Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar /3-amyloid (A~3)

Islet amyloidosis is characterized by the deposition and accumulation of amylin in pancreatic beta-cells and is observed in 90% of patients with type 2 diabetes. Previous studies have also revealed the presence of the specific heparan sulfate proteoglycan, perlecan, colocalized to islet amyloid deposits, similar to perlecan's known involvement with other amyloid proteins. In the present study, perlecan purified from the Engelbreth-Holm-Swarm (EHS) tumor was used to define perlecan's interactions with amylin (i.e., islet amyloid polypeptide) and its effects on amylin fibril formation. Using a solid phase-binding immunoassay, human amylin, but not rat amylin, bound immobilized EHS perlecan with a single dissociation constant (Kd) = 2.75 x 10(-6) mol/l. The binding of human amylin to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans (GAGs), and was completely abolished by 10 micromol/l heparin. Using thioflavin T fluorometry, Congo red staining, and electron microscopy methodology, intact perlecan was found to enhance amylin fibril formation in a dosage-dependent manner, with the majority of these effects attributed to the heparan sulfate GAG chains of perlecan. Other sulfated GAGs and related macromolecules were also effective in the enhancement of amylin fibril formation in the order of heparin > heparan sulfate > chondroitin-4-sulfate = dermatan sulfate = dextran sulfate > pentosan polysulfate, implicating the importance of the specific GAG/carbohydrate backbone. The sulfate content of heparin/heparan sulfate was also important for the enhancement of amylin fibril formation in the order of heparin > N-desulfated N-acetylated heparin > completely desulfated N-sulfated heparin > completely desulfated N-acetylated heparin. These studies suggest that the enhancement effects of perlecan on amylin fibril formation are mediated primarily by both specific GAG chain backbone and GAG sulfate content, and implicate perlecan as an important macromolecule that is likely involved in the pathogenesis of islet amyloidosis.

Brain aging and Alzheimer’s disease both demonstrate the accumulation of beta-amyloid protein containing “plaques” and tau protein containing “tangles” that contribute to accelerated memory loss and cognitive decline. In the present investigation we identified a specific plant extract and its constituents as a potential alternative natural solution for preventing and reducing both brain “plaques and tangles”. PTI-00703 cat’s claw (Uncaria tomentosa from a specific Peruvian source), a specific and natural plant extract from the Amazon rain forest, was identified as a potent inhibitor and reducer of both beta-amyloid fibrils (the main component of “plaques”) and tau protein paired helical filaments/fibrils (the main component of “tangles”). PTI-00703 cat’s claw demonstrated both the ability to prevent formation/aggregation and disaggregate preformed Aβ fibrils (1–42 and 1–40) and tau protein tangles/filaments. The disaggregation/dissolution of Aβ fibrils occurred nearly instantly when PTI-00703 cat’s claw and Aβ fibrils were mixed together as shown by a variety of methods including Thioflavin T fluorometry, Congo red staining, Thioflavin S fluorescence and electron microscopy. Sophisticated structural elucidation studies identified the major fractions and specific constituents within PTI-00703 cat’s claw responsible for both the observed “plaque” and “tangle” inhibitory and reducing activity. Specific proanthocyanidins (i.e. epicatechin dimers and variants thereof) are newly identified polyphenolic components within Uncaria tomentosa that possess both “plaque and tangle” reducing and inhibitory activity. One major identified specific polyphenol within PTI-00703 cat’s claw was epicatechin-4β-8-epicatechin (i.e. an epicatechin dimer known as proanthocyanidin B2) that markedly reduced brain plaque load and improved short-term memory in younger and older APP “plaque-producing” (TASD-41) transgenic mice (bearing London and Swedish mutations). Proanthocyanidin B2 was also a potent inhibitor of brain inflammation as shown by reduction in astrocytosis and gliosis in TASD-41 transgenic mice. Blood-brain-barrier studies in Sprague-Dawley rats and CD-1 mice indicated that the major components of PTI-00703 cat’s claw crossed the blood-brain-barrier and entered the brain parenchyma within 2 minutes of being in the blood. The discovery of a natural plant extract from the Amazon rain forest plant (i.e. Uncaria tomentosa or cat’s claw) as both a potent “plaque and tangle” inhibitor and disaggregator is postulated to represent a potential breakthrough for the natural treatment of both normal brain aging and Alzheimer’s disease.

Co-infusion of the specific heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (A beta) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar A beta amyloid in brain [Snow, A.D. et al. (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a approximately 220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HCl, followed by anion-exchange and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with A beta (1-40) into rodent hippocampus, led to deposition of fibrillar A beta amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of A beta amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules.

