Mary Sundsmo

Secreted form of amyloid β protein precursor is involved in the growth regulation of fibroblasts

Fibroblasts that harbor an antisense construct of amyloid beta protein precursor (ABPP) cDNA, A-1, produced less ABPP mRNA and ABPP and grew poorly. Normal growth was restored when either parent cell conditioned medium (CM) or purified ABPP was provided. The capacity of the CM to restore cell growth was abolished by passage through an anti-ABPP immunoaffinity column; the activity was in the bound fraction. A Mr 90,000 protein recognized by the anti-ABPP antibody was diminished in the CM of A-1. CM from ABPP cDNA-transfected cells expressing high levels of ABPP was more potent than that from non-transfected parent cells in restoring A-1 growth. These results indicate that ABPP is released from cells into the medium and has an autocrine function in growth regulation.

Oxidative stress induces amyloid-like aggregate formation of Nacp/α-synuclein in vitro

The precursor of non-amyloid beta protein component of Alzheimers disease amyloid (NACP/alpha-synuclein), found in Lewy bodies of Parkinsons disease (PD), is a presynaptic protein genetically linked to some familial types PD. Mechanisms of abnormal NACP/alpha-synuclein aggregation in neurodegenerative diseases are unclear. Since oxidative stress might play a role in PD pathogenesis, we investigated the role of iron and peroxide in NACP/alpha-synuclein aggregation. Immunoblot analysis showed that human NACP/alpha-synuclein (but not beta-synuclein) aggregated in the presence of ferric ion and was inhibited by the iron chelator deferoxamine. Ferrous ion was not effective by itself, but it potentially aggregated NACP/alpha-synuclein in the presence of hydrogen peroxide. NACP/ alpha-synuclein aggregates displayed strong thioflavine-S and congo-red reactivity, reminiscent of amyloid. This study suggests that NACP/alpha-synuclein aggregation might be closely related to oxidative reactions which may play a critical role in neurodegeneration in disorders with Lewy bodies.

Human recombinant NACP/α-synuclein is aggregated and fibrillated in vitro: Relevance for Lewy body disease

The precursor of non-amyloid beta protein component of Alzheimers disease amyloid (NACP/alpha-synuclein) is aggregated and fibrillated under certain conditions, i.e., increasing time lag, high temperature and low pH. These in vitro aggregates form Thioflavine-S-positive filamentous structures, reminiscent of amyloid-like fibrils. Since some Lewy bodies in Parkinsons disease display Thioflavine-S reactivity, our results may suggest that amyloidogenic properties of NACP/alpha-synuclein may play a crucial role in pathogenesis of disorders with Lewy bodies such as Parkinsons disease.

Immunoreactivity of CD45, a protein phosphotyrosine phosphatase, in Alzheimer's disease.

SummaryBoth protein kinases and phosphoprotein phosphatases are important components of signal transduction systems in cells. Recent studies in Alzheimers disease (AD) have shown abnormal protein phosphorylation in the cortex suggesting an alteration in these enzymes. In the present study, an antibody against CD45 was used to analyze the status of this protein phosphotyrosine phosphatase in AD. We studied and quantified the immunohistochemical and immunochemical distribution of this integral membrane protein in control and AD brain. We found that anti-CD45 immunostained the great majority of microglia, both resting and activated. These cells were Ricinus communis agglutinin I positive and glial fibrillary acidic protein and neurofilament negative. The AD frontal cortex showed a 35% (P<0.01) increase in the number of anti-CD45 immunoreactive microglia as compared with controls. These results were consistent with the immunoblot quantification of CD45 immunoreactivity following native gel electrophoresis. In AD, 30% of the CD45-immunostained microglia were clustered in the neuritic plaques (about six per plaque) while the remaining 70% were scattered in the neuropil. The AD hippocampus showed an increase in CD45-immunoreactive microglia in the molecular layer of the dentte gyrus. At the ultrastructural level, CD45 immunoreactivity was localized exclusively to the plasma membrane of the microglia. The presence of the anti-CD45 immunoreactivity in microglia suggests the possibility that they may require the presence of CD45 as a cell surface receptor which may regulate cell function through modulation of intracellular signaling.

