Gerardo Castillo

Perlecan Binds to the β-Amyloid Proteins (Aβ) of Alzheimer's Disease, Accelerates Aβ Fibril Formation, and Maintains Aβ Fibril Stability

Abstract: Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar β‐amyloid (Aβ) deposits of Alzheimers disease. Perlecan purified from the Engelbreth‐Holm‐Swarm tumor was used to define perlecans 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 perlecans ability to maintain amyloid fibril stability. Perlecans 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 perlecans 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 Alzheimers disease.

Laminin inhibition of β-amyloid protein (Aβ) fibrillogenesis and identification of an Aβ binding site localized to the globular domain repeats on the laminin A chain

β‐Amyloid protein (Aβ) is a major component of neuritic plaques and cerebrovascular amyloid deposits in the brains of patients with Alzheimers disease (AD). Inhibitors of Aβ fibrillogenesis are currently sought as potential future therapeutics for AD and related disorders. In the present study, the basement membrane protein laminin was found to bind Aβ 1–40 with a single dissociation constant, Kd = 2.7 × 10–9 M, and serve as a potent inhibitor of Aβ fibril formation. 25 μM of Aβ 1–40 was incubated at 37°C for 1 week in the presence of 100 nM of laminin or other basement membrane components, including perlecan, type IV collagen, and fibronectin to determine their effects on Aβ fibril formation as evaluated by thioflavin T fluorometry. Of all the basement membrane components tested, laminin demonstrated the greatest inhibitory effect on Aβ‐amyloid fibril formation, causing a ninefold inhibition at 1 and 3 days and a 21‐fold inhibition at 1 week. The inhibitory effects of laminin on Aβ fibrillogenesis occurred in a dose‐dependent manner and were still effective at lower concentrations. The inhibitory effects of laminin on Aβ 1–40 fibril formation was confirmed by negative stain electron microscopy, whereby laminin caused an almost complete inhibition of Aβ fibril formation and assembly by 3 days, resulting in the appearance of primarily amorphous nonfibrillar material. Laminin also caused partial disassembly of preformed Aβ‐amyloid fibrils following 4 days of coincubation. Laminin was not effective as an inhibitor of islet amyloid polypeptide fibril formation, suggesting that laminins amyloid inhibitory effects were Aβ‐specific. To identify a potential Aβ‐binding site(s) on laminin, laminin was first digested with V8, trypsin, or elastase. An Aβ‐binding elastase digestion product of ∼120–130 kDa was found. In addition, a ∼55 kDa fragment derived from V8 and elastase‐digested laminin interacted with biotinylated Aβ 1–40. Amino acid sequencing of the ∼55 kDa fragment identified a conformationally dependent Aβ‐binding site within laminin localized to the globular repeats on the laminin A chain. These studies demonstrate that laminin not only binds Aβ with relatively high affinity but is a potent inhibitor of Aβ‐amyloid fibril formation. In addition, further identification of an Aβ‐binding domain within the globular repeats on the laminin A chain may lead to the design of new therapeutics for the inhibition of Aβ fibrillogenesis. J. Neurosci. Res. 62:451–462, 2000.

Localization of perlecan (or a perlecan‐related macromolecule) to isolated microglia in vitro and to microglia/macrophages following infusion of beta‐amyloid protein into rodent hippocampus

The origin of the heparan sulfate proteoglycan (PG), perlecan, in beta‐amyloid protein (Aβ)‐containing amyloid deposits in Alzheimers disease (AD) brain is not known. In the present investigation we used indirect immunofluorescence, SDS‐PAGE, and Western blotting with a specific perlecan core protein antibody to identify possible cell candidates of perlecan production in both primary cell cultures and in a rat infusion model. Double and triple‐labeled indirect immunofluorescence was performed on dissociated primary rat septal cultures using antibodies for specific identification of cell types and for perlecan core protein. In mixed cultures of both embryonic day 18 (containing neurons and glia) and postnatal day 2–3 (devoid of neurons), microglia identified by labeling with OX‐42 or anti‐ED1 were the only cell type also double labeled with an affinity‐purified polyclonal antibody against perlecan core protein. Similar immunolabeling of microglia with the anti‐perlecan antibody was also observed in purified cultures of post‐natal rat microglia. Analyses of PGs from cultured postnatal rat microglia by Western blotting using a polyclonal antibody against perlecan core protein revealed an ∼400 kDa band in cell layer, which was intensified following heparitinase/heparinase digestion, suggestive of perlecan core protein. Other lower Mr bands were also found implicating either degradation of the 400 kDa core protein or the presence of separate and distinct gene products immunologically related to perlecan. Reverse transcription followed by polymerase chain reaction using human perlecan domain I specific primers demonstrated perlecan mRNA in cultured human microglia derived from postmortem normal aged and AD brain. Following a 1‐week continuous infusion of Aβ (1–40) into rodent hippocampus, immunoperoxidase immunocytochemistry and double‐labeled immunofluorescent studies revealed perlecan accumulation primarily localized to microglia/macrophages within the Aβ infusion site. These studies have identified microglia/macrophages as one potential source of perlecan (or a perlecan‐related macromolecule) which may be important for the ongoing accumulation of both perlecan and Aβ in the amyloid deposits of AD. GLIA 21:228–243, 1997.

