Neurotoxins released from interferon-gamma-stimulated human astrocytes.

Astrocytes become activated in degenerative neurological diseases. In order to gain a greater understanding of the inflammatory factors released upon activation, we stimulated adult human astrocytes with interferon-gamma and examined the resultant conditioned medium (CM) for toxicity against differentiated human neuroblastoma SH-SY5Y cells. Cell death was measured by lactate dehydrogenase release assay. We then used various treatments of the media to determine the distribution and nature of the toxic components. Removal of interleukin-6 by a specific antibody reduced the toxicity by 22%. Blockade of proteases with an inhibitor cocktail reduced it by a further 22%. When oxygen-free radical production was blocked with NADPH oxidase inhibitors, the toxicity was reduced by 15.4%. When prostaglandin production was blocked by cyclooxygenase inhibitors, the toxicity of the CM was reduced by 14.5%. When glutamate was removed by treatment with glutamate decarboxylase, the toxicity was reduced by 10.3%. When the inhibitors were added together to the astrocyte culture, the total toxicity of the CM was reduced by 91%. This was in reasonable agreement with the 85.37% total obtained by adding the individual components. The data show that activated astrocytes release a specific combination of neurotoxic compounds. They suggest that effective anti-inflammatory treatment of such neurodegenerative diseases as Alzheimer's disease, Parkinson's disease and Amyotrophic lateral sclerosis could be improved by using an appropriate combination of anti-inflammatory agents instead of relying on any single agent.

Converging perturbed microvasculature and microglial clusters characterize Alzheimer disease brain.

We have investigated physical properties of microvasculature and vessel association with microglial clusters in cortical tissue from Alzheimer disease individuals, classified as severe (ADsev) or mild (ADmild), and nondemented controls (ND). Immunostaining with laminin or von Willerbrand factor demonstrated numbers of microvessels and microvascular density were significantly higher in ADsev cases compared with levels in ADmild or ND cases suggesting proangiogenic activity in ADsev brain. Evidence for extravascular laminin immunoreactivity was found in ADsev tissue and was largely absent in ADmild and ND cases suggesting vascular remodeling in ADsev brain included abnormalities in blood vessels. Microgliosis was progressively increased from ND to ADmild to ADsev with the latter demonstrating areas of clustered microglia (groupings of three or more cells) rarely observed in ADmild or ND cases. Microglial clusters in ADsev brain were in close proximity with extravascular laminin and also plasma protein, fibrinogen, implicating vascular perturbation as a component of inflammatory reactivity. ADsev brain also exhibited elevated levels of the pro-inflammatory/angiogenic factors tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) in association, relative to non-association, with microglial clusters. The presence of extravascular laminin and fibrinogen and the vascular modifying factors, TNF-α and VEGF in localization with clusters of activated microglia, is consistent with microglial-induced vascular remodeling in ADsev brain. Microglial-vascular reciprocal interactions could serve a critical role in the amplification and perpetuation of inflammatory reactivity in AD brain.

Lrrk2 interaction with alpha-synuclein in diffuse Lewy body disease.

Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of genetically inherited Parkinson's disease (PD) and its more severe variant diffuse Lewy body disease (DLB). Pathological mutations in Lrrk2 are autosomal dominant, suggesting a gain of function. Mutations in alpha-synuclein also produce autosomal dominant disease. Here we report an interaction between Lrrk2 and alpha-synuclein in a series of diffuse Lewy body (DLB) cases and in an oxidative stress cell based assay. All five cases of DLB, but none of five controls, showed co-immunoprecipitation of Lrrk2 and alpha-synuclein in soluble brain extracts. Colocalization was also found in pathological deposits in DLB postmortem brains by double immunostaining. In HEK cells transfected simultaneously with plasmids expressing Lrrk2 and alpha-synuclein, co-immunoprecipitation of Lrrk2 and alpha-synuclein was detected when they were exposed to oxidative stress by H(2)O(2). Taken together, these results suggest the possibility that in PD and related synucleinopathies, oxidative stress upregulates alpha-syn and Lrrk2 expression, paving the way for pathological interactions. New therapeutic approaches to PD and the synucleinopathies may result from limiting the interaction between Lrrk2 and alpha-synuclein.

Tauopathies with parkinsonism: clinical spectrum, neuropathologic basis, biological markers, and treatment options.

