Mitochondrial respiratory chain deficiency correlates with the severity of neuropathology in sporadic Creutzfeldt-Jakob disease.

Mitochondrial dysfunction has been implicated in multiple neurodegenerative diseases but remains largely unexplored in Creutzfeldt-Jakob disease. Here, we characterize the mitochondrial respiratory chain at the individual neuron level in the MM1 and VV2 common molecular subtypes of sporadic Creutzfeldt-Jakob disease. Moreover, we investigate the associations between the mitochondrial respiratory chain and neuropathological markers of the disease.Brain tissue from individuals with sporadic Creutzfeldt-Jakob disease and age-matched controls were obtained from the brain collection of the Austrian Creutzfeldt-Jakob Surveillance. The mitochondrial respiratory chain was studied through a dichotomous approach of immunoreactivities in the temporal cortex and the hippocampal subregions of CA4 and CA3.We show that profound deficiency of all mitochondrial respiratory complexes (I-V) occurs in neurons of the severely affected temporal cortex of patients with Creutzfeldt-Jakob disease. This deficiency correlates strongly with the severity of neuropathological changes, including vacuolation of the neuropil, gliosis and disease associated prion protein load. Respiratory chain deficiency is less pronounced in hippocampal CA4 and CA3 regions compared to the temporal cortex. In both areas respiratory chain deficiency shows a predilection for the MM1 molecular subtype of Creutzfeldt-Jakob disease.Our findings indicate that aberrant mitochondrial respiration could be involved early in the pathogenesis of sporadic Creutzfeldt-Jakob disease and contributes to neuronal death, most likely via ATP depletion. Based on these results, we propose that the restricted MRI diffusion profile seen in the brain of patients with sporadic Creutzfeldt-Jakob disease might reflect cytotoxic changes due to neuronal respiratory chain failure and ATP loss.

Immunohistochemical and ultrastructural evidence for the pathogenesis of white matter degeneration in patients with panencephalopathic-type Creutzfeldt-Jakob disease: Inducible nitric oxide synthase overexpression in bizarre astrocytes.

Excessive production of nitric oxide (NO) due to the overinduction of inducible nitric oxide synthase (iNOS) has a severe cytotoxic effect, which may relate to the pathogenesis of neurodegenerative disorders. In this study, we report the novel finding that iNOS is overinduced in a large number of bizarre astrocytes in the white matter of patients with panencephalopathic (PE)-type Creutzfeldt-Jakob disease (CJD). This study was carried out on brain tissue from seven patients with PE-type CJD. As controls, 12 normal individuals and nine patients with cerebral infarction were examined. We identified a large number of bizarre astrocytes in the degenerative cerebral white matter in PE-type CJD. Using immunohistochemistry, only bizarre astrocytes in PE-type CJD showed strong immunoreactivity for both iNOS and superoxide dismutase 1 (SOD1). Ultrastructural examination demonstrated that these bizarre astrocytes contained many free polyribosome-like granules. No significant iNOS immunoreactivity was observed in either the astrocytes of patients with cerebral infarcts or in the normal controls. This study suggests that the iNOS-overexpressing astrocytes, especially iNOS-overexpressing bizarre astrocytes, could play an important role in the development of white matter lesions in PE-type CJD. Our data also suggest that the bizarre astrocytes could be protecting themselves from the cytotoxicity of NO by producing SOD1. These immunohistochemical findings are supported by the ultrastructural observation of numerous polyribosome granules restricted to the cytoplasm of these bizarre astrocytes.

NADPH oxidases as drug targets and biomarkers in neurodegenerative diseases: What is the evidence?

Neurodegenerative disease are frequently characterized by microglia activation and/or leukocyte infiltration in the parenchyma of the central nervous system and at the molecular level by increased oxidative modifications of proteins, lipids and nucleic acids. NADPH oxidases (NOX) emerged as a novel promising class of pharmacological targets for the treatment of neurodegeneration due to their role in oxidant generation and presumably in regulating microglia activation. The unique function of NOX is the generation of superoxide anion (O2•-) and hydrogen peroxide (H2O2). However in the context of neuroinflammation, they present paradoxical features since O2•-/H2O2 generated by NOX and/or secondary reactive oxygen species (ROS) derived from O2•-/H2O2 can either lead to neuronal oxidative damage or resolution of inflammation. The role of NOX enzymes has been investigated in many models of neurodegenerative diseases by using either genetic or pharmacological approaches. In the present review we provide a critical assessment of recent findings related to the role of NOX in the CNS as well as how the field has advanced over the last 5 years. In particular, we focus on the data derived from the work of a consortium (Neurinox) funded by the European Commission's Programme 7 (FP7). We discuss the evidence gathered from animal models and human samples linking NOX expression/activity with neuroinflammation in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease as well as autoimmune demyelinating diseases like multiple sclerosis (MS) and chronic inflammatory demyelinating polyneuropathy (CIDP). We address the possibility to use measurement of the activity of the NOX2 isoform in blood samples as biomarker of disease severity and treatment efficacy in neurodegenerative disease. Finally we clarify key controversial aspects in the field of NOX, such as NOX cellular expression in the brain, measurement of NOX activity, impact of genetic deletion of NOX in animal models of neurodegeneration and specificity of NOX inhibitors.

