human tissues that express high levels of ALDP, such as dorsal root ganglia, adrenal cortex, distal tubules of kidney, and skin. In contrast to the peptide hormones ß-LPH and ß-endorphin, which are derived from the C terminus of POMC, the N-terminal peptides ACTH, α-MSH, and γ-MSH were never detected in peroxisomes. This novel peroxisomal localization of ß-endorphin and ß-LPH in ALDP-positive cells was confirmed by costaining with ALDP and the peroxisomal marker catalase. Moreover, peroxisomal sorting of ß-LPH could be modeled in HeLa cells by ectopic expression of a POMC variant, modified to allow cleavage and release of ß-LPH within the secretory pathway. Although ß-LPH and ß-endorphin were only associated with peroxisomes in cells that normally express ALDP, the transporter activity of ALDP is not necessary for the peroxisomal localization, as demonstrated in tissues of X-linked adrenoleukodystrophy patients lacking functional ALDP. It remains to be elucidated whether and how the peroxisomal localization of POMC-derived hormones has a role in the endocrine dysfunction of peroxisomal disease.

Creutzfeldt-Jakob disease in Austria: an autopsy-controlled study.

BACKGROUND: Definite diagnosis of prion diseases or transmissible spongiform encephalopathies (TSEs) requires neuropathology, usually at autopsy. Epidemiology of human TSEs has relied on definite as well as 'probable' cases in which neuropathological confirmation is lacking, usually because of low autopsy rates in most countries. METHODS: In Austria, an active surveillance program for human prion diseases was established in 1996. Since then, more than 900 referrals were analyzed. Postmortem investigation of brain tissue is mandatory in every suspect case of TSE. Thus, epidemiological data on TSEs from Austria may serve as autopsy-controlled reference for countries with lower autopsy rates. RESULTS: The total number of TSE cases in Austria since 1969 is 206. The average yearly mortality for the active surveillance period from 1996 to 30 June 2006 is 1.39 per million, with the highest rates for Vienna (2.37) compared with other provinces. Eighty-five percent of definite TSEs were classified as sporadic Creutzfeldt-Jakob disease (sCJD). We observed a significant linear increase in the mean age at death of 0.6 years per calendar year. Clinical diagnostic surveillance criteria had a sensitivity and specificity of 82.7 and 80.0% for probable CJD, respectively, and a positive predictive value of 80.5% for probable and 38.9% for 'possible' CJD. Alternative neuropathological diagnoses in suspect cases included Alzheimer's disease with or without Lewy body pathology, vascular encephalopathy, metabolic encephalopathies and viral or limbic encephalitis. CONCLUSION: The steady increase in mortality rates, especially in old age groups, most likely reflects improved case ascertainment due to active surveillance causing higher awareness of the medical community. In comparison with other European countries, it is reassuring to note that the overall death rate of TSEs does not differ from the Austrian autopsy-controlled data, thus confirming the value of clinical surveillance criteria.

Asymptomatic persistence of anti-Yo antibodies for 5 years without relapse of malignancy.

Neurological paraneoplastic syndromes (NPS) are rare disorders in association with malignancy and the presence of various antineuronal antibodies. The persistence of antineuronal antibodies for years after the eradication of the tumor without clinical manifestations of a NPS has not been reported. In a 64-year-old woman with a history of gynecologic malignancy, treated by surgery and chemotherapy with docetaxel and carboplatin, ptosis, hypertelorism, dysarthria, short stature, upper and lower limb weakness, exaggerated tendon reflexes and recurrent creatine-kinase elevation were found. Nerve conduction studies disclosed polyneuropathy and muscle biopsy nonspecific myopathic features. Though anti-Yo antibodies were repeatedly positive, the clinical findings and polyneuropathy were rather attributed to a suspected metabolic myopathy or chemotherapy than a NPS. Anti-Yo antibodies remained positive at 5 further determinations. During the 5 years of follow up there was improvement of polyneuropathy and no relapse of the malignancy, as assessed clinically, by tumor markers, computed tomography scans and 18-fluorodeoxyglucose positron emission tomography. Anti-Yo antibodies may occur without the typical clinical manifestations of a NPS and may persist for years without the development of a NPS or relapse of the tumor.

