Library

Notes: Abstract: Our previous studies have implicated perlecan, a specific heparan sulfate proteoglycan, in the pathogenesis of fibrillar β-amyloid protein (Aβ) accumulation and persistence in Alzheimer's disease (AD) brain. In the present investigation, we determined if perlecan mRNA was present in rodent and human brain tissue and whether perlecan persistence in Aβ amyloid deposits in AD hippocampus may be partly due to increased perlecan expression and/or decreased perlecan degradation. To detect and to quantify low-abundance perlecan mRNA in rodent and postmortem human brain tissue, regions of perlecan domain I (503 and 366 bp) containing the unique heparan sulfate glycosaminoglycan attachment sites were analyzed by reverse transcription (RT) and polymerase chain reaction (PCR). Perlecan mRNA was detected in rodent brain, kidney, and liver and in human AD and normal aged frontal cortex. Different-size transcripts of perlecan domain I were found, suggesting the existence of alternatively spliced variants of perlecan or closely related gene products. Quantitative competitive RT-PCR using a mutant perlecan domain I internal standard was used to determine perlecan mRNA levels in total RNA isolated from the hippocampus of 10 AD (mean ± SEM duration of illness, 11.3 ± 1.4 years) and 10 normal aged controls. No significant difference in perlecan mRNA levels from the hippocampus of AD (1.12 ± 0.29 amol/500 ng of total RNA) versus normal aged controls (1.09 ± 0.30 amol/500 ng of total RNA) was found, indicating that perlecan expression remained at steady-state levels. These results therefore suggest that perlecan persistence in Aβ-amyloid deposits in late-stage AD may be primarily due to decreased perlecan degradation and removal.

Notes: Abstract: Expression of the genes encoding the β/A4 amyloid protein precursor (APP) and microtubule-associated protein τ was studied in an embryonal carcinoma cell line (P19) that differentiates in vitro into cholinergic neurons after treatment with retinoic acid. Expression of APP increased 34- (mRNA) and 50-fold (protein) during neuronal differentiation; APP-695 accounted for most of this increase. These remarkable increases in APP expression coincided with a proliferation of neuronal processes and with an increase in content of τ mRNA. Moreover, subsequent decreases in the levels of APP and τ mRNA coincided with the onset of the degeneration of the neuronal processes. Immunocytochemical staining suggested that 〉85% of the P19-derived neurons are cholinergic and that APP is present in the neuronal processes and cell bodies. These results suggest that APP may play an important role in construction of neuronal networks and neuronal differentiation and also indicate that this embryonal carcinoma cell line provides an ideal model system to investigate biological functions of APP and the roles of APP and τ protein in development of Alzheimer's disease in cholinergic neurons.

Notes: Abstract: Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar β-amyloid (Aβ) deposits of Alzheimer's disease. Perlecan purified from the Engelbreth-Holm-Swarm tumor was used to define perlecan's interactions with Aβ and its effects on Aβ fibril formation. Using a solid-phase binding immunoassay, freshly solubilized full-length Aβ peptides bound immobilized perlecan at two sites, representing both high-affinity [KD = ∼5.8 × 10−11M for Aβ (1–40); KD = ∼6.5 × 10−12M for Aβ (1–42)] and lower-affinity [KD = 3.5 × 10−8M for Aβ (1–40); KD = 4.3 × 10−8M for Aβ (1–42)] interactions. An increase in the binding capacity of Aβ (1–40) to perlecan correlated with an increase in Aβ amyloid fibril formation during a 1-week incubation period. The high-capacity binding of Aβ (1–40) to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans and was completely abolished by heparin, but not by chondroitin-4-sulfate. Using a thioflavin T fluorometry assay, perlecan accelerated the rate of Aβ (1–40) amyloid fibril formation, causing a significant increase in Aβ fibril assembly over a 2-week incubation period at 1 h (2.8-fold increase), 1 day (3.6-fold increase), and 3 days (2.8-fold increase) in comparison with Aβ (1–40) alone. Perlecan also initially accelerated the formation of Aβ (1–42) fibrils within 1 h and maintained significantly higher levels of Aβ (1–42) thioflavin T fluorescence throughout a 2-week experimental period in comparison with Aβ (1–42) alone, suggesting perlecan's ability to maintain amyloid fibril stability. Perlecan's effects on Aβ (1–40) fibril formation and maintenance of Aβ (1–42) fibril stability occurred in a dose-dependent manner and was also mediated primarily by perlecan's glycosaminoglycan chains. Perlecan was the most effective enhancer and accelerator of Aβ fibril formation when compared directly with other amyloid plaque components, including apolipoprotein E, α1-antichymotrypsin, P component, C1q, and C3. This study, therefore, demonstrates that perlecan not only binds to the predominant isoforms of Aβ, but also accelerates Aβ fibril formation and stabilizes amyloid fibrils once formed, confirming pivotal roles for perlecan in the pathogenesis of Aβ amyloidosis in Alzheimer's disease.

Notes: Abstract: Our previous studies have demonstrated that perlecan and perlecan-derived glycosaminoglycans (GAGs) not only bind β-amyloid protein (Aβ) 1-40 and 1-42, but are also potent enhancers of Aβ fibril formation and stabilize amyloid fibrils once formed. However, it was not determined which moieties in perlecan heparan sulfate GAG chains may be responsible for the observed effects and whether other GAGs were also capable of a similar enhancement of Aβ fibril formation as observed with perlecan GAGs. In the present study, thioflavin T fluorometry (over a 1-week period) was used to extend our previous studies and to test the hypothesis that the sulfate moiety is critical for the enhancing effects of heparin/heparan sulfate GAGs on Aβ 1-40 fibrillogenesis. This hypothesis was confirmed when removal of all sulfates from heparin (i.e., completely desulfated N-acetylated heparin) led to a complete loss in the enhancement of Aβ fibrillogenesis as demonstrated in both thioflavin T fluorometry and Congo red staining studies. On the other hand, removal of O-sulfate from heparin (i.e., completely desulfated N-sulfated heparin), and to a lesser extent N-sulfate (i.e., N-desulfated N-acetylated heparin), resulted in only a partial loss of the enhancement of Aβ 1-40 fibril formation. These studies indicate that the sulfate moieties of GAGs are critical for enhancement of Aβ amyloid fibril formation. In addition, other sulfated molecules such as chondroitin-4-sulfate, dermatan sulfate, dextran sulfate, and pentosan polysulfate all significantly enhanced (greater than twofold by 3 days) Aβ amyloid fibril formation. These latter findings indicate that deposition and accumulation of other GAGs at sites of Aβ amyloid deposition in Alzheimer’s disease brain may also participate in the enhancement of Aβ amyloidosis.