Benzothiazole aniline tetra(ethylene glycol) and 3-amino-1,2,4-triazole inhibit neuroprotection against amyloid peptides by catalase overexpression in vitro.

Alzheimer's disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-ß (Aß), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aß, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aß, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45-50 had additive neuroprotective actions against the Aß peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects.

Variably protease-sensitive prionopathy in the UK: a retrospective review 1991-2008.

Variably protease-sensitive prionopathy is a newly described human prion disease of unknown aetiology lying out with the hitherto recognized phenotypic spectrum of Creutzfeldt-Jakob disease. Two cases that conform to the variably protease-sensitive prionopathy phenotype have been identified prospectively in the U.K. since the first description of the condition in 2008 in the U.S.A. To determine the incidence and phenotype of variably protease-sensitive prionopathy within a single well-defined cohort, we have conducted a retrospective review of patients referred to the National Creutzfeldt-Jakob Disease Research & Surveillance Unit during the period 1991-2008. The approach taken was to screen frozen brain tissue by western blotting for the form of protease-resistant prion protein that characterizes variably protease-sensitive prionopathy, followed by neuropathological and clinical review of candidate cases. Cases diagnosed as sporadic Creutzfeldt-Jakob disease with atypical neuropathology were also reviewed. Four hundred and sixty-five cases were screened biochemically, yielding four candidate cases of variably protease-sensitive prionopathy. One was discounted on pathological and clinical grounds, and one was a known case of variably protease-sensitive prionopathy previously reported, leaving two new cases, which were confirmed biochemically and neuropathologically as variably protease-sensitive prionopathy. A third new case that lacked frozen tissue was recognized retrospectively on neuropathological grounds alone. This means that five cases of variably protease-sensitive prionopathy have been identified (prospectively and retrospectively) during the surveillance period 1991-2011 in the U.K. Assuming ascertainment levels equivalent to that of other human prion diseases, these data indicate that variably protease-sensitive prionopathy is a rare phenotype within human prion diseases, which are themselves rare. Biochemical investigation indicates that the abnormal protease-resistant prion protein fragment that characterizes variably protease-sensitive prionopathy is detectable at low levels in some cases of sporadic Creutzfeldt-Jakob disease and conversely, that the form of abnormal prion protein that characterizes sporadic Creutzfeldt-Jakob disease can be found in certain brain regions of cases of variably protease-sensitive prionopathy, indicating molecular overlaps between these two disorders.

A low-molecular-weight ferroxidase is increased in the CSF of sCJD cases: CSF ferroxidase and transferrin as diagnostic biomarkers for sCJD.

AIMS: Most biomarkers used for the premortem diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) are surrogate in nature, and provide suboptimal sensitivity and specificity. RESULTS: We report that CJD-associated brain iron dyshomeostasis is reflected in the cerebrospinal fluid (CSF), providing disease-specific diagnostic biomarkers. Analysis of 290 premortem CSF samples from confirmed cases of CJD, Alzheimer's disease, and other dementias (DMs), and 52 non-DM (ND) controls revealed a significant difference in ferroxidase (Frx) activity and transferrin (Tf) levels in sporadic Creutzfeldt-Jakob disease (sCJD) relative to other DM and ND controls. A combination of CSF Frx and Tf discriminated sCJD from other DMs with a sensitivity of 86.8%, specificity of 92.5%, accuracy of 88.9%, and area-under-the receiver-operating-characteristic (ROC) curve of 0.94. This combination provided a similar diagnostic accuracy in discriminating CJD from rapidly progressing cases who died within 6 months of sample collection. Surprisingly, ceruloplasmin and amyloid precursor protein, the major brain Frxs, displayed minimal activity in the CSF. Most of the Frx activity was concentrated in the <3-kDa fraction in normal and diseased CSF, and resisted heat and proteinase-K treatment. INNOVATION: (i) A combination of CSF Frx and Tf provides disease-specific premortem diagnostic biomarkers for sCJD. (ii) A novel, nonenzymatic, nonprotein Frx predominates in human CSF that is distinct from the currently known CSF Frxs. CONCLUSION: The underlying cause of iron imbalance is distinct in sCJD relative to other DMs associated with the brain iron imbalance. Thus, change in the CSF levels of iron-management proteins can provide disease-specific biomarkers and insight into the cause of iron imbalance in neurodegenerative conditions.

A common BACE1 polymorphism is a risk factor for sporadic Creutzfeldt-Jakob disease.

The ß site APP cleaving enzyme 1 (BACE1) is the rate-limiting ß-secretase enzyme in the amyloidogenic processing of APP and Aß formation, and therefore it has a prominent role in Alzheimer's disease (AD) pathology. Recent evidence suggests that the prion protein (PrP) interacts directly with BACE1 regulating its ß-secretase activity. Moreover, PrP has been proposed as the cellular receptor involved in the impairment of synaptic plasticity and toxicity caused by Aß oligomers. Provided that common pathophysiologic mechanisms are shared by Alzheimer's and Creutzfeldt-Jakob (CJD) diseases, we investigated for the first time to the best of our knowledge a possible association of a common synonymous BACE1 polymorphism (rs638405) with sporadic CJD (sCJD). Our results indicate that BACE1 C-allele is associated with an increased risk for developing sCJD, mainly in PRNP M129M homozygous subjects with early onset. These results extend the very short list of genes (other than PRNP) involved in the development of human prion diseases; and support the notion that similar to AD, in sCJD several loci may contribute with modest overall effects to disease risk. These findings underscore the interplay in both pathologies of APP, Aß oligomers, ApoE, PrP and BACE1, and suggest that aging and perhaps vascular risk factors may modulate disease pathologies in part through these key players.

Altered Prion protein expression pattern in CSF as a biomarker for Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are not fully understood, however neuronal loss followed by cell lysis is assumed to cause the increase in 14-3-3 levels, which also occurs in conditions such as brain ischemia. Here we investigated the relation between the levels of 14-3-3 protein, Lactate dehydrogenase (LDH) activity and expression of the prion protein (PrP) in CSF of sporadic and familial CJD cases. Unexpectedly, we found normal levels of LDH activity in CJD cases with moderate levels of 14-3-3 protein. Increased LDH activity was only observed in a percentage of the CSF samples that also exhibited high 14-3-3 levels. Analysis of the PrP expression pattern in CSF revealed a reduction in PrP levels in all CJD cases, as well as marked changes in its glycosylation pattern. PrP present in CSF of CJD cases was sensitive to proteases. The alterations in PrP expression observed in CJD cases were not detected in other pathologies affecting the nervous system, including cases of dementia and tropical spastic paraparesis/HTLV-1 associated myelopathy (HAM/TSP). Time course analysis in several CJD patients revealed that 14-3-3 levels in CSF are dynamic and show a high degree of variability during the end stage of the disease. Post-mortem analysis of brain tissue also indicated that 14-3-3 protein is upregulated in neuronal cells, suggesting that its expression is modulated during the course of the disease. These results suggest that a combined analysis of 14-3-3 and PrP expression pattern in CSF is a reliable biomarker to confirm the clinical diagnosis of CJD patients and follow disease progression.

Protease-resistant PrP and PrP oligomers in the brain in human prion diseases after intraventricular pentosan polysulfate infusion.

Intraventricular infusion of pentosan polysulfate (PPS) as a treatment for various human prion diseases has been applied in Japan. To evaluate the influence of PPS treatment we performed pathological examination and biochemical analyses of PrP molecules in autopsied brains treated with PPS (one case of sporadic Creutzfeldt-Jakob disease (sCJD, case 1), two cases of dura mater graft-associated CJD (dCJD, cases 2 and 4), and one case of Gerstmann-Sträussler-Scheinker disease (GSS, case 3). Six cases of sCJD without PPS treatment were examined for comparison. Protease-resistant PrP (PrP(res) ) in the frontal lobe was evaluated by Western blotting after proteinase K digestion. Further, the degree of polymerization of PrP molecules was examined by the size-exclusion gel chromatography assay. PPS infusions were started 3-10 months after disease onset, but the treatment did not achieve any clinical improvements. Postmortem examinations of the treated cases revealed symmetrical brain lesions, including neuronal loss, spongiform change and gliosis. Noteworthy was GFAP in the cortical astrocytes reduced in all treated cases despite astrogliosis. Immunohistochemistry for PrP revealed abnormal synaptic deposits in all treated cases and further plaque-type PrP deposition in case 3 of GSS and case 4 of dCJD. Western blotting showed relatively low ratios of PrP(res) in case 2 of dCJD and case 3 of GSS, while in the treated sCJD (case 1), the ratio of PrP(res) was comparable with untreated cases. The indices of oligomeric PrP were reduced in one sCJD (case 1) and one dCJD (case 2). Although intraventricular PPS infusion might modify the accumulation of PrP oligomers in the brains of patients with prion diseases, the therapeutic effects are still uncertain.

