Melatonin Treatment Enhances Aß Lymphatic Clearance in a Transgenic Mouse Model of Amyloidosis.

BACKGROUND: It has been postulated that inadequate clearance of the amyloid ß protein (Aß) plays an important role in the accumulation of Aß in sporadic late onset Alzheimer's disease (AD). While the blood brain barrier (BBB) has taken the center stage in processes involving Aß clearance, little information is available about the role of the lymphatic system. We previously reported that Aß is cleared through the lymphatic system. We now assessed lymphatic Aß clearance by treating a mouse model of AD amyloidosis with melatonin, an Aß aggregation inhibitor and immuno-regulatory neurohormone. OBJECTIVE: To confirm and expand our initial finding that Aß is cleared through the lymphatic system. Lymphatic clearance of metabolic and cellular "waste" products from the brain into the peripheral lymphatic system has been known for a long time. However, except for our prior report, there is no additional experimental data published about Aß being cleared into peripheral lymph nodes. METHODS: For these experiments, we used a transgenic mouse model (Tg2576) that over-expresses a mutant form of the Aß precursor protein (APP) in the brain. We examined levels of Aß in plasma and in lymph nodes of transgenic mice as surrogate markers of vascular and lymphatic clearance, respectively. Aß levels were also measured in the brain and in multiple tissues. RESULTS: Clearance of Aß peptides through the lymphatic system was confirmed in this study. Treatment with melatonin led to the following changes: 1-A statistically significant increase in soluble monomeric Aß40 and an increasing trend in Aß42 in cervical and axillary lymph nodes of treated mice. 2- Statistically significant decreases in oligomeric Aß40 and a decreasing trend Aß42 in the brain. CONCLUSION: The data expands on our prior report that the lymphatic system participates in Aß clearance from the brain. We propose that abnormalities in Aß clearance through the lymphatic system may contribute to the development of cerebral amyloidosis. Melatonin and related indole molecules (i.e., indole- 3-propionic acid) are known to inhibit Aß aggregation although they do not reverse aggregated Aß or amyloid fibrils. Therefore, these substances should be further explored in prevention trials for delaying the onset of cognitive impairment in high risk populations.

A unique case of limb-girdle muscular dystrophy type 2A carrying novel compound heterozygous mutations in the human CAPN3 gene.

A unique sib pair afflicted by limb girdle muscular dystrophy type 2A (LGMD2A) is described showing a slowly progressive autosomal recessive type of muscular dystrophy with onset in the third and fourth decades. The patients had early asymmetric muscle involvement characterized by prominent biceps brachii atrophy with sparing of the knee extensors. Additional findings included elevation of serum creatine kinase level, myopathic EMG changes and dystrophic type of pathology on muscle biopsy. Asymmetrical wasting of muscles in the extremities exhibited uniform and highly selective CT imaging patterns. RNA and DNA analyses confirmed novel compound heterozygous mutations (R147X/L212F) in the human CAPN3 gene.

The amyloid beta protein induces oxidative damage of mitochondrial DNA.

Multiple lines of evidence suggest involvement of oxidative stress in the pathogenesis of Alzheimer disease (AD). The finding that amyloid beta peptide (A beta) has neurotoxic properties and that such effects are mediated in part by free-radicals has provided an avenue to explore new therapeutic strategies. In this study, we showed that exposure of PC 12 cells to an A beta fragment induces oxidative damage of mitochondrial DNA. Cells were exposed for 24 h to 50 microM A beta (25-35) or to 50 microM of a control peptide with a scrambled sequence. Oxidative damage of mitochondrial DNA (mtDNA) was assessed using a Southern blot technique and an mtDNA-specific probe recognizing a 13.5-kilobase restriction fragment. Treatment of DNA with NaOH was used to reveal abasic sites and single strand breaks. Treatment with endonuclease III or FAPy glycosylase was used to detect pyrimidine or purine lesions, respectively. Cells exposed to A beta exhibited marked oxidative damage of mtDNA as evidenced by characteristic changes on Southern blots. Cells exposed to the scrambled peptide did not show such modifications. Simultaneous addition of the pineal hormone melatonin consistently prevented the A beta-induced oxidative damage to mtDNA. Mitochondrial dysfunction in AD has been demonstrated by several laboratories. This study provides experimental evidence supporting a causative role of A beta in mitochondrial lesions of AD.

