Bozena Mazur-Kolecka

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.

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.

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.

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.

Abnormal Intracellular Accumulation and Extracellular Aβ Deposition in Idiopathic and Dup15q11.2-q13 Autism Spectrum Disorders

Background It has been shown that amyloid ß (Aβ), a product of proteolytic cleavage of the amyloid β precursor protein (APP), accumulates in neuronal cytoplasm in non-affected individuals in a cell type–specific amount. Methodology/Principal Findings In the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15) and autism spectrum disorder (ASD). In spite of interindividual differences within each examined group, levels of intraneuronal Aβ load were significantly greater in the dup(15) autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test). In eight regions, intraneuronal Aβ load differed significantly between idiopathic autism and control groups (p<0.0001). The intraneuronal Aβ was mainly N-terminally truncated. Increased intraneuronal accumulation of Aβ17–40/42 in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aβ accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aβ1–40/42 detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aβ and an extracellular deposition of full-length Aβ in nonfibrillar plaques. Conclusions/Significance The higher prevalence of excessive Aβ accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15) compared to subjects with idiopathic ASD, supports the concept of mechanistic and functional links between autism, epilepsy and alterations of APP processing leading to neuronal and astrocytic Aβ accumulation and diffuse plaque formation.

Accumulation of Alzheimer amyloid-β peptide in cultured myocytes is enhanced by serum and reduced by cerebrospinal fluid

Smooth muscle cells cultured from leptomeningeal vessels from old dogs with amyloid-angiopathy accumulate intracellular deposits that are immunoreactive for amyloid-β peptide (Aβ). We used this cellular model in the present study to examine the influence of sera and cerebrospinal fluid on intracellular accumulation of Aβ-immunoreactive deposits and on secretion of soluble Aβ into culture media. We found that sera from old dogs significantly increased the percentage of Aβ-positive smooth muscle cells in culture. The enhanced accumulation of Aβ was associated with (a) lower secretion of Aβ into media, (b) altered maturation of amyloid-β-precursor protein (AβPP) into AβPP751–770 with faster electrophoretic mobility, (c) increased accumulation of C-terminal fragments of AβPP (12–15 kD, 10kD and less), and (d) increased secretion of AβPP into culture media. These findings suggest that age- or disease-related serum factors increase accumulation of Aβ by affecting production and processing of AβPP. In contrast, cerebrospinal fluids reduced accumulation of Aβ. Involvement of Aβ-carrier proteins—apolipoprotein E and transthyretin—in accumulation of Aβ is demonstrated. Accumulation of Aβ in cultured smooth muscle cells—a model of β-amyloidosis—may be regulated by factors that alter production and processing of AβPP as well as the fate of soluble Aβ in extracellular space.

Lysosomal deposition of Aβ in cultures of brain vascular smooth muscle cells is enhanced by iron

Recently, we found that brain vascular smooth muscle cells from Tg2576 mice over-expressed the APP transgene in culture, secreted amyloid-beta peptide (Abeta) and accumulated Abeta intracellularly. Now we detected this intracellular Abeta inside lysosomes, which were also rich in C-terminal domain of APP, but not in endoplasmic reticulum, Golgi apparatus, or trans-Golgi network. Treatment of cultures with ferrous ions (50-150 microM) increased the proportion of muscle cells with Abeta immunoreactive granules and the amounts of intracellular Abeta1-40 and Abeta1-42 in a dose-dependent manner. This increase of intracellular Abeta1-40 by iron was inhibited by alpha-tocopherol, but not by a water-soluble antioxidant melatonin. The increase of intracellular Abeta1-42 by iron was not inhibited by alpha-tocopherol or melatonin. Cell treatment with iron did not alter the lysosomal localization of Abeta immunoreactivity. Cell treatment with iron (II and III), copper (II), zinc (II) and aluminum (III) increased cellular levels of carbonyls. However, the effect of zinc on Abeta accumulation in cultures was weak, and there were no effects of copper and aluminum. The data suggest that iron may be the factor that triggers vascular amyloidosis. Lysosomal accumulation of APP and Abeta initiates deposition of amyloid in blood vessels in Tg2576 mice.

Effect of DYRK1A activity inhibition on development of neuronal progenitors isolated from Ts65Dn mice.

Overexpression of dual‐specificity tyrosine‐(Y)‐phosphorylation‐regulated kinase 1A (DYRK1A), encoded by a gene located in the Down syndrome (DS) critical region, is considered a major contributor to developmental abnormalities in DS. DYRK1A regulates numerous genes involved in neuronal commitment, differentiation, maturation, and apoptosis. Because alterations of neurogenesis could lead to impaired brain development and mental retardation in individuals with DS, pharmacological normalization of DYRK1A activity has been postulated as DS therapy. We tested the effect of harmine, a specific DYRK1A inhibitor, on the development of neuronal progenitor cells (NPCs) isolated from the periventricular zone of newborn mice with segmental trisomy 16 (Ts65Dn mice), a mouse model for DS that overexpresses Dyrk1A by 1.5‐fold. Trisomy did not affect the ability of NPCs to expand in culture. Twenty‐four hours after stimulation of migration and neuronal differentiation, NPCs showed increased expression of Dyrk1A, particularly in the trisomic cultures. After 7 days, NPCs developed into a heterogeneous population of differentiating neurons and astrocytes that expressed Dyrk1A in the nuclei. In comparison with disomic cells, NPCs with trisomy showed premature neuronal differentiation and enhanced γ‐aminobutyric acid (GABA)‐ergic differentiation, but astrocyte development was unchanged. Harmine prevented premature neuronal maturation of trisomic NPCs but not acceleration of GABA‐ergic development. In control NPCs, harmine treatment caused altered neuronal development of NPCs, similar to that in trisomic NPCs with Dyrk1A overexpression. This study suggests that pharmacological normalization of DYRK1A activity may have a potential role in DS therapy.