ERK2 is increased in cerebrospinal fluid of Creutzfeldt-Jakob disease patients.

The clinical diagnosis of Creutzfeldt-Jakob disease (CJD) can be supported by several biochemical markers in cerebrospinal fluid (CSF) such as 14-3-3 proteins and tau protein. Unfortunately, none of the currently known markers are suited for screening or seems to be directly related to the pathophysiological process. A marker fulfilling these criteria might facilitate the early detection and might also serve in monitoring drug efficacy. Recently, the extracellular signal-regulated kinase ERK1/2 was detected in CSF of patients with neuropsychiatric disorders. Furthermore, ERK1/2 was reported to be activated in brains of animals infected with pathological prion protein. Therefore, we investigated CSF of 19 patients with CJD, 23 patients with other dementias including patients with Alzheimer's disease, and 12 patients with other neurological disorders. The measurement of ERK1/2 in the CSF samples was performed with an electrochemiluminescence assay and Western immunoblot. ERK1/2 and doubly phosphorylated ERK1/2 (pERK1/2) were detected in all patient groups. Significantly elevated mean levels of total ERK1/2 and pERK1/2 were found in the CJD patients. This increase was also observed in a CJD case that was negative for 14-3-3 protein or in CJD cases that had low levels of tau protein. Western immunoblot analysis suggested that ERK2 was the predominant form of ERKs present in our CSF samples. This pilot study suggests that ERK1/2 is a potential CSF biomarker for CJD, directly associated with the pathophysiological processes. Analysis of larger sample cohorts including other diseases with rapid neurodegeneration are required to confirm our findings.

Codon 129 polymorphism specific cerebrospinal fluid proteome pattern in sporadic Creutzfeldt-Jakob disease and the implication of glycolytic enzymes in prion-induced pathology.

Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which can reflect a physiological and pathological state of the central nervous system. In our present study, we show CSF protein patterns from patients with the two most frequent subtypes of sporadic Creutzfeldt-Jakob disease (sCJD) defined by the codon 129 genotype (MM, MV, and VV) and the protease-resistant form of prion protein (type 1 and type 2). The densitometric analysis of 2D gels showed up-regulation of 27 and down-regulation of 3 proteins in the MM-sCJD as well as the up-regulation of 24 proteins in the VV-sCJD as compared to nondemented control. Almost 40% of sCJD specific regulated proteins in CSF are involved in glucose metabolism, regardless of the codon 129 polymorphism. The increase in CSF levels of lactate dehydrogenase (LDH), glucose-6-phosphate isomerase (G6PI), and fructose-bisphosphate aldolase A (ALDOA) were validated on a larger group of sCJD patients including three possible codon 129 polymorphism carriers and three control groups consisting of nondemented, neurological cases as well as patients suffering from Alzheimer's disease or vascular dementia. Subsequently, the abundance of these glycolytic enzymes in the brain as well as their cellular localization were determined. This study demonstrates for the first time the implication of G6PI in prion-induced pathology as well as its cellular translocalization in sCJD. The identification of sCJD-regulated proteins in CSF of living symptomatic patients in our study can broaden our knowledge about pathological processes occurring in sCJD, as they are still not fully understood.

Neuronal phosphorylated RNA-dependent protein kinase in Creutzfeldt-Jakob disease.