Neuronal oxidative stress precedes amyloid-beta deposition in Down syndrome.

The predictable chronological sequence of pathological events in Down syndrome (DS) provides the opportunity to rigorously investigate the relationship between oxidative stress and amyloid-beta (Abeta) deposition. In this study, we report a marked accumulation of oxidized nucleic acid, 8-hydroxyguanosine (8OHG), and oxidized protein, nitrotyrosine, in the cytoplasm of cerebral neurons in DS with the levels of nucleic acid and protein oxidation paralleling each other. Relative density measurements of neuronal 8OHG immunoreactivity showed that there was a significant increase (p < 0.02) in DS (n = 22, ages 0.3-65 yr) compared with age-matched controls (n = 10, ages 0.3-64 yr). As a function of age, 8OHG immunoreactivity increased significantly in the teens and twenties (p < 0.04), while Abeta burden only increased after age 30 (p < 0.0001). In 9 cases of DS bearing Abeta deposition, the extent of deposits of Abeta ending at amino acid 42 (Abeta42) was actually associated with a decrease in relative 8OHG (r = -0.79, p < 0.015) while Abeta40 was not. These findings suggest that in brains of patients with DS, increased levels of oxidative damage occur prior to the onset of Abeta deposition.

Catecholamines inhibit lipid peroxidation in young, aged, and Alzheimer's disease brain.

Some catecholamines and indolamines inhibit lipid peroxidation. Recent studies indicate that catecholaminergic inhibition of lipid peroxidation may be receptor mediated in vivo and in cell cultures. Because oxidative stress is one of the hypothesized pathogenic mechanisms for neurodegenerative diseases, including Alzheimer's disease (AD), we hypothesized that catecholaminergic and indolaminergic inhibition of lipid peroxidation would be altered in AD as compared to age-matched non-AD. To test this hypothesis we studied the effect of a variety of neurotransmitters and their antagonists on ascorbate-stimulated lipid peroxidation in membrane fragment preparations derived from postmortem human brain. In this in vitro system, the inhibition of lipid peroxidation by dopamine and serotonin did not appear to be receptor mediated. Further, our findings indicate that there is no apparent effect of age or AD on the inhibition of lipid peroxidation by catecholaminergic and indolaminergic agents.

Complement depletion affects demyelination and inflammation in experimental allergic neuritis.

The effect of systemic complement depletion by cobra venom factor (CVF) on experimental allergic neuritis (EAN) was studied in rats immunized with variable amounts of bovine peripheral nerve myelin. Low-dose myelin EAN rats treated with CVF i.p. (n = 10) had lower clinical scores (0.3 +/- 0.7 vs. 1.1 +/- 1.1), less demyelination (0.4 +/- 0.8 vs. 1.9 +/- 1.1) and inflammation (0.6 +/- 1.2 vs. 2 +/- 1) than EAN animals treated with i.p. saline (n = 10). Endoneurial infiltrates had fewer ED1-positive (phagocytic) macrophages (0.4 +/- 0.5 vs. 1.6 +/- 1.1) and CD11bc-positive (expressing iC3b receptor or CR3) cells (1 +/- 0.8 vs. 2.5 +/- 0.8) (mean +/- S.D.) detected by immunocytochemistry. This effect was partially abrogated by immunizing animals with a higher dose of myelin. Our studies suggest that complement may play a role in the recruitment of macrophages into the endoneurium and in opsonizing myelin for phagocytosis.

Oral administration of type I interferon modulates the course of experimental allergic neuritis.

We investigated the effect of oral administration of type I interferon (IFN) in experimental allergic neuritis (EAN) in Lewis rats immunized with bovine peripheral nerve myelin. Starting at 7 days preceding immunization, rats were fed daily until sacrifice either with 5000 U rat IFN-alpha/beta or mock-IFN. The clinical severity of EAN was significantly reduced in IFN-alpha/beta fed animals compared to mock-IFN fed controls. Demyelination, but not inflammation, was decreased in IFN-alpha/beta fed compared to mock-IFN fed rats at day 20 after immunization. In situ IFN-gamma production and inflammation were reduced when evaluated by immunocytochemistry at day 13 after immunization. Spleen cells from IFN-alpha/beta fed compared to mock-IFN fed EAN rats showed significantly reduced proliferation to stimulation with Con A or peripheral nerve myelin. IFN-gamma production in draining lymph node cells was significantly reduced after stimulation with bovine peripheral nerve myelin. Our data suggest that oral administration of IFN-alpha/beta reduces the severity of EAN, possibly by a reduction in IFN-gamma production.

Colloid (hyaline) inclusion bodies in the central nervous system: their presence in the substantia nigra is diagnostic of Parkinson's disease.

Intracytoplasmic "colloid" inclusions have been described within neurons of several discrete central nervous system nuclei in a variety of entities. Although they lack specificity for any particular disease, they are believed to represent one of the morphologic changes of neuronal aging. Because premature aging of the substantia nigra has been one of the claimed mechanisms occurring in Parkinson's disease, the prevalence of colloid inclusions was studied within the substantia nigra in 15 patients with Parkinson's disease, 15 age-matched controls, 50 "normal" individuals, 10 patients with dementia of Alzheimer's type, and two patients with amyotrophic lateral sclerosis. Colloid bodies were found in the substantia nigra of all patients with Parkinson's disease and were virtually absent in the other populations. Histochemical and ultrastructural analyses showed that colloid bodies differ from early and mature Lewy bodies. They may represent the "pale" inclusions rarely mentioned in the literature and often mistaken for early Lewy bodies. "Colloid" bodies in the substantia nigra are diagnostic of Parkinson's disease. These findings support the theory of "premature" aging of the substantia nigra in this disease.

Concurrent neuroborreliosis and Alzheimer's disease: analysis of the evidence.

Several recent reports have claimed a possible association between Borrelia burgdorferi infection and Alzheimer's disease (AD). Herein, we describe our search for additional evidence of neuroborreliosis in AD. Brain tissue from neuropathologically confirmed cases of AD was cultured for B burgdorferi using standard microbiologic methods. Material derived from culture was further examined using electron microscopy, direct immunofluorescence and acridine orange fluorescence. Previous studies have shown high titers of antiborrelia antibodies in CSF in all cases of confirmed neuroborreliosis; therefore, we tested CSF from neuropathologically confirmed cases of AD by indirect immunofluorescence and enzyme-linked immunoassay. In addition, imprint preparations from AD and control brain tissues were studied by direct immunofluorescence using a monoclonal antiborrelia antibody. Finally, a Western blot method was used to analyze protein extracts from cultures and AD brain tissue for the presence of borrelia antigen. Contrary to previous studies, our results do not support an association between infection with B burgdorferi and AD.

Microscopic polyangiitis with ocular involvement.

Microscopic polyangiitis is an exclusively small-vessel (arterioles, capillaries, or venules) vasculitis that primarily involves the kidney and often involves the lungs, skin, or nervous system. Characteristic features include focal segmental glomerulonephritis, nongranulomatous necrotizing vasculitis, and serum positive for perinuclear-staining antineutrophil cytoplasmic antibodies (P-ANCA). We report a case of microscopic polyangiitis with previously unreported eyelid and conjunctival manifestations that responded well to immunosuppressive therapy.

Indole-3-propionate: a potent hydroxyl radical scavenger in rat brain.

The hydroxyl radical scavenging activity of indole-3-propionate was evaluated by kinetic competition studies with the hydroxyl radical trapping reagent 2,2'-azino-bis-(3-ethyl-benz-thiazoline-6-sulfonic acid) (ABTS) and by measuring hydroxyl radical-initiated lipid peroxidation in the rat striatum. Using ABTS, the indole was shown to act as a potent hydroxyl radical scavenger with a rate constant of 7.8x1010 mol l-1 s-1. Hydroxyl radical-initiated lipid peroxidation, determined by measuring tissue malondialdehyde formation, was inhibited dose-dependently both in vitro and in vivo. Indole-3-propionate reacts with hydroxyl radicals at a diffusion controlled rate and can thereby provide on-site protection against the oxidative damage of biomolecules induced by these highly reactive and toxic oxygen intermediates. While it remains to be established if endogenous brain tissue levels of indole-3-propionate are sufficiently high to have a significant impact on total antioxidative capacity, the compound itself or a structurally related agent may be useful as an antioxidant adjuvant to combat hydroxyl radical-mediated oxidative stress.

Heat-shock induces abnormalities in the cellular distribution of amyloid precursor protein (APP) and APP fusion proteins.

The heat shock or stress response may play a role in the pathogenesis of Alzheimer's disease. We conducted experiments to visualize microscopically the distribution of wild type amyloid precursor protein (APP) and the behavior of an APP deletion mutant under stress. This was achieved by heat-shock treatment of cells expressing fusion recombinant APP proteins tagged with secreted placental alkaline phosphatase (SEAP). The fusion proteins were cleaved and secreted in a manner similar to wild type APP in unstressed control cells. SEAP activity was detected by cytochemical methods within the cytoplasm in less than 10% of transfected unstressed cells. Heat shocked cells showed a striking difference from the control cells in that over 90% of the stressed cells displayed strong intracytoplasmic SEAP activity occurring with Golgi-like pattern and/or membranous distribution. The effects of heat shock were not due to a peculiar behavior of the clones and depended on the APP portion of the constructs. This study shows miscompartmentalization of APP under stress. Such cellular changes may bear important implications in the processing of APP.

Image analysis microspectroscopy of senile plaque capillary amyloid in Alzheimer's disease. A preliminary study.

The relationship between cerebral amyloid and the microvasculature in senile plaques was studied with image analysis microspectroscopy. This imaging technique permitted topographic quantification of amyloid concentration within microscopic subregions of single senile plaques. The highest concentration of amyloid was measured at the walls of intraplaque capillaries. Spectroscopic images revealed a decreasing gradient of amyloid concentration with increasing distance from intraplaque capillaries. The concentration of amyloid in nonplaque capillaries was lower than that measured in capillaries within well-developed amyloid plaques. These findings support the premise that plaque amyloid deposition starts at the level of the capillary.

Lewy bodies of Parkinson's disease. Immune electron microscopic demonstration of neurofilament antigens in constituent filaments.

Recently it has been shown by light microscopic immunochemistry that Lewy bodies (LBs) react with antibodies raised against neurofilament proteins (NFPs). Because of the ubiquity of the NFPs within neurons, the heterogeneous makeup of the inclusions, and the varying patterns of immunolabeling, we undertook to determine whether the labeled elements are indeed constituent filaments. Employing a preembedding technique, we investigated sections of the same LBs by light and electron immunochemistry. Decoration of the filaments was obtained with a monoclonal anti-NFP antibody. Whereas cores of mature LBs were unreactive by light microscopy, these same cores yielded a positive reaction at the ultrastructural level. Early LBs were intensely labeled in both the core and periphery. These results demonstrate that the filamentous profiles that form the LBs are antigenically identical to neurofilaments and suggest a posttranslational modification of the filaments as they "age" within the inclusion.

Computerized image-analysis microspectroscopy of tissue sections.

A simple technique using monochromatic light was applied to computer-assisted image analysis of tissue sections. The method consists of the determination of the spectral characteristics of different elements of interest and selective monochromatic illumination of a microscopic field followed by image analysis. Results show a dramatic increase in resolution and contrast. By means of an image subtraction technique at two wavelengths, it was possible to achieve specific optical isolation and quantification of image components.

PAS reaction stains phagocytosed atypical mycobacteria in paraffin sections.

In 4% formaldehyde-fixed, paraffin-embedded tissues, PAS negativity for mycobacteria in the literature has been axiomatic. However, recent observations of disseminated human mycobacteriosis have shown that intracellular (phagocytosed) organisms stained strongly positive with the routine PAS technique. This staining was abolished by a sequential hydrolytic procedure, which suggests that the carbohydrate residues of the mycobacterial peptidoglycolipids are responsible for the reaction. This staining characteristic in tissue sections is of diagnostic importance, since few bacteria of medical relevance are concomitantly acid-fast and PAS positive. The nature of this affinity is for the aqueous form of basic fuchsin.

Two distinct types of intraneuronal neurofibrillary tangles highlighted by polarized light after silver impregnation with a modified Bielschowsky method.

Neurofibrillary tangles (NFTs) are one of the morphological hallmarks of Alzheimer's disease. The birefringency and dichroism of NFTs following congo red staining have long been known. Herein, we report the observation that a subset of NFTs show distinct birefringency induced by a modified Bielschowsky silver impregnation method. Birefringency of NFTs could not be elicited after silver impregnation with one other version of the Bielschowsky method or with the Bodian technique. To our knowledge, these properties of NFTs after metal impregnation have not been previously documented.