Tauopathies with parkinsonism represent a spectrum of disease entities unified by the pathologic accumulation of hyperphosphorylated tau protein fragments within the central nervous system. These pathologic characteristics suggest shared pathogenetic pathways and possible molecular targets for disease-modifying therapeutic interventions. Natural history studies, for instance, in progressive supranuclear palsy, frontotemporal dementia with parkinsonism linked to chromosome 17, corticobasal degeneration, and Niemann-Pick disease type C as well as in amyotrophic lateral sclerosis/Parkinson-dementia complex permit clinical characterization of the disease phenotypes and are crucial to the development and validation of biological markers for differential diagnostics and disease monitoring, for example, by use of neuroimaging or proteomic approaches. The wide pathologic and clinical spectrum of the tauopathies with parkinsonism is reviewed in this article, and perspectives on future advances in the understanding of the pathogenesis are given, together with potential therapeutic strategies.

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions.

Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27 - 30 detectable by the 3F4 antibody against human PrP109 - 112. We recently identified a new PK-resistant PrP species, designated PrP*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97 - 108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP*20 but also a small amount of 3F4-detected PK-resistant PrP27 - 30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP*20 to the occurrence of the 3F4-detected PrP27 - 30 was observed. Our study suggests that an increase in the level of PrP*20 characterizes the early stages of prion diseases.

Autosomal dominant dystonia-plus with cerebral calcifications.

OBJECTIVE: To report genealogic, clinical, imaging, neuropathologic, and genetic data from a Canadian kindred with dystonia and brain calcinosis originally described in 1985. METHODS: The authors performed clinical examinations and CT and PET studies of the head and analyzed blood samples. One autopsy was performed. RESULTS: The family tree was expanded to 166 individuals. No individuals were newly affected with dystonia, but postural tremor developed in two. The mean age at symptom onset was 19 years. Eight individuals had dystonia: three focal, one segmental, one multifocal, and three generalized. Seven displayed additional signs: chorea, intellectual decline, postural tremor, and dysarthria. CT studies were performed on five affected and 10 at-risk family members. All affected individuals and eight at-risk individuals had brain calcinosis. PET scans in two individuals showed reduced D(1)- and D(2)-receptor binding and reduced uptake of 6-[(18)F]fluoro-l-dopa. Autopsy of one affected individual showed extensive depositions of calcium in the basal ganglia, thalamus, cerebral white matter, and cerebellum. No specific immunohistochemistry abnormalities were seen. Genome search data showed no evidence of linkage to the previously described loci IBGC1, DYT1, and DYT12. CONCLUSIONS: The phenotype of this family consists of dystonia-plus syndrome. Brain calcium deposits vary in severity and distribution, suggesting that calcifications alone are not entirely responsible for the observed clinical signs. Further studies are needed to elucidate the etiology of this heterogeneous group of disorders.

Role of ICAM-1 in persisting inflammation in Parkinson disease and MPTP monkeys.

It has been established that neuroinflammation is present in the substantia nigra (SN) of Parkinson disease (PD) cases but the factors responsible are as yet unknown. One contributing protein may be the intercellular adhesion molecule-1 (ICAM-1, CD54). ICAM-1 with its counter receptor, the lymphocyte function-associated antigen 1 (LFA-1) is known to play a key role in inflammatory processes and in T-cell mediated host defense mechanisms. We detected large numbers of ICAM-1-positive reactive astrocytes in the SN of a series of 14 patients with neuropathologically confirmed PD, including 3 of familial origin, compared with 11 age-matched controls. In PD SN, these ICAM-1-positive reactive astrocytes were particularly concentrated around many residual neurons in areas of heavy neuronal loss and extracellular melanin accumulation. LFA-1-positive reactive microglia gathered in areas of intense ICAM-1 expression, and LFA-1-positive leukocytes were identified infiltrating the tissue. Double immunostaining for ICAM-1 and LFA-1 revealed aggregates of reactive microglia embedded in areas of diffuse ICAM-1. Leukocyte counts were 5 fold higher in PD SN compared to controls (P < 0.001). Similar over-expression of ICAM-1 was found in monkeys that had been exposed to MPTP from 5.5 to 14 years previously compared with control monkeys. The presence of ICAM-1-positive reactive astrocytes in Parkinson disease and MPTP-treated monkeys is indicative of a sustained inflammatory process and suggests that antiinflammatory agents may have a place in PD therapy.

Genome-wide analysis of the parkinsonism-dementia complex of Guam.

