Janusz Frackowiak

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.

Cerebral amyloid angiopathy plays a direct role in the pathogenesis of Alzheimer's disease. Pro-CAA position statement

For the purposes of this debate here we argue the case that cerebral amyloid angiopathy (CAA) has a direct role in the pathogenesis of Alzheimers disease (AD). Firstly, there is a very close relationship between CAA and AD and they share genetic risk factors. Secondly, we propose a specific mechanism which puts age-related cerebrovascular degeneration at a crucial point in the pathogenesis of AD as follows. Amyloid beta-protein (Abeta) is normally eliminated from the brain along with extracellular fluid by bulk flow along the perivascular pathway. Age-related fibrosis of cerebral cortical and meningeal arteries leads to impaired drainage of Abeta along the perivascular pathway and, together with the production of Abeta by smooth muscle cells and perivascular cells, is responsible for accumulation of Abeta as CAA. Reduced elimination leads to increased concentration of soluble Abeta in the extracellular fluid of the brain parenchyma. Increased concentration of soluble Abeta leads to the formation of insoluble Abeta plaques, other features of AD pathology, and dementia.

Non-Fibrillar β-Amyloid Protein is Associated with Smooth Muscle Cells of Vessel Walls in Alzheimer Disease

Abstract. Meningeal blood vessels were studied in Alzheimer disease (AD) and control brain specimens obtained from autopsies within 16 hours after death. Serial sections were stained with thioflavine S and Congo red and immunostained for the presence of β-amyloid precursor protein (βPP) and β-protein and for smooth muscle-specific proteins myosin, α-actin, and desmin. Isolated blood vessels were studied by immunoblotting for the presence of βPP, fragments of βPP, and β-protein. The arteries that were strongly immunopositive for β-protein in all layers of the walls were also positive for amyloid fibrils on thioflavine S and Congo red stainings. The focal immunostaining for β-protein in less affected vessels was located in the tunica media in the cytoplasm of smooth muscle cells or formed granules between myocytes. The cytoplasmic p-protein and some of the small deposits present between cells were negative for amyloid fibrils. The vessels isolated from specimens containing β-protein-immunoreactive material contained 3 kD, 4.2–4.5 kD, 8.5–9 kD, and 17.5 kD β-protein-immunoreactive bands. These bands were not found in the samples assessed as β-protein-negative by immunocytochemistry. These data indicate that during formation of amyloid in AD vessel walls, nonfibrillar, monomeric, and oligomeric β-protein accumulate.

In vitro production of β-amyloid in smooth muscle cells isolated from amyloid angiopathy-affected vessels ☆

Our recent results indicate that in Alzheimers disease (AD) amyloid angiopathy, smooth muscle cells are responsible for beta-amyloid deposition in the vascular wall. Aged dogs have been shown to develop beta-amyloid angiopathy similar to that in AD. Thus, we used brain and peripheral vessels from aged and young dogs to isolate cells of the vascular wall: smooth muscle cells, fibroblasts, and endothelial cells, and to study their ability to produce beta-protein. We demonstrate that only myocytes from aged animals cultured for up to 4 weeks accumulate beta-protein-immunoreactive material intracellularly, in the form of fibrillar and amorphous deposits.

The role of overexpressed DYRK1A protein in the early onset of neurofibrillary degeneration in Down syndrome

The gene encoding the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A (DYRK1A) is located in the Down syndrome (DS) critical region of chromosome 21. The third copy of DYRK1A is believed to contribute to abnormal brain development in patients with DS. In vitro studies showing that DYRK1A phosphorylates tau protein suggest that this kinase is also involved in tau protein phosphorylation in the human brain and contributes to neurofibrillary degeneration, and that this contribution might be enhanced in patients with DS. To explore this hypothesis, the brain tissue from 57 subjects including 16 control subjects, 21 patients with DS, and 20 patients with sporadic Alzheimer’s disease (AD) was examined with two antibodies to the amino-terminus of DYRK1A (7F3 and G-19), as well as two polyclonal antibodies to its carboxy-terminus (X1079 and 324446). Western blots demonstrated higher levels of full-length DYRK1A in the brains of patients with DS when compared to control brains. Immunocytochemistry revealed that DYRK1A accumulates in neurofibrillary tangles (NFTs) in subjects with sporadic AD and in subjects with DS/AD. Overexpression of DYRK1A in patients with DS was associated with an increase in DYRK1A-positive NFTs in a gene dosage-dependent manner. Results support the hypothesis that overexpressed DYRK1A contributes to neurofibrillary degeneration in DS more significantly than in subjects with two copies of the DYRK1A gene and sporadic AD. Immunoreactivity with antibodies against DYRK1A not only in NFTs but also in granules in granulovacuolar degeneration and in corpora amylacea suggests that DYRK1A is involved in all three forms of degeneration and that overexpression of this kinase may contribute to the early onset of these pathologies in DS.

Apolipoproteins E3 and E4 induce, and transthyretin prevents accumulation of the Alzheimer's β-amyloid peptide in cultured vascular smooth muscle cells

Cultured brain vascular smooth muscle cells (SMCs) accumulate beta-peptide in intracytoplasmic granules [9,10,33]. We show here that apoE3 and E4 induces the intracytoplasmic beta-peptide accumulation in cultured human and canine SMCs. The induction is dose-dependent and the accumulated granules also contain apoE and some were thioflavine S-positive. The deposits induced with apoE3 were more abundant though less stable than with apoE4. Transthyretin at physiological concentrations blocked the effects of apoE3/E4. Thus, accumulation of beta-peptide appears to be regulated by beta-peptide carrier proteins.

