Rosacea: Alzheimer’s of the Face

Is Rosacea Caused by Amyloid LL-37, as Alzheimer’s Is Caused by Anti-microbial Abeta?
A recent article in PLoS One (Thanks Daniel!) suggests that the amyloid beta (Abeta) proteins that aggregate to form fibrous plaques in the brain tissue of Alzheimer victims, function as typical defensive anti-microbial peptides (AMPs), similar to the LL-37 cathelicidin implicated in facial tissue in rosacea.  The structural and functional similarities of Abeta and LL-37 suggest to me that Alzheimer’s and rosacea may also be similar in initiation and treatment.  Let’s compare amyloids and AMPs.

[The figure shows a model protein (from ref.) used to examine stain binding to amyloids.  The stains appear to bind to aromatic amino acids spaced evenly between adjacent proteins, but adjacent basic amino acids (blue) are spaced the same way and provide sites for heparin binding.]

Amyloids:

• Amyloid proteins/peptides align into stacks and fibers
• Stacked beta sheets bind amyloid stains: Congo Red, Thioflavin-T
• Fibers form on anionic polymers: heparin, DNA
• Short amyloid stacks are toxic to cells
• Proteases produce multiple sizes of amyloid peptides

Anti-microbial Peptides:

• AMPS typically contain heparin-binding domains -- basic peptides/ plus charge
• Some AMPs, e.g. LL-37, form fibers on DNA, heparin (stain with amyloid stains)
• Toxic to cell membranes
• Kallikrein stimulated by gut flora migrates to face and clips LL-37 to a smaller peptide that binds to host DNA and stimulates the TLR receptor to produce inflammation
• Stomach pepsin hydrolyzes dietary proteins into anti-microbial peptides (heparin is secreted by mast cells onto to the intestinal surface to protect from any amyloid-like effects)
• Defensins, cathelicidins and other AMPs are under transcriptional control of vitamin D receptor

Abeta Is Anti-microbial Like LL-37

Amyloid beta is the well-known source of the fibrous plaques forming brain lesions in Alzheimer’s disease.  The normal function of Abeta has not been firmly established.  The recent article shows data to support Abeta as an anti-microbial peptide comparable to LL-37 against several pathogenic bacteria and yeast.  Knock-out mice deprived of a gene corresponding to Abeta are susceptible to bacterial infections.  The anti-microbial activity present in extracts from Alzheimer’s disease brains was inactivated by anti-Abeta antibodies.

Implications of Abeta as an AMP Like LL-37

The similarities between AMPs and amyloid peptides suggest some implications for both Alzheimer’s disease and rosacea.  Vitamin D is a hormone that binds to a cytoplasmic receptor and the vitD/receptor complex then acts as a transcription factor that controls the expression of defensins in the intestines, LL-37 in facial skin and perhaps Abeta in brains.

Amyloids form fibers on a scaffolding of heparan sulfate (HS).  There is usually an excess of HS on the surface of cells and the HS is rapidly recycled back into cells.  During inflammation, mast cells release heparin, short fragments of HS, that should also inhibit amyloid fiber formation on HS.   Chronic inflammation, however, reduces HS production and may set the stage for amyloid fiber formation.  HS metabolism of the brain may be vitally important to the development of Alzheimer’s disease, especially since the increasing chronic inflammation of aging people should deplete brain HS.

LL-37 forms complexes with DNA from damaged host cells in rosacea skin.  The LL-37/DNA complexes trigger TLRs and inflammation.  LL-37 may normally bind to cell surface HS and chronic inflammation of the skin may cause the shift to pathogenic autoinflammation.  Topical application or perhaps low dose IV heparin may be effective in disrupting the autoinflammation due to LL-37.  Part of the toxicity of LL-37 in the skin may be due to amyloid like structures that could form with inadequate HS and overabundant LL-37 production.  Vitamin D metabolism should also be very important, since LL-37 synthesis is controlled by vitamin D.  This is consistent with the benefits that some rosaceans observe with high doses of vitamin D3 supplements.

references:
Soscia SJ, Kirby JE, Washicosky KJ, Tucker SM, Ingelsson M, Hyman B, Burton MA, Goldstein LE, Duong S, Tanzi RE, Moir RD.  The Alzheimer's disease-associated amyloid beta-protein is an antimicrobial peptide.  PLoS One. 2010 Mar 3;5(3):e9505.

Abedini A, Tracz SM, Cho JH, Raleigh DP.  Characterization of the heparin binding site in the N-terminus of human pro-islet amyloid polypeptide: implications for amyloid formation.  Biochemistry. 2006 Aug 1;45(30):9228-37.