BACKGROUND: Parkinsonism-dementia complex (PDC) is a neurofibrillary tangle degeneration involving the deposition of Alzheimer-type tau, predominantly in the mesial temporal cortex, brainstem, and basal ganglia. It occurs in focal geographic isolates, including Guam and the Kii peninsula of Japan. The familial clustering of the disease has suggested that a genetic factor could be important in its etiology. OBJECTIVE: To determine whether a genetic locus could be identified, linked, or associated with PDC. DESIGN AND PATIENTS: We performed a genome-wide association study of 22 Guamanian PDC and 19 control subjects using 834 microsatellite markers with an approximate genome-wide marker density of 4.4 centimorgans. RESULTS: Two-point association analysis identified 17 markers (P<.015). Each of these markers then underwent conventional linkage analysis in 5 families with PDC. One marker, D20S103, generated a logarithm of odds score of greater than 1.5. Multipoint association analysis also highlighted 2 other areas on chromosome 14q (adjacent to D14S592, 59.2 megabases [M]) and chromosome 20 (adjacent to D20S470, 17.4 M) with multipoint association logarithm of the odds scores of greater than 2. The areas around D20S103, D14S592, and D20S470 were further analyzed by association using additional microsatellite markers and by conventional linkage analysis. This did not provide further evidence for the role of these areas in PDC. CONCLUSIONS: This study has not identified a single gene locus for PDC, confirming the impression of a geographic disease isolate with a complex genetic, a genetic/environmental etiology, or a purely environmental etiology.

Inflammatory processes in amyotrophic lateral sclerosis.

Neuroinflammation is a characteristic of pathologically affected tissue in several neurodegenerative disorders. These changes can be observed in the brainstem and spinal cord of amyotrophic lateral sclerosis (ALS) cases and in mouse models of the disease. They include an accumulation of large numbers of activated microglia and astrocytes, as well as small numbers of T cells, mostly adhering to postcapillary venules. Accompanying biochemical alterations include the appearance of numerous molecules characteristic of free-radical attack, the occurrence of proteins associated with activation of the complement cascade, and a sharp upregulation of the enzyme cyclooxygenase 2 (COX-2). Anti-inflammatory agents may have a role to play in treating ALS. COX-2 is a particularly attractive target because of its marked increase in ALS spinal cord.

Analysis of tau haplotypes in Pick's disease.

Pick's disease (PiD) is characterized by the deposition of tau protein as three-repeat tau Pick bodies, whereas progressive supranuclear palsy (PSP) involves the deposition of four-repeat tau neurofibrillary tangles. PSP is associated with the tau H1 haplotype. The authors investigated a possible association between PiD and the tau H1 or H2 haplotype. There was no difference between the tau H2 haplotype or H2H2 genotype frequency in PiD cases and control subjects. No tau mutations were identified in pathologically typical cases of PiD, with antibody 12-E8-negative Pick bodies.

High stability of mRNAs postmortem and protocols for their assessment by RT-PCR.

Measurement of gene expression is a major area of brain research. We report on the remarkable postmortem stability of a selection of brain mRNAs in both fresh and frozen brain tissue. We describe techniques for extracting total RNA, synthesizing cDNAs from the mRNAs, amplifying specific cDNAs by the polymerase chain reaction technique, and quantitating the products. We chose five genes to study: the housekeeping gene cyclophilin; the complement components C3 and C4; the microtubule associated protein-2 (MAP-2); and the strongly inducible cyclooxygenase COX-2. We found little deterioration in total RNA or in any of the mRNAs in postmortem tissue up to 96 h. When tissue was frozen, stored at -70 degrees C for 15 years and then thawed, there was no evidence of deterioration from storage, but there was gradual deterioration post thawing. All the mRNAs were stable for 1-2 h at 4 degrees C following thawing. Cyclophilin, C3 and C4 mRNAs were still stable after 8 h, MAP-2 and COX-2 mRNAs showed significant deterioration between 2 and 4 h, and COX-2 mRNA showed drastic deterioration between 4 and 8 h. The data give no indication of rapid postmortem degeneration of RNA. Reliable mRNA values may be obtained from postmortem brain with long autolysis times provided the tissue has been kept in the cold, and from frozen tissues for 1-2 h after thawing.

Polymorphisms in inflammatory genes and the risk of Alzheimer disease.