Diagnosis of X-linked adrenoleukodystrophy in blood leukocytes.

OBJECTIVES: Our aim was to replace cultured skin fibroblasts in the diagnosis of X-linked adrenoleukodystrophy (X-ALD) by peripheral blood cells. DESIGN AND METHODS: Very long chain fatty acids (VLCFAs) were analyzed in leukocytes from X-ALD patients, heterozygotes, and controls using gas chromatography-mass spectrometry (GC-MS). Immunofluorescence for adrenoleukodystrophy protein (ALDP) was performed in mononuclear blood cell preparations of X-ALD patients known to be ALDP negative in fibroblasts, heterozygote relatives of these patients, and controls. RESULTS: All X-ALD patients were distinguishable from controls by VLCFA analysis in leukocytes. 91.7% of heterozygotes were identified by combined VLCFA analysis in leukocytes and plasma. All patients investigated lacked ALDP immunoreactivity in mononuclear cells, while heterozygotes showed mosaic patterns of positive and negative cells. CONCLUSION: Determination of VLCFAs by GC-MS in combination with ALDP immunofluorescence in peripheral blood cells provides a fast and minimally invasive diagnostic method for X-ALD, which, in contrast to plasma analysis, is independent of alimentary influences. Notably, joint evaluation of leukocytes and plasma considerably improves the identification of heterozygotes.

Prominent corticosteroid disturbance in experimental prion disease.

Prion diseases comprise a group of neurodegenerative disorders that invariably lead to death in affected individuals. The most prominent event in these diseases is a rapid and pronounced neuronal loss, although the cause and the precise mechanisms of neuronal cell death have not been identified so far. Recently, it has been suggested that corticosteroids might play a role in the pathogenesis of neurodegenerative disorders in general, as the regulation of these hormones was found to be disturbed in Alzheimer's and Parkinson's disease, as well as in a transgenic mouse model of Alzheimer's disease. To evaluate the possible corticosteroid disturbances in prion diseases, we determined the concentration of corticosterone metabolites in the faeces of scrapie-inoculated mice during the course of the clinical disease. We observed markedly elevated concentrations of the metabolites during the last 5 weeks of the disease, as well as a severe disturbance of circadian periodicity of corticosterone excretion as much as 2 weeks before this elevation. A simultaneous downregulation of cerebral neuronal glucocorticoid receptors was not detectable by immunohistochemistry, indicating that increased corticosteroids can elicit their effects in mouse scrapie freely. The dysregulation of corticosteroid excretion might act as a further cofactor in the pathogenesis of scrapie, for example by preconditioning nerve cells to disease-immanent neurotoxic stimuli, such as oxidative stress, and to apoptosis.

Endoplasmic reticulum stress features are prominent in Alzheimer disease but not in prion diseases in vivo.

Prion diseases and Alzheimer disease (AD) share a variety of clinical and neuropathologic features (e.g. progressive dementia, accumulation of abnormally folded proteins in diseased tissue, and pronounced neuronal loss) as well as pathogenic mechanisms like generation of oxidative stress molecules and complement activation. Recently, it was suggested that neuronal death in AD may have its origin in the endoplasmic reticulum (ER). Cellular stress conditions can interfere with protein folding and subsequently cause accumulation of unfolded or misfolded proteins in the ER lumen. The ER responds to this by the activation of adaptive pathways, which are termed unfolded protein response (UPR). The UPR transducer PERK, which launches the most immediate response to ER stress (i.e. the transient attenuation of mRNA translation), and the downstream effector of PERK, eIF2alpha, were shown to be activated in AD. We demonstrate that neither in sporadic nor in infectiously acquired or inherited human prion diseases can the activated forms of PERK and eIF2alpha be detected, except when concomitant neurofibrillary pathology is present; whereas the distribution of phosphorylated PERK correlates with abnormally phosphorylated tau in AD. In brains of scrapie-affected mice and mice infected with sporadic or variant Creutzfeldt-Jakob disease, activated PERK is only very faintly expressed. The lack of prominent activation of the PERK-eIF2alpha pathway in prion diseases suggests that, in contrast to AD, ER stress does not play a crucial role in neuronal death in prion disorders.