Biancalana M, Makabe K, Koide A, Koide S. Molecular mechanism of thioflavin-T binding to the surface of beta-rich peptide self-assemblies.  J Mol Biol. 2009 Jan 30;385(4):1052-63. Epub 2008 Nov 14.

Bacterial Amyloid (Curli Fibers) Forms Biofilms

E. coli Curli Stacks in Congo Red Staining Fibers

We can’t cure diseases, because we don’t understand basic chemistry (what is hydrophobic) and biology (which came first the biofilm or the bacterial cell wall?)  Let’s look at a fundamental biological process, how bacteria form biofilms.

Biofilm Formation from Secreted Proteins and Polysaccharides

Investigators passed some E. coli through a special slide chamber so they could watch at high magnification as a single bacterium attached to the surface, divided to produce a colony of a few bacteria and then began to secrete proteins (curli fibers) and polysaccharides (colanic acid and cellulose) to make the biofilm matrix.  The matrix stained red with Congo Red.

Congo Red Stains Amyloids, Cellulose and Rare LPS



Staining with Congo Red shows that the spacing of hydrophobic patches on the surface of the biofilm matrix matches the flat hydrophobic, aromatic rings of the dye, Congo Red.  This particular dye is important, because Congo Red also specifically stains amyloid, e.g. beta amyloid of Alzheimer’s disease.  But Congo Red also binds to cellulose, a linear beta 1,4-glucan polysaccharide.  This seems paradoxical, because we are taught that the sugars of which a polysaccharide are made are hydrophilic.  That turns out to be a half-truth. 

Faces of Sugars Are Hydrophobic

The hydrogen bonding hydroxyl groups that make sugars water soluble and hydrophilic, radiate from a ring of carbons, and the faces of that ring cannot make hydrogen bonds.  The faces of sugars are hydrophobic and in most cases will bind to hydrophobic surfaces, such as aromatic amino acids, e.g. tryptophan, tyrosine and phenylalanine.  Thus, carbohydrate binding enzymes, such as shown in the figure bind cellulose (in grey and red) in a groove lined with aromatic amino acids (yellow and orange) so that each sugar orients over and sometimes sandwiched between aromatic amino acid residues.  This also explains why Congo Red binds to cellulose, since the aromatic rings of the dye bind to neighboring glucose residues along the relatively flat cellulose strand.  Most other polysaccharides and smaller sugars lack this spacing of sugars and they don’t stain red with Congo Red.

Basic Amino Acids Bind Hydrophobically

Another misperception is that basic amino acids, positively charged lysine and arginine, are hydrophilic.  The nitrogen atoms that make these amino acids positively charged, can form hydrogen bonds, but the hydrocarbon tails that have these nitrogenous tips, are hydrophobic.  Thus, basic amino acids and aromatic amino acids can stack to form tryptophan/arginine ladders in which they alternate.  A prominent example of these interdigitations are the way that nuclear localization signals, a quartet of basic amino acids, bind to importin via its projecting, spaced tryptophans and drag proteins through pores into the nucleus.  Similarly, the basic amino acids of heparin-binding domains extend across the hydrophobic faces of the sugars of heparin and hydrogen bond with their tips to the sulfates of the heparin.  In each of these binding examples the binding is primarily hydrophobic.

Amyloid Binds Congo Red by Stacked Heparin-Binding Domains

Amyloids are proteins that stack together like stacking chairs, so that each protein is oriented in the same way all along the stack.  In the case of the beta amyloid that makes up the toxic plaque in Alzheimer’s disease, each amyloid peptide is stacked like a hair pin on top of the next to make a fiber.  At the bend in beta amyloid, is a basic amino acid and the amyloid fiber has a band of basic amino acids along its length.  The spacing between the basic amino acids in an amyloid stack is just spanned by Congo Red, so amyloids are diagnostically stained red.  This same spacing of basic amino acids fits the sugars in heparin.  Thus, heparin can catalyze amyloid formation and is abundant in amyloid plaques in Alzheimer’s

Bacterial Biofilms Form from Amyloids and Polysaccharides

The E. coli cells that formed the biofilms that started this article secrete a protein, curli, that stacks as an amyloid into fibers.  These fibers stained by Congo Red and bind to the cellulose that is also produced by the E. coli.  It should not be surprising that biofilm formation is catalyzed by heparin and biofilm formation is a major problem in catheter infection, since heparin is used to coat catheters to keep them from forming blood clots.  Amyloids are also formed from stacked seminal acid phosphatase proteins that form fibers in the presence of heparin and facilitate HIV infection.

Do Biofilms Foment Amyloid Production?

Basic amino acids, sugars, aromatic amino acids and plant phytochemicals all bind each other via their hydrophobic surfaces.  It would not be surprising that bacteria that produce proteins and acidic polysaccharides that interact hydrophobically would also interact with host molecules with a similar spacing of hydrophobic surfaces, which are common in heparin-binding interactions and nucleic acid interactions.  The bacteria in biofilms produce both proteins and polysaccharides that may catalyze amyloid production.  The acidic biofilm polysaccharide, colanic acid, may replace heparan sulfate and curli should bind to heparin.

Berberine Binds Heparin and Blocks Amyloids and Biofilms

Just as bacterial products may compete for host heparin and heparin-binding domains, phytochemicals that interact with heparin, such as the phytochemical berberine, should disrupt heparin mediated molecular interactions, and by extension also biofilms.  There is experimental evidence for berberine both disrupts amyloid formation and biofilm assembly.

Synuclein and Amyloid Diseases

NSAIDs, such as ibuprofen and aspirin are possible treatments to inhibit the aggregation of proteins (synuclein, beta amyloid) on charged polymers in amyloid diseases, such as Parkinson’s disease, Alzheimer’s disease, etc. Contradictory studies show that intracellular aggregate formation may be protective, since dimers are more toxic than aggregates.

The list of amyloid diseases is long and there are few effective treatments. In each case a protein starts to accumulate in fibers that form amyloid plaques inside or outside the cells. The large aggregates outside are toxic. Inside it appears that the large aggregates are not as toxic as small clumps, oligomers, of the protein.

The amyloid proteins are stacked up in the fibers in a very organized way, so that the same portions of the protein are lined up on each side of the fibers. Outside the cell, the regions with basic amino acids interact with heparin, and in Alzheimer’s disease, for example, the beta amyloid plaque is half heparin. In test tube experiments, fiber formation from protein solutions is accelerated by adding heparin.

Amyloid fibers also form inside cells in the case of the tau fibers of Alzheimer’s disease or the synuclein aggregates in Parkinson’s disease. In theses cases, there should not be any intracellular heparin, and it is not known what polyanion (RNA?) serves to accelerate fiber formation in these cases.

Non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and aspirin, reduce the incidence of Parkinson’s and Alzheimer’s diseases. It has recently been shown that in test tube experiments, NSAIDs also decrease the formation of amyloid fibers from synuclein.

Amyloid fibers can be stained by Congo Red and thioflavin. Curcumin is the active component of tumeric and it has a structure related to Congo Red. Curcumin has been shown in recent studies to block synuclein amyloid formation.

In addition, the heparin in the fiber complexes can be stained with berberine. Berberine is a traditional herbal treatment for arthritis. It would not be surprising if it was also effective against Alzheimer’s amyloid plaque.

The large extracellular plaque aggregates appear to be toxic, but the small, oligomeric aggregate of protein appear to be the toxic form in cells. Recent experiments show that facilitating the formation of large intracellular aggregates minimizes the toxicity in animal models of Huntington’s and Parkinson’s diseases. It appears that the large visible aggregates are not the form that kills the cell.

For the time being, the only safe treatments that focus on amyloid fiber formation are the NSAIDs, curcumin and perhaps berberine.

references:
Hirohata M, Ono K, Morinaga A, Yamada M. 2008. Non-steroidal anti-inflammatory drugs have potent anti-fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils in vitro. Neuropharmacology 54(3):620-7.

Pandey N, Strider J, Nolan WC, Yan SX, Galvin JE. 2008. Curcumin inhibits aggregation of alpha-synuclein. Acta Neuropathol. 115(4):479-89.

Bodner RA, Outeiro TF, Altmann S, Maxwell MM, Cho SH, Hyman BT, McLean PJ, Young AB, Housman DE, Kazantsev AG. 2006. Pharmacological promotion of inclusion formation: a therapeutic approach for Huntington's and Parkinson's diseases. Proc Natl Acad Sci U S A. 103(11):4246-51.

Outeiro TF, Kontopoulos E, Altmann SM, Kufareva I, Strathearn KE, Amore AM, Volk CB, Maxwell MM, Rochet JC, McLean PJ, Young AB, Abagyan R, Feany MB, Hyman BT, Kazantsev AG. 2007. Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson's disease. Science. 317(5837):516-9.