The mechanisms of neuronal apoptosis in Creutzfeldt-Jakob disease (CJD) and their relationship to accumulated prion protein (PrP) are unclear. A recent cell culture study showed that intracytoplasmic PrP may induce phosphorylated RNA-dependent protein kinase (PKR(p))-mediated cell stress. The double-stranded RNA protein kinase PKR is a proapoptotic and stress kinase that accumulates in degenerating neurons in Alzheimer disease. To determine whether neuronal apoptosis in human CJD is associated with activation of the PKR(p) signaling pathway, we assessed in situ end labeling and immunocytochemistry for PrP, glial fibrillary acidic protein, CD68, activated caspase 3, and phosphorylated PKR (Thr451) in samples of frontal, occipital, and temporal cortex, striatum, and cerebellum from 6 patients with sporadic CJD and 5 controls. Neuronal immunostaining for activated PKR was found in all CJD cases. The most staining was in nuclei and, in contrast to findings in Alzheimer disease, cytoplasmic labeling was not detected. Both the number and distribution of PKR(p)-positive neurons correlated closely with the extent of neuronal apoptosis, spongiosis, astrocytosis, and microglial activation and with the phenotype and disease severity. There was no correlation with the type, topography, or amount of extracellular PrP deposits. These findings suggest that neuronal apoptosis in human CJD may result from PKR(p)-mediated cell stress and are consistent with recent studies supporting a pathogenic role for intracellular or transmembrane PrP.

Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin.

Disease-related PrP(Sc) [pathogenic PrP (prion protein)] is classically distinguished from its normal cellular precursor, PrP(C)(cellular PrP) by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease-resistant fragments of PrP(Sc) using PK (proteinase K), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrP(Sc) in its full-length form. In the present study, we show that thermolysin can degrade PrP(C) while preserving both PK-sensitive and PK-resistant isoforms of disease-related PrP in both rodent and human prion strains. For mouse RML (Rocky Mountain Laboratory) prions, the majority of PK-sensitive disease-related PrP isoforms do not appear to contribute significantly to infectivity. In vCJD (variant Creutzfeldt-Jakob disease), the human counterpart of BSE (bovine spongiform encephalopathy), up to 90% of total PrP present in the brain resists degradation with thermolysin, whereas only approximately 15% of this material resists digestion by PK. Detection of PK-sensitive isoforms of disease-related PrP using thermolysin should be useful for improving diagnostic sensitivity in human prion diseases.

Role of COX-2 in inflammatory and degenerative brain diseases.

In the last decade, the potential role of cyclooxygenase-2 (COX-2) and prostaglandins (PGs) in brain diseases has been extensively studied. COX-2 over-expression has been associated with neurotoxiticy in acute conditions, such as hypoxia/ischemia and seizures, as well as in inflammatory chronic diseases, including Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease (AD). However, the role played by COX-2 in neurodegenerative diseases is still controversial and further clinical and experimental studies are warranted. In addition, the emerging role of COX-2 in behavioural and cognitive functions strongly indicates that studies aimed at improving our knowledge of the physiological role of COX-2 in the central nervous system are crucial to fully understand the pros and cons of its manipulation in disabling neurological diseases.

Altered glycosylation of acetylcholinesterase in the Creutzfeldt-Jakob cerebrospinal fluid.

Several neurodegenerative disorders present deficiencies in the cholinergic system. Scarce research on prion encephalopathies has examined the levels of cholinergic pathway-related enzymes. Acetylcholinesterase (AChE) is expressed as several molecular forms. The potential importance of these variants is increased by the possibility that AChE has roles other than acetylcholine hydrolysis. We investigated the levels of AChE, its molecular forms, and glycosylation in the cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients.

Reduced expression of NO-sensitive guanylyl cyclase in reactive astrocytes of Alzheimer disease, Creutzfeldt-Jakob disease, and multiple sclerosis brains.

In Alzheimer's disease (AD) brains increased NO synthase (NOS) expression is found in reactive astrocytes surrounding amyloid plaques. We have recently shown that treatment with beta-amyloid peptides or IL-1beta down-regulates NO-sensitive soluble guanylyl cyclase (sGC) in cultured astrocytes and in adult rat brain. In this work, we have examined sGC activity and expression in postmortem brain tissue of AD patients and matched controls. No significant alteration was observed in basal or NO-stimulated sGC activity, nor in sGC beta1 and alpha1 subunit levels in cortical extracts of AD brains. Immunohistochemistry showed intense and widespread labeling of sGC beta1 in cortical and hippocampal neurons and white matter fibrillar astrocytes, while grey matter astrocytes were faintly stained. In AD, expression of sGC in neurons and fibrillar astrocytes is not altered but is markedly reduced in reactive astrocytes surrounding amyloid plaques. Immunostaining for sGC beta1 was also lacking in reactive astrocytes in cortex and subcortical white matter in Creutzfeldt-Jakob disease brains and in subacute and chronic plaques in multiple sclerosis (MS) brains. Thus, induction of astrocyte reactivity is associated with decreased capacity to generate cGMP in response to NO both in vitro and in vivo. This effect may be related to the development of the astroglial inflammatory response.