The concept of inflammation as a major factor in Alzheimer disease (AD) has heretofore been based on postmortem findings of autodestructive changes associated with the lesions coupled with epidemiological evidence of a protective effect of anti-inflammatory agents. Now there is evidence that the risk of AD is substantially influenced by a total of 10 polymorphisms in the inflammatory agents interleukin 1alpha, interleukin 1beta, interleukin 6, tumor necrosis factor alpha, alpha(2)-macroglobulin, and alpha(1)-antichymotrypsin. The polymorphisms are all common ones in the general population, so there is a strong likelihood that any given individual will inherit 1 or more of the high-risk alleles. The overall chances of an individual developing AD might be profoundly affected by a "susceptibility profile" reflecting the combined influence of inheriting multiple high-risk alleles. Since some of the polymorphisms in question have already been linked to peripheral inflammatory disorders, such as juvenile rheumatoid arthritis, myasthenia gravis, and periodontitis, associations between AD and several chronic degenerative diseases may eventually be demonstrated. Such information could lead to strategies for therapeutic intervention in the early stages of such disorders.

Relationship between beta amyloid peptide generating molecules and neprilysin in Alzheimer disease and normal brain.

beta-Amyloid peptide (Abeta) is generated by two cleavages of amyloid precursor protein (APP). The initial cleavage by BACE is followed by gamma-secretase cleavage of the C-terminal APP fragment. Presenilin-1 (PS-1) is intimately related to gamma-secretase. Once formed, Abeta is mainly broken down by neprilysin. To estimate vulnerability to Abeta senile plaque formation, we measured the relative mRNA levels of APP695, APP751, APP770, BACE, presenilin-1 (PS-1) and neprilysin in nine brain areas and in heart, liver, spleen and kidney in a series of Alzheimer disease (AD) and control cases. Each of the mRNAs was expressed in every tissue examined. APP695 was the dominant APP isoform in brain. Compared with controls, APP695 and PS-1 mRNA levels were significantly elevated in high plaque areas of AD brain, while neprilysin mRNA levels were significantly reduced. BACE levels were not significantly different in AD compared with control brain. In peripheral organs, there were no significant differences in any of the mRNAs between AD and control cases. APP isoforms were differently expressed in the periphery than in brain, with APP 751>770>695. Neprilysin mRNA levels were much higher, while APP695 and PS-1 mRNA levels were much lower in the periphery than in brain. The data suggest that, in the periphery, the capacity to degrade Abeta is srong, accounting for the failure of Abeta deposits to form. In plaque prone areas of AD brain, the capacity to degrade Abeta is weak, while the capacity to generate Ab is upregulated. In plaque resistant areas of brain, a closer balance exists, but there is some tendency towards lower degrading and higher synthesizing capacity in AD brain compared with control brain. Overall, the data indicate that effectiveness of degradation by neprilysin may be a key factor in determining whether Abeta deposits develop.

Inflammatory cytokine levels are influenced by interactions between THP-1 monocytic, U-373 MG astrocytic, and SH-SY5Y neuronal cell lines of human origin.

We measured the secretion of interleukin (IL)1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha) from human monocytic (THP-1), astrocytic (U-373 MG) and neuronal (SH-SY5Y) cell lines alone and in co-culture, with and without stimulation by a combination of lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) or amyloid beta peptide 1-40 (Abeta). LPS+IFN-gamma stimulation increased IL-1beta secretion 16-fold from THP-1 cells. It increased IL-6 secretion 23-fold from THP-1 cells and 2.5-fold from U-373 MG cells. It increased TNF-alpha secretion 3.4-fold from THP-1 cells, but did not influence its secretion from U-373 MG cells. It did not affect the secretion of any of the cytokines from SH-SY5Y cells. Abeta stimulation increased IL-6 secretion 2.3-fold from U-373 MG cells but did not influence secretion of IL-1beta or TNF-alpha. Abeta stimulation also failed to influence secretion of any of the cytokines from THP-1 or SH-SY5Y cells. When THP-1 and U-373 MG cells were cocultured, IL-1beta and IL-6 secretion, but not TNF-alpha secretion, were significantly reduced from the levels obtained in independent cultures, suggesting that a mutual suppressive action may occur between microglia and astrocytes.

3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA in Alzheimer and control brain.

Statins are widely used pharmaceutical agents which lower plasma cholesterol by inhibiting the rate controlling enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase. One epidemiological study suggests that statin therapy may provide protection against Alzheimer disease (AD). The aim of the present study was to determine the relative expression of HMG-CoA reductase mRNAs in various areas of brain as well as in peripheral organs and to compare values in AD and control cases. High levels of the mRNA were found in all areas of brain but no obvious differences were found between AD and controls. We conclude that brain has a robust capacity to synthesize cholesterol which appears to be unaffected by AD pathology.

A clinical and pathological study of motor neurone disease on Guam.

Despite over 40 years of intensive study, the cause of the high incidence of motor neurone disease (MND) on Guam, and the relationship between this disease and MND seen in the rest of the world are still uncertain. We present a series of 45 cases of Guamanian MND, which reaffirm the clinical similarity between this disease and MND seen in other countries. However, the occurrence of MND among the indigenous Chamorros of Guam is distinguished by four factors: (i) high prevalence; (ii) frequent familial occurrence; (iii) co-occurrence with the parkinsonism-dementia complex (PDC); and (iv) association with an unusual and distinctive linear retinopathy termed Guam retinal pigment epitheliopathy (GRPE). These distinguishing factors were not present in four non-Chamorros who resided on Guam when their MND symptoms occurred. Pathologically, the classical features of MND were seen in Guamanian Chamorro cases including ubiquitin inclusions. Neurofibrillary tangles were frequently seen. The neurofibrillary tangles appeared in the same distribution as described in the PDC but, unlike classical PDC, they were not usually associated with cell loss and occurred less frequently. While neurofibrillary tangle formation and the clinicopathological syndrome of MND may occur in parallel, observations from this series suggest that pathologically classical MND on Guam may occur independently of neurofibrillary degeneration and the clinical features of PDC.

Marked increase in cyclooxygenase-2 in ALS spinal cord: implications for therapy.

OBJECTIVE: To evaluate the hypothesis that cyclooxygenase-2 (COX-2) is linked to the pathology of ALS by determining whether COX-2 mRNA levels are upregulated in ALS spinal cord. METHODS: Spinal cord from 11 ALS cases and 27 controls consisting of 15 cases of Alzheimer disease (AD), six cases of Parkinson disease (PD), three cases of cerebrovascular disease, and three control cases were analyzed. Total RNA was extracted and reverse transcriptase-PCR analysis performed for the mRNA of COX-2, COX-1, the microglial marker CD11b, and the housekeeping gene cyclophilin. RESULTS: In ALS compared with non-ALS spinal cord, COX-2 mRNA was upregulated 7.09-fold (p < 0.0001), COX-1 1.14-fold (p = 0.05), and CD11b 1.85-fold (p = 0.0012). COX-2 mRNA levels in AD, PD, cerebrovascular disease, and control cases were each significantly lower than in ALS and were not significantly different from each other. Western blots of the protein products were in general accord with the mRNA data, with COX-2 protein levels being upregulated 3.79-fold compared with non-ALS cases (p = 0.015). CONCLUSIONS: The strong upregulation of COX-2 mRNA in ALS is in accord with studies in the superoxide dismutase transgenic mouse model in which COX-2 upregulation occurs. Taken in conjunction with evidence of a neuroprotective effect of COX-2 inhibitors in certain animal models and in organotypic cultures, the data are supportive of a possible future role for COX-2 inhibitors in the treatment of ALS.

Complement components, but not complement inhibitors, are upregulated in atherosclerotic plaques.

Complement activation occurs in atherosclerotic plaques. The capacity of arterial tissue to inhibit this activation through generation of the complement regulators C1 inhibitor, decay accelerating factor, membrane cofactor protein (CD46), C4 binding protein (C4BP), and protectin (CD59) was evaluated in pairs of aortic atherosclerotic plaques and nearby normal artery from 11 human postmortem specimens. All 22 samples produced mRNAs for each of these proteins. The ratios of plaque versus normal artery pairs was not significantly different from unity for any of these inhibitors. However, in plaques, the mRNAs for C1r and C1s, the substrates for the C1 inhibitor, were increased 2.35- and 4.96-fold, respectively, compared with normal artery; mRNA for C4, the target for C4BP, was elevated l.34-fold; and mRNAs for C7 and C8, the targets for CD59, were elevated 2.61- and 3.25-fold, respectively. By Western blotting and immunohistochemistry, fraction Bb of factor B, a marker of alternative pathway activation, was barely detectable in plaque and normal arterial tissue. These data indicate that it is primarily the classical, not the alternative pathway, that is activated in plaques and that key inhibitors are not upregulated to defend against this activation.