The updated “Unifying Hypothesis of Alzheimer’s disease” (AD) is described that links all the observed neuropathology in AD brain (i.e., plaques, tangles, and cerebrovascular amyloid deposits), as well as inflammation, genetic factors (involving ApoE), “AD-in-a-Dish” studies, beta-amyloid protein (Aβ) as a microbial peptide; and theories that bacteria, gut microflora, gingivitis and viruses all play a role in the cause of AD. The common link is the early accumulation of heparan sulfate proteoglycans (HSPGs) and heparan sulfate glycosaminoglycans (GAGs). HS GAG accumulation and/or decreased HS GAG degradation is postulated to be the key initiating event. HS GAGs and highly sulfated macromolecules induce Aβ 1–40 (but not 1–42) to form spherical congophilic maltese-cross star-like amyloid core deposits identical to those in the AD brain. Heparin/HS also induces tau protein to form paired helical filaments (PHFs). Increased sulfation and/or decreased degradation of HSPGs and HS GAGs that occur due to brain aging leads to the formation of plaques and tangles in AD brain. Knockout of HS genes markedly reduce the accumulation of Aβ fibrils in the brain demonstrating that HS GAGs are key. Bacteria and viruses all use cell surface HS GAGs for entry into cells, including SARS-CoV-2. Bacteria and viruses cause HS GAGs to rapidly increase to cause near-immediate aggregation of Aβ fibrils. “AD-in-a-dish” studies use “Matrigel” as the underlying scaffold that spontaneously causes plaque, and then tangle formation in a dish. Matrigel mostly contains large amounts of perlecan, the same specific HSPG implicated in AD and amyloid disorders. Mucopolysaccharidoses caused by lack of specific HS GAG enzymes lead to massive accumulation of HS in lysosomal compartments in neurons and contribute to cognitive impairment in children. Neurons full of HS demonstrate marked accumulation and fibrillization of Aβ, tau, α-synuclein, and prion protein (PrP) in mucopolysaccharidosis animal models demonstrating that HS GAG accumulation is a precursor to Aβ accumulation in neurons. Brain aging leads to changes in HSPGs, including newly identified splice variants leading to increased HS GAG sulfation in the AD brain. All of these events lead to the new “Unifying Hypothesis of Alzheimer’s disease” that further implicates HSPGs /HS GAGs as key (as first hypothesized by Snow and Wight in 1989).

The origin of the heparan sulfate proteoglycan (PG), perlecan, in beta‐amyloid protein (Aβ)‐containing amyloid deposits in Alzheimer's 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. © 1997 Wiley‐Liss, Inc.