Defective neurite extension is caused by a mutation in amyloid β/A4 (Aβ) protein precursor found in Familial Alzheimer's Disease

Clonal central nervous system neuronal cells, B103, do not synthesize detectable endogenous APP or APLP. B103 cells transfected with both wild-type (B103/APP) and mutant APP construct (B103/APP delta NL) secreted comparable amounts of soluble forms of APP (sAPP). B103/APP cells produced sAPP and cleaved at amyloid beta/A4 (A beta) 16, the alpha-secretase site, and B103/APP delta NL cells produced sAPP beta cleaved at A beta 1, the beta-secretase site. B103/APP delta NL cells developed fewer neurites than B103/APP cells in a serum-free defined medium. Neurite numbers of parent B103 cells were increased by the 50% conditioned medium (CM) from B103/APP cells but reduced by the CM from B103/APP delta NL cells. Chemically synthesized A beta at concentration levels higher than 1 nM reduced numbers of neurites from B103 or B103/APP delta NL cells. However, A beta at 1-100 nM could not reduce the neurite number of B103/APP cells. The protective activity against A betas deleterious effect to reduce neurite numbers was attributed to sAPP alpha in the CM. Although sAPP alpha could block the effect of A beta, sAPP beta could not do so under the identical condition, suggesting the importance of the C-terminal 15-amino acid sequence in sAPP alpha. Nevertheless, sAPP alphas protective activity required the N-terminal sequence around RERMS, previously identified to be the active domain of sAPP beta. The overall effect of APP mutation which overproduced A beta and sAPP beta and underproduced sAPP alpha was a marked decline in the neurotrophic effect of APP. We suggest that the disruption of balance between the detrimental effect of A beta and the trophic effect of sAPP may be important in the pathogenesis of AD caused by this pathogenic APP mutation.

The synaptic protein NACP is abnormally expressed during the progression of Alzheimer' s disease

NACP, the precursor of non-A beta component of Alzheimers disease (AD) amyloid (NAC), is a synaptic protein that could potentially be involved in AD. We studied, by dot-blot, NACP levels in the frontal cortex of AD cases staged according to severity of disease and correlated them with cognitive performance and neuropathological markers. Early AD cases showed one fold higher levels of NACP immunoreactivity (IR) compared to moderate and severe AD. Levels of NACP-IR were correlated with tangle counts (r = -0.305, P = 0.04) and Blessed score (r = -0.356, P = 0.01), but not with plaque counts (r = 0.132, P = 0.39). This study suggests that the abnormal accumulation of NACP during the early stages of AD might play an important role in the mechanisms of neurodegeneration and synaptic damage in AD.

Neurofibrillary tangle-associated alteration of stathmin in Alzheimer's disease

Stathmin (p19), a 19-kDa cytosolic phosphorotein, plays a key role in converting extracellular signals into intracellular biochemical changes. Antibodies and cDNA specific for stathmin were used to study its levels and localization in normal and Alzheimers disease (AD) brain tissue. The stathmin protein concentration was reduced in AD neocortex as assessed by Western blotting, whereas the concentration of its mRNA detected by both in situ hybridization and slot blot were increased in AD. The alteration of the stathmin protein concentration was negatively correlated with neurofibrillary tangle numbers but not with plaque numbers. Immunoreactivity was evenly localized to the cytoplasm of neurons in control cortical sections, whereas in AD it was preferentially localized to some of the neurofibrillary tangle-bearing neurons. Numbers of stathmin-positive neurons were inversely correlated with tangle numbers but not with plaque numbers in the frontal cortex of AD patients.

Degradation of proteins in the membrane-cytoskeleton complex in Alzheimer's disease. Might amyloidogenic APP processing be just the tip of the iceberg?

Many transmembrane proteins are linked to cytoskeletal proteins and form the membrane-cytoskeleton complex (FIG. 1). Membrane proteins, such as band-3, Na+/K+ ATPase, Na+ channel, and amyloid protein precursor (APP), are likely to be anchored to elements of the cytoskeleton, such as microfilaments, intermediate filaments, and microtubules via interactions with ankyrin, spectrin, and various yet unidentified proteins.’.* APP is abnormally degraded in Alzheimer’s disease (AD). Here we present evidence that other membrane and cytoskeletal proteins including band-3 and spectrin may also be abnormally proteolyzed. A single abnormal protease may be responsible for this rather general proteolysis in AD, but another possibility worth considering is that the altered membrane structure in AD3 may affect the interaction of membranecytoskeleton complex proteins with the membrane, thereby exposing previously hidden sites on these proteins, and making them susceptible to proteolytic activity. For example, recent studies show APP mutations near or within the transmembrane regions of the m ~ l e c u l e , ~ ~ ~ which might alter the interaction of APP with the membrane and increase its vulnerability to amyloidogenic proteolysis. However, mutations in APP are rare and only segregate with certain families that are susceptible to early-onset AD.6 Thus, in the majority of AD cases, the aberrant amyloidogenic degradation of APP might be a result ofaltered membrane properties rather than mutations within the APP molecule. The altered membrane properties could be a result of mutated cytoskeletal proteins. A recent study in a dystrophin-deficient mouse genetic model of Duchenne