Pathogenesis of prion diseases.

Prion diseases are rare neurological disorders that may be of genetic or infectious origin, but most frequently occur sporadically in humans. Their outcome is invariably fatal. As the responsible pathogen, prions have been implicated. Prions are considered to be infectious particles that represent mainly, if not solely, an abnormal, protease-resistant isoform of a cellular protein, the prion protein or PrP(C). As in other neurodegenerative diseases, aggregates of misfolded protein conformers are deposited in the CNS of affected individuals. Pathogenesis of prion diseases comprises mainly two equally important, albeit essentially distinct, topics: first, the mode, spread, and amplification of infectivity in acquired disease, designated as peripheral pathogenesis. In this field, significant advances have implicated an essential role of lymphoid tissues for peripheral prion replication, before a likely neural spread to the CNS. The second is the central pathogenesis, dealing, in addition to spread and replication of prions within the CNS, with the mechanisms of nerve cell damage and death. Although important roles for microglial neurotoxicity, oxidative stress, and complement activation have been identified, we are far from complete understanding, and therapeutic applications in prion diseases still need to be developed.

Activity-dependent autocrine-paracrine activation of neuronal P2Y receptors.

Activation of P2Y receptors by released nucleotides subserves important autocrine-paracrine functions in various non-neural tissues. To investigate how P2Y receptors are activated in a neuronal environment, we used PC12 cells in which nucleotides were found to elicit increases in inositol phosphates via P2Y2 and decreases in cAMP via P2Y12 receptors. Depolarization of PC12 cells raised inositol phosphates, and blockade of voltage-gated Ca2+ channels by Cd2+ or degradation of extracellular nucleotides by apyrase prevented this effect. In nondepolarized cells, apyrase did not affect inositol phosphates. Depolarization of PC12 cells also reduced the A2A receptor-mediated synthesis of cAMP. This effect was again prevented by Cd2+ or apyrase, but apyrase enhanced the synthesis of cAMP even in nondepolarized cells. Overexpression of rat P2Y2 receptors increased the nucleotide-dependent inositol phosphate accumulation and enhanced the effect of K+ depolarization. Nevertheless, apyrase still failed to alter spontaneous inositol phosphate accumulation. Expression of rat P2Y1 receptors, in contrast, led to huge increases in spontaneous inositol phosphate accumulation, which was reduced by a receptor antagonist or by apyrase. This increased synthesis of inositol phosphates could not be further enhanced by depolarization or receptor agonists, but when endogenous nucleotides were removed by superfusion, recombinant P2Y1 receptors could be activated to mediate an inhibition of M-type K+ channels. These results indicate that nucleoside diphosphate-sensitive (P2Y12 and P2Y1) receptors are activated by spontaneous nucleotide release, whereas triphosphate-sensitive (P2Y2) receptors require an excess of depolarization-evoked release to become activated.

Inhibition of adenylyl cyclase by neuronal P2Y receptors.

P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y(12). Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. ADP and ATP (0.1 - 100 microM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A(2A) or recombinant beta(2) receptors. Forskolin-dependent cyclic AMP production was reduced by ADPbetaS (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, or adenosine-3'-phosphate-5'-phosphate, but attenuated by reactive blue 2, ATP(alpha)S, and 2-methylthio-AMP. RT - PCR demonstrated the expression of P2Y(2), P2Y(4), P2Y(6), and P2Y(12), but not P2Y(1), receptors in PC12 cells. In Northern blots, only P2Y(2) and P2Y(12) were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged. We conclude that P2Y(12) receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity.