George Merz

Ultrastructure of the microglia that phagocytose amyloid and the microglia that produce β-amyloid fibrils

SummaryThe function of microglia associated with β-amyloid deposits still remains a controversial issue. On the basis of recent ultrastructural data, microglia were postulated to be cells that form amyloid fibrils, not phagocytes that remove amyloid deposits. In this electron microscopic study, we examined the ability of microglia to ingest and digest exogenous amyloid fibrils in vitro. We demonstrate that amyloid fibrils are ingested by cultured microglial cells and collected and stored in phagosomes. The ingested, nondegraded amyloid remains within phagosomes for up to 20 days, suggesting a very limited effectiveness of microglia in degrading β-amyloid fibrils. On the other hand, we showed that in microglial cells of classical plaques in brain cortex of patients with Alzheimers disease, amyloid fibrils appear first in altered endoplasmic reticulum and deep infoldings of cell membranes. These differences in intracellular distribution of amyloid fibrils in microglial cells support our observations that microglial cells associated with amyloid plaques are engaged in production of amyloid, but not in phagocytosis.

Methylation regulates the intracellular protein-protein and protein-RNA interactions of FMRP.

FMRP, the fragile X mental retardation protein, is an RNA-binding protein that interacts with ∼4% of fetal brain mRNA. We have recently shown that a methyltransferase (MT) co-translationally methylates FMRP in vitro and that methylation modulates the ability of FMRP to bind mRNA. Here, we recapitulate these in vitro data in vivo, demonstrating that methylation of FMRP affects its ability to bind to FXR1P and regulate the translation of FMRP target mRNAs. Additionally, using double-label fluorescence confocal microscopy, we identified a subpopulation of FMRP-containing small cytoplasmic granules that are distinguishable from larger stress granules. Using the oxidative-stress induced accumulation of abortive pre-initiation complexes as a measure of the association of FMRP with translational components, we have demonstrated that FMRP associates with ribosomes during initiation and, more importantly, that methylation regulates this process by influencing the ratio of FMRP-homodimer-containing mRNPs to FMRP-FXR1P-heterodimer-containing mRNPs. These data suggest a vital role for methylation in normal FMRP functioning.

Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to Icelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA‐I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half‐life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, ≥70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway. J. Cell. Physiol. 173:423–432, 1997.

Nature of the scrapie agent: current status of facts and hypotheses.

Scrapie is a slow infection affecting the central nervous system (CNS) of sheep and goats (Dickinson, 1976). Experimentally the agent has been passaged in mice, hamsters and a number of other laboratory animal species. Kuru and Creutzfeldt-Jakob disease (CJD) are related diseases occurring in humans (Gajdusek, 1977). These diseases are caused by agents that have unconventional characteristics compared to those of known animal viruses (Gajdusek, 1977). The unusual physical, chemical and biological properties of the scrapie agent have led to an abundance of theories concerning its nature. Each of the major classes of macromolecules has been suggested as an important component of the scrapie agent. Hypotheses have suggested that scrapie agent is composed exclusively of protein (Griffith, 1967), exclusively of polysaccharide (Gibbons & Hunter, 1967), or exclusively of nucleic acid (Diener, 1972). Other hypotheses have suggested that it is a replicating membrane component (Gibbons & Hunter, 1967) or a filterable virus (Eklund et al., 1963). This is but a partial list of the theoretical constructs that have been proposed.

Altered binding of mutated presenilin with cytoskeleton-interacting proteins1

The majority of familial Alzheimers disease (AD) cases are linked to mutations on presenilin 1 and 2 genes (PS1 and PS2). The normal function of the proteins and the mechanisms underlying early‐onset AD are currently unknown. To address this, we screened an expression library for proteins that bind differentially to the wild‐type PS1 and mutant in the large cytoplasmic loop (PS1L). Thus we isolated the C‐terminal tail of the 170 kDa cytoplasmic linker protein (CLIP‐170) and Reed–Sternberg cells of Hodgkins disease‐expressed intermediate filament‐associated protein (Restin), cytoplasmic proteins linking vesicles to the cytoskeleton. PS1L binding to CLIP‐170/restin requires Ca2+. Treating cells with thapsigargin or ionomycin increased the mutated PS1 in CLIP‐170 immunoprecipitates. Further, PS1 and CLIP‐170 co‐localize in transfected cells and neuronal cultures.

Alteration of astrocytes and Wnt/β-catenin signaling in the frontal cortex of autistic subjects.

BackgroundAutism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. To date the etiology of this disorder is poorly understood. Studies suggest that astrocytes play critical roles in neural plasticity by detecting neuronal activity and modulating neuronal networks. Recently, a number of studies suggested that an abnormal function of glia/astrocytes may be involved in the development of autism. However, there is yet no direct evidence showing how astrocytes develop in the brain of autistic individuals.MethodsStudy subjects include brain tissue from autistic subjects, BTBR T + tfJ (BTBR) and Neuroligin (NL)-3 knock-down mice. Western blot analysis, Immunohistochemistry and confocal microscopy studies have be used to examine the density and morphology of astrocytes, as well as Wnt and β-catenin protein expression.ResultsIn this study, we demonstrate that the astrocytes in autisitcsubjects exhibit significantly reduced branching processes, total branching length and cell body sizes. We also detected an astrocytosis in the frontal cortex of autistic subjects. In addition, we found that the astrocytes in the brain of an NL3 knockdown mouse exhibited similar alterations to what we found in the autistic brain. Furthermore, we detected that both Wnt and β-catenin proteins are decreased in the frontal cortex of autistic subjects. Wnt/β-catenin pathway has been suggested to be involved in the regulation of astrocyte development.ConclusionsOur findings imply that defects in astrocytes could impair neuronal plasticity and partially contribute to the development of autistic-like behaviors in both humans and mice. The alteration of Wnt/β-catenin pathway in the brain of autistic subjects may contribute to the changes of astrocytes.Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. To date the etiology of this disorder is poorly understood. Studies suggest that astrocytes play critical roles in neural plasticity by detecting neuronal activity and modulating neuronal networks. Recently, a number of studies suggested that an abnormal function of glia/astrocytes may be involved in the development of autism. However, there is yet no direct evidence showing how astrocytes develop in the brain of autistic individuals. Study subjects include brain tissue from autistic subjects, BTBR T + tfJ (BTBR) and Neuroligin (NL)-3 knock-down mice. Western blot analysis, Immunohistochemistry and confocal microscopy studies have be used to examine the density and morphology of astrocytes, as well as Wnt and β-catenin protein expression. In this study, we demonstrate that the astrocytes in autisitcsubjects exhibit significantly reduced branching processes, total branching length and cell body sizes. We also detected an astrocytosis in the frontal cortex of autistic subjects. In addition, we found that the astrocytes in the brain of an NL3 knockdown mouse exhibited similar alterations to what we found in the autistic brain. Furthermore, we detected that both Wnt and β-catenin proteins are decreased in the frontal cortex of autistic subjects. Wnt/β-catenin pathway has been suggested to be involved in the regulation of astrocyte development. Our findings imply that defects in astrocytes could impair neuronal plasticity and partially contribute to the development of autistic-like behaviors in both humans and mice. The alteration of Wnt/β-catenin pathway in the brain of autistic subjects may contribute to the changes of astrocytes.

Neuropathology of the Aging Brain and Dementia of the Alzheimer Type

The two most prominent histopathological changes found in high numbers in the Alzheimer’s disease (AD) brain are the neurofibrillary tangle and the neuritic and amyloid plaque. Each of these lesions is characterized by an accumulation of proteinaceous fibrous profiles (the paired helical filaments) and amyloid fibers, respectively. These structures are not only distinguishable from each other but from all of the normal cellular filaments as well. Both lesions are also found, in low numbers, in the brains of aged, but otherwise clinically normal, individuals. This quantitative difference between age-associated pathology and the AD brain implies that the cause(s) responsible for the lesions may be fundamentally the same and differ only in degree.

Isolation and characterization of macrophages from scrapie-infected mouse brain

SummaryWe have isolated and characterized a population of brain macrophages from normal and scrapieinfected mice. The cells are phagocytic, possess Fc-IgG receptors, Mac-1 surface antigen and proliferate in the presence of macrophage colony stimulating factor. They resemble microglia in that they have a plasmalemmal distribution of the enzyme nucleoside diphosphatase, a property that is characteristic of microglia in situ. In two of the three combinations of scrapie agent and mouse strain examined, the number of brain macrophages was several fold higher than in normal control mice. The increase was not observed in mice infected intraperitoneally or in control mice inoculated with normal brain homogenate. The increase is detectable as early as 3–5 weeks postinoculation. The agent/host combination that failed to show an increase in brain macrophages is one that develops large numbers of amyloid plaques. These observations suggest that these cells are closely associated with the scrapie pathogenic process in the CNS. The failure of these cells to increase in the plaque forming model of scrapie disease also suggests that they play a role in the control of CNS amyloidogenesis.

Abnormal cell properties and down-regulated FAK-Src complex signaling in B lymphoblasts of autistic subjects.

Recent studies suggest that one of the major pathways to the pathogenesis of autism is reduced cell migration. Focal adhesion kinase (FAK) has an important role in neural migration, dendritic morphological characteristics, axonal branching, and synapse formation. The FAK-Src complex, activated by upstream reelin and integrin β1, can initiate a cascade of phosphorylation events to trigger multiple intracellular pathways, including mitogen-activated protein kinase-extracellular signal-regulated kinase and phosphatidylinositol 3-kinase-Akt signaling. In this study, by using B lymphoblasts as a model, we tested whether integrin β1 and FAK-Src signaling are abnormally regulated in autism and whether abnormal FAK-Src signaling leads to defects in B-lymphoblast adhesion, migration, proliferation, and IgG production. To our knowledge, for the first time, we show that protein expression levels of both integrin β1 and FAK are significantly decreased in autistic lymphoblasts and that Src protein expression and the phosphorylation of an active site (Y416) are also significantly decreased. We also found that lymphoblasts from autistic subjects exhibit significantly decreased migration, increased adhesion properties, and an impaired capacity for IgG production. The overexpression of FAK in autistic lymphoblasts countered the adhesion and migration defects. In addition, we demonstrate that FAK mediates its effect through the activation of Src, phosphatidylinositol 3-kinase-Akt, and mitogen-activated protein kinase signaling cascades and that paxillin is also likely involved in the regulation of adhesion and migration in autistic lymphoblasts.

Microtubular interactions of presenilin direct kinesis of Aβ peptide and its precursors

In our previous study we demonstrated that presenilin 1 (PS1) interacts with cytoplasmic linker protein 170/Restin (CLIP‐170/Restin). Herein we show that disruption of the interaction of these proteins within neuronal cell‐lines (SY5Y and N2a) can be accomplished by the transfection of vectors that drive the expression of peptide fragments corresponding to their binding domains (BDPs). Interestingly, the disruption of the PS1/CLIP‐170 complex is associated with both decreased secretion of endogenous Aβ and decreased uptake of exogenous Aβ from the medium. BDP‐expressing cells were also more resistant to surges of Aβ secretion induced by thapsigargin and ionomycin (that elevate intracellular calcium concentrations) and mutations in PS1 linked to familial Alzheimers disease. Uptake of Aβ by SY5Y cells was amplified when preincubated with ApoE and was mediated through lipoprotein receptor‐related protein (LRP). BDP‐expressing cells or cells treated with PS1 anti‐sense oligonucleotides were less capable of taking up Aβ from the medium compared with controls, indicating that the PS1/CLIP‐170 interaction is involved and that PS1 cannot be substituted. In this study, we also mapped the minimum binding domains (mBDPs) of PS1 and CLIP‐70 to regions corresponding to the N‐terminal end of the large cytoplasmic loop of PS1 and the metal binding motif‐containing C‐terminal end of CLIP‐170. Further, our data obtained from experiments involving in vitro taxol‐polymerization of tubulin and confocal immmunofluorescence suggest that PS1, via CLIP‐170, may serve as an anchor to the microtubules for specific subcellular fractions containing amyloidogenic fragments. Interestingly, Notch is absent from this population of microtubule binding subcellular fractions and its cleavage was unaffected in cells transfected with the PS1‐based BDP. This raises the possibility that the interaction of PS1 with CLIP‐170 could provide the conceptual basis for anti‐amyloidogenic therapeutic strategies with improved specificity. However, this approach may be hampered by a low efficiency, because it may also block Aβ clearance from the interstitial space of the CNS.