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Memory loss is primarily caused by the accumulation of both brain plaques [(consisting of beta-amyloid protein (Aβ) 1–42)] and neurofibrillary tangles (consisting of paired helical and straight filaments containing tau protein). Neuroinflammation is the third key and important factor that leads to accelerated memory loss and eventual dementia. Brain plaques, tangles and inflammation is the trilogy mainly responsible for causing memory loss that has now been documented for over 20 years in the scientific literature. The present investigation used in vitro quantitative methods to directly compare the ability of major memory-support dietary supplements to reduce pre-formed Aβ 1–42 fibrils (21 supplements tested) and tau protein paired helical/straight filaments (13 supplements tested)—two of the three most important targets for memory loss. Additionally, 18 different manufacturers of cat’s claw (Uncaria tomentosa) were directly compared for their ability to inhibit/reduce Aβ 1–42 fibrils and/or tau paired helical/straight filaments based on recent findings that PTI-00703 cat’s claw is a specific and potent inhibitor/reducer of all three targets -brain plaques, tangles and inflammation (Snow et al. in Sci Rep 9:561, 2019). In the present investigation quantitative Thioflavin T fluorometry was used on a comparative weight-to-weight basis at increasing concentrations with ingredients tested from the actual capsules the consumer ingests. Major memory-support dietary supplements were directly compared for their ability to inhibit and disaggregate/reduce both Aβ 1–42 fibrils and/or tau paired helical/straight filaments. Dietary supplements touted to enhance memory comparatively tested included Prevagen, FOCUSfactor, PROCERA AVH, Alpha Brain, NAD+OVIM, BRAIN JUICE, Cebria, EXCELEROL, NOOCUBE, US Doctor’s Clinical Brain Power ADVANCED, healthycell pro, LUMONOL, Brain Awake, BRAIN ARMOR, brainMD (BRAIN & MEMORY POWER BOOST), Brain Support, Clarity (BRAIN HEALTH FORMULA), brainMD (NEUROVITE PLUS), neuriva (Original and Plus) and percepta. This is the first paper to actually comparatively test these memory-support supplements for their ability to reduce Aβ fibrils and tau protein tangles. Percepta (PTI-00703 cat’s claw and a specific oolong tea extract) was determined to be the most effective and potent memory support dietary supplement to disaggregate/disrupt Aβ 1–42 fibrils (range of 25–89%) and tau paired helical/straight filaments (range of 26–86%) at all 3–4 doses tested in comparison to other major memory-support dietary supplements tested. This was at least more than double (> 50%) for percepta reducing Aβ 1–42 fibrils and in comparison to the other 20 memory-support dietary supplements tested. The ranking order for memory-support supplement effects based on reducing Aβ 1–42 fibrils (Aβ 1–42: memory-support supplement at 1:0.1 weight-to-weight in a 3-day study) was percepta (69.6% reduction) >>> Alpha Brain (34.9% reduction) = US Doctor’s Clinical Brain Power ADVANCED (32.4%) = BRAIN JUICE (30.1%) = neuriva Plus (27%) = neuriva Original (27%) > NEUROVITE PLUS (22.9%) = NOOCUBE (19.9%) = EXCELEROL (17.3%) = healthycell pro (17.2%) > Prevagen (12.9%) > PROCERA AVH (6.5%) = FOCUSfactor (5.5%) > Cebria (0%) = Brain Awake (0%) = Brain Support (0%) = brainMD (BRAIN & MEMORY POWER BOOST) (0%) = NAD+OVIM (0%) = BRAIN ARMOR (0%) = LUMONOL (0%). The ranking order for memory support supplement effects on reducing tau paired helical/straight filaments (tau:memory supplement at 1:1 weight-to-weight at 3 days) was percepta (85.7% reduction) >>> neuriva Plus (57.9%) >> BRAIN JUICE (41.9%) = EXCELEROL (41.0%) = neuriva Original (38.4%) = US Doctor’s Clinical Brain Power ADVANCED (38.3%) = healthycell pro (37.6%) >> Alpha Brain (27.9%) >> NOOCUBE (17.6%) >> FOCUSfactor (8.7%) > Cebria (3.6%) = PROCERA AVH (0%) = Prevagen (0%). Congo red staining, Thioflavin S fluorescence, circular dichroism (CD) spectroscopy and electron microscopy confirmed the positive results observed with the supplement percepta. CD spectroscopy demonstrated that percepta caused a marked inhibition of beta-sheet secondary folding of tau protein into paired helical filaments. PTI-00703 cat’s claw (main ingredient in percepta) was also identified as the most potent cat’s claw bark powder (Uncaria tomentosa) to reduce and inhibit Aβ 1–42 fibrils and tau tangles in comparison to 17 other manufacturers of cat’s claw extracts. Although there are thousands of brain memory-support dietary supplements in the marketplace today, none of them have been directly compared and analyzed for their ability to reduce and/or inhibit two major targets of memory loss i.e. Aβ 1–42 fibrils and tau paired helical/straight filaments (major constituents of brain plaques and tangles). In our comparison studies, we show that percepta has the most potent ability to disaggregate/reduce Aβ 1–42 fibrils and tau protein paired helical/straight filaments as demonstrated by a variety of methods most likely due to the specific polyphenol content in PTI-00703 cat’s claw (i.e. polyphenols and proanthocyanidins) as we have previously shown (Snow et al. in Sci Rep 9:561, 2019). Memory-support dietary supplements tested that also contained polyphenols and/or cat’s claw in their product demonstrated some Aβ fibril and tau protein tangle reducing activity, but were much less effective than percepta. Percepta’s main ingredient, PTI-00703 cat’s claw, has previously been shown to reduce brain amyloid plaques and Aβ 1–42/40 insoluble/soluble levels in brain (in plaque-producing transgenic mice) with marked concurrent memory improvements (shown by Morris water maze testing) (Snow et al. in Sci Rep 9:561, 2019). The present investigation further confirms that percepta is one of the best dietary supplements that causes a marked reduction and inhibition of Aβ fibrils and tau tangle filaments -two important major targets for memory-support. In addition, PTI-00703 cat’s claw was the most effective cat’s claw (Uncaria tomentosa) ingredient for reducing /disaggregating and inhibiting Aβ 1–42 fibrils and tau protein paired helical/straight filaments in comparison to 17 other manufacturers of cat’s claw extracts tested.