Secretion and accumulation of Alzheimer's β-protein by cultured vascular smooth muscle cells from old and young dogs

Cultured smooth muscle cells isolated from beta-amyloid-affected blood vessels from old dogs accumulate beta-protein at early passages [5,24]. Now, we show that smooth muscle cells derived from amyloid-free brain blood vessels and peripheral arteries from old and young animals are induced by culture conditions to deposit intracellularly fibrillar and non-fibrillar beta-protein. Accumulation of beta-protein is associated with a higher secretion of beta-protein, but not with a higher secretion of beta-amyloid precursor protein (beta APP) or higher cellular content of beta APP. Gradual cessation of proliferative activity was observed in cultures that accumulate beta-protein.

Link Between DYRK1A Overexpression and Several-Fold Enhancement of Neurofibrillary Degeneration With 3-Repeat Tau Protein in Down Syndrome

Triplication of chromosome 21 in Down syndrome (DS) results in overexpression of the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A gene (DYRK1A). DYRK1A phosphorylates cytoplasmic tau protein and appears in intraneuronal neurofibrillary tangles (NFTs). We have previously shown significantly more DYRK1A-positive NFTs in DS brains than in sporadic Alzheimer disease (AD) brains. This study demonstrates a gene dosage-proportional increase in the level of DYRK1A in DS in the cytoplasm and the cell nucleus, and enhanced cytoplasmic and nuclear immunoreactivity of DYRK1A in DS. The results suggest that overexpressed DYRK1A may alter both phosphorylation of tau and alternative splicing factor (ASF). Two-dimensional electrophoresis revealed modification of ASF phosphorylation in DS/AD and AD in comparison to controls. Altered phosphorylation of ASF by overexpressed nuclear DYRK1A may contribute to the alternative splicing of the tau gene and an increase by 2.68× of the 3R/4R ratio in DS/AD, and a several-fold increase in the number of 3R tau-positive NFTs in DS/AD subjects compared with that in sporadic AD subjects. These data support the hypothesis that phosphorylation ofASF by overexpressed DYRK1A may contribute to alternative splicing of exon 10, increased expression of 3R tau, and early onset of neurofibrillary degeneration in DS.

Cell type- and brain structure-specific patterns of distribution of minibrain kinase in human brain

The minibrain kinase (Mnb/Dyrk1A) gene is localized in the Down syndrome (DS) critical region of chromosome 21. This gene encodes a proline-directed serine/threonine protein kinase (minibrain kinase-Mnb/Dyrk1A), which is required for the proliferation of distinct neuronal cell types during postembryonic neurogenesis. To study the distribution of Mnb/Dyrk1A during human brain development and aging, we raised Mnb/Dyrk1A-specific antibody (mAb 7F3) and examined 22 brains of normal subjects from 8 months to 90 years of age. We found that neurons were the only cells showing the presence of 7F3-positive product in both cell nucleus and cytoplasm. Nuclear localization supports the concept that Mnb/Dyrk1A may be involved in control of gene expression. Synaptic localization of Mnb/Dyrk1A also supports our previous studies suggesting that Mnb/Dyrk1A is a regulator of assembly of endocytic apparatus and appears to be involved in synaptic vesicle recycling and synaptic signal transmission. Accumulation of numerous 7F3-positive corpora amylacea in the memory and motor system subdivisions in subjects older than 33 years of age indicates that Mnb/Dyrk1A is colocalized with markers of astrocyte and neuron degeneration. Differences in the topography and the amount of Mnb/Dyrk1A in neurons, astrocytes, and ependymal and endothelial cells appear to reflect cell type- and brain structure-specific patterns in trafficking and utilization of Mnb/Dyrk1A.

Amyloid-β impairs development of neuronal progenitor cells by oxidative mechanisms

Neuronal progenitor cells (NPCs) are being considered for treatment of neurodegenerative diseases associated with beta-amyloidosis: Alzheimers disease (AD) and Down syndrome (DS). However, the neurotoxic properties of amyloid-beta peptide (Abeta) may impair survival and differentiation of transplanted NPCs. Hence, we studied the influence of Abeta on development of human NPCs--proliferation, migration, formation of colonies of neurons, formation processes--in culture. Pre-fibrillized human Abeta1-40 blocked development of neuronal colonies. NPC development was impaired in the presence of soluble Abeta1-40 (1.75-7 microM), and NPC differentiation into large and small neurons was altered, as demonstrated by morphometry. Antioxidant vitamin E partially abolished these effects, but not the reduced formation of neuronal processes. NPCs cultured with 7 microM Abeta1-40 accumulated Abeta monomers and oligomers and contained higher levels of protein carbonyls and lipid peroxidation products HNE and MDA. We suggest that Abeta1-40 impairs development of NPCs by oxidative damage. Hence, a prerequisite of successful neuroreplacement therapy using NPCs in AD and DS/AD may be removal of amyloid-beta and antioxidative treatment.