CSF lactate dehydrogenase activity in patients with Creutzfeldt-Jakob disease exceeds that in other dementias.

The diagnosis of Creutzfeldt-Jakob disease (CJD) is still made by exclusion of other dementias. We now evaluated lactate dehydrogenase (LDH) in the cerebrospinal fluid (CSF) as a possible additional diagnostic tool. CSF LDH levels of patients with CJD (n = 26) were compared with those in other dementias (n = 28). LDH isoenzymes were determined in a subset (n = 9). Total LDH and isoenzyme LDH-1 were significantly higher, whereas the fractions of LDH-2 and LDH-3 were significantly lower in CJD patients. We conclude that in addition to established CSF parameters, LDH and its isoenzymes might serve as a further help to discriminate between CJD and other dementias.

Matrix metalloproteinase profile in patients with Creuztfeldt-Jakob disease.

Preliminary findings suggest that abnormalities in matrix metalloproteinase (MMP) activity may be found in the cerebrospinal fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). In this study of 16 subjects with CJD and 16 age-, and sex-matched controls, we determined the presence of MMP-2 and MMP-9 in their active and proenzyme forms, the relative levels of MMP-3 and four inhibitors of MMP activity (TIMP-1, TIMP-2, TIMP-3 and TIMP-4), and the concentration of 4-3-3 protein. The methodology used involved zymography and immunological techniques. The results indicate that, compared with controls, CJD patients have a significantly higher positive frequency of pro-MMP-9 and of the active form of MMP-2, along with significantly higher levels of TIMP-1 and TIMP-2, classical inhibitors of MMP-9 and MMP-2, respectively. We also found a positive correlation between 14-3-3 protein concentration and that of TIMP-1 and TIMP-2 levels (correlation coefficients of 0.793 and 0.798, respectively). These results suggest that abnormalities in MMP and TIMP profiles may be helpful in the biochemical characterisation of CJD.

Cyclooxygenase-1 and -2 in brains of patients who died with sporadic Creutzfeldt-Jakob disease.

Cyclooxygenases (COXs) mediate inflammation, immunomodulation, blood flow, apoptosis, and fever in various diseases of the brain. Whereas COX-2 is cytokine inducible, COX-1 is expressed by macrophages/microglial cells that accumulate in pathological foci. We analyzed the localization of COX-1 and COX-2 in postmortem cortex slices of eight patients who died with sporadic Creutzfeldt-Jakob disease (CJD) and four neuropathologically unaltered controls by immunohistochemical double-labeling, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blotting experiments. In healthy brains, COX-1 was expressed by single macrophages/microglial cells and COX-2 by disseminated neurons. In patients with CJD, significantly (p = 0.0195) more COX-1-expressing macrophages/microglial cells were detected adjacent to neurons. COX-2 expression was predominantly observed in neurons, and their number was significantly higher (p < 0.0001) compared to controls. RT-PCR and Western blotting revealed more COX-1 and COX-2 mRNA and protein in one CJD patient than in one control patient. These data show that accumulation of COX-1-expressing macrophages/microglial cells and COX-2-expressing neurons might represent important regulatory mechanisms in the complex process of neuronal degeneration in CJD patients.

Overexpressed protein disulfide isomerase in brains of patients with sporadic Creutzfeldt-Jakob disease.

Earlier studies have failed to detect covalent modifications in beta-sheet-rich scrapie isoform prion protein (PrP(Sc)) and have concluded that the conversion of alpha-helix-rich cellular form prion protein (PrP(C)) to PrP(Sc) represents purely conformational transition not involving chemical reactions. However, recent studies have shown that the intradisulfide bond of PrP(C) can play an important role for instability and conformational change to PrP(Sc). Interestingly, we found overexpressed protein disufide isomerase (PDI) in brains of sporadic Creutzfeldt-Jakob disease (sCJD, human prion disease) patients using two dimensional electrophoresis and Western blot analysis but not in other neurodegenerative disorders as Down Syndrome and Alzheimer's disease. However, proteinase K digestion and plasminogen binding assay of brain homogenates incubated with PDI suggest that PDI has no effect on either proteinase resistance or conformational change of PrP. Overexpression of PDI protein in sCJD brain may simply reflect a cellular defense response against the altered prion protein.