Fiber Diffraction As a Screen for Amyloid Inhibitors

Targeting the initial formation of amyloid assemblies is a preferred approach to therapeutic intervention in amyloidoses, which include such diseases as Alzheimers, Parkinsons, Huntingtons, etc., as the early-stage, oligomers that form before the development of beta-conformation-rich fibers are thought to be toxic. X-ray patterns from amyloid assemblies always show two common intensity maxima: one at 4.7 A corresponding to the hydrogen-bonding spacing between the beta-chains, and the other at approximately 10 A corresponding to the spacing between beta-pleated sheets. We report here the application of fiber x-ray diffraction to monitor these structural indicators of amyloid fiber assembly in the presence of small, aromatic molecules, some of which have been assessed by other techniques as being inhibitory. The compounds included butylated hydroxytoluene, chloramphenicol, cotinine, curcumin, diphenylalanine (FF), ethyl 3-aminobenzoate methane sulfonate, hexachlorophene, melatonin, methylpyrrolidine, morin, nicotine, phenolphthalaine, PTI-00703 (Cats claw), pyridine, quinine, sulfadiazine, tannic acid, tetracaine, tetrachlorosalicylanilide, and tetracycline. Their effects on the aggregation of Abeta1-40, Abeta11-25, Abeta12-28, Abeta17-28, Abeta16-22, and Abeta16-22[methylated] analogues were characterized in terms of the integral widths and integrated intensities of the two characteristic reflections. Peptide Abeta11-25 with or without small molecules showed varying relative intensities but similar coherent lengths of 28-49 A in the intersheet and 171-221 A in the H-bonding directions. PTI-00703, however, abolished the H-bonding reflection. Among previously reported aromatic inhibitors for Abeta11-25, PTI-00703, tannic acid, and quinine were more effective than curcumin, morin, and melatonin based on the criterion of crystallite volume. For the N-methylated and control samples, there were no substantial differences in spacings and coherent lengths; however, the relative volumes of the beta-crystallites, which were calculated from the magnitude of the intensities, decreased with increase in concentration of Abeta16-22Me. This may be accounted for by the binding of Abeta16-22Me to the monomer or preamyloid oligomer of Abeta16-22. The fiber diffraction approach, which can help to specify whether an amyloidophilic compound acts by impeding hydrogen-bonding or by altering intersheet interactions, may help provide a rationale basis for the development of other therapeutic reagents.

P4-320: Development of novel disease-modifying small peptides that cause a reduction of brain amyloid plaque load and improved memory in a transgenic mouse model of Alzheimer’s disease

Our previous work identified a region within the globular domain of laminin that binds effectively to the beta-amyloid protein of Alzheimer’s disease (AD), inhibits beta-amyloid protein fibril formation, and disrupts pre-formed beta-amyloid protein fibrils. Screening of over 300 synthetic peptides (12and 13-mers) derived from globular domain regions of various laminin A chains resulted in the identification of 19 peptides with marked beta-amyloid protein-fibril disrupting activity. Novel 6-9 mer peptide analogs were designed that were derived from their parent 12-13 mer peptide sequences, and confirmed to maintain beta-amyloid fibril disrupting ability by a number of different in vitro screening assays. In the present study the effects of four of the top peptides on potential reduction of brain amyloid load and improved memory was assessed in a transgenic model of AD. Groups of 6-6.5month old APP mice (containing the London and Swedish mutations) were injected daily i.p. (at 50mg/kg/day) for 90 days with either saline, or four different novel 6-9mer peptides (as described above). The results demonstrated that two of these novel peptides significantly reduced fibrillar beta-amyloid protein load in brain of APP mice (as assessed by image analysis quantitation of Thioflavin S fluorescence and Congo red staining) by 38-63%, and one of these peptides also caused a marked and significant improvement (by 52%) in hippocampus dependent memory (i.e. spatial acquisition) as determined by Morris water maze testing. These results suggest that small novel peptides show promise for the development of new disease-modifying therapeutics for the prevention and treatment of AD and related disorders.