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The Microbiota in Neurodegeneration and the Prospects for Gene Therapy Robert P. Friedland MD [email protected] Seventh International Conference on Alzheimer’s Disease and Related Disorders in the Middle East (ICAD-ME) Collaborators University of Louisville • D. Demuth, PhD • C. Dryden, MD • A. Gadre, MD • A. Kumar, PhD • R. Kerber, PhD • K. LaFaver, MD • R. Liu, PhD • J. McMillan, MA • R. Prough PhD • V. Stribinskis, PhD Oral Health Medicine Surgery (ENT) Anatomy and Neurobiology Public Health Neurology Neurology Anatomy and Neurobiology Biochemistry Biochemistry Others • S. Chen PhD • M. Ihara, MD • E. Masliah, MD • N. Kuriyama • K. Nakano, DDS, PhD • P. O’Toole, PhD • R. Raha-Chowdhury, PhD CWRU, Cleveland, Ohio National Cardiovascular Center, Osaka National Institute on Aging, NIH Kyoto Prefectural Univ. Univ. of Osaka School of Dentistry Univ. College, Cork, Ireland Univ. of Cambridge, UK Acknowledgements • • • • Michael J. Fox Foundation University of Louisville Mary and Mason Rudd Family M.Chapman (U. Michigan) Mechanisms of molecular mimicry involving the microbiota in neurodegeneration • Outline • What is molecular mimicry? • What are the issues/problems? • What are the data and hypotheses? • Bacterial amyloid project in rats and C elegans • Further considerations (pathogenesis, therapy and prevention) The story • Why do people get sporadic Parkinson’s, Alzheimer’s or other related neurodegenerations? • The microbiota (i.e., gut bacteria) have been shown to have important roles in human metabolism and immunity • It is known that gut exposure to amyloid proteins can cause noncatalytic seeding of endogenous protein misfolding resulting in neurodegeneration (BSE) Low hanging fruit PD AD LT IBS AD Alzheimer PD Parkinson IBS Irritable Bowel LT Lactose intolerance Low hanging fruit PD AD LT IBS AD Alzheimer PD Parkinson IBS Irritable Bowel LT Lactose intolerance LT Chief questions 1. What initiates misfolding in the protein folding disorders (Alzheimer, Parkinson’s and others)? • Is it random? Prusiner, 2013 • Or induced by environmental factors? 2. What initiates and sustains inflammation in the brain in these disorders? Both protein misfolding as amyloid and inflammation may be needed for disease What might be the trigger/s of misfolding? Microbiota, microbiome, metagenome • The greatest opportunity for humans to be exposed to foreign antigens is through the microbiota (the microbe population in and on our bodies) • 80% not yet cultivated • the most densely popualtied microbial ecosystem • Microbiome or metagenome: The combined genetic material of the microorganisms in a particular environment • Fungi, bacteria, Archaea, animal and plant viruses, and other larger life-forms • Largest population of microbiota reside in the gut: mouth, nose, pharynx, & intestines Bacteria make amyloid proteins • • • • • • • • Bacillus subtilis Escherichia coli Klebsiella pneumoniae Mycobacterium tuberculosis Salmonella enterica, typhimurium Staphlococcus aureus Streptococcus mutans, coelicolor Xanthomonas axonopodis E coli with curli fibers [The list is incomplete] • Genes for producing amyloids are also found in other phylum of bacteria: Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteoabacter, Thermodesulfobacteria Hufnagel et al, 2013; Schwartz et al, 2013; Bieler et al, 2005 Photo courtesy of Chapman lab What are the precedents for an oral factor to induce an amyloid misfolding disease? • Mad cow disease [bovine spongiform encephalopathy (BSE)] in cattle & new variant Creutzfeldt Jakob disease (nvCJD) in humans • Scrapie in sheep • Chronic wasting disease in elk and deer • Serum AA and foie gras feeding in mice (Solomon et al, 2007) Curli contains short peptide repeats similar to yeast and human prions E coli curli Salmonella fimbrin PrP Yeast Cherny et al, 2005 Cross-seeding involving AB, AS, Tau, PrP, curli • “Aβ and αS acted as seeds and affected each other's aggregation pathways in vitro” Ono et al, 2012 • AS seeds misfolding of Tau Guo et al, 2011 • Prion and AD model mice Morales et al 2010 • Curli from E coli can trigger murine experimental serum AA amyloidosis Lundmark et al, 2005 Could the microbiota influence inflammation in the brain in neurodegeneration? Bacterial amyloid and inflammation • TLR2/1 is the primary system for bacterial amyloid recognition by the inmate immune system • TLR2 is a primary pathway for Aβ peptide and alpha synuclein to trigger neuroinflammatory activation • This involves enhanced expression of cytokines, & interferons, involving CD14, NFkB, INOS Madan, Amar 2008, Liu et al, 2012, Tukel et al, 2010; Kim et al 2013, Rapinski et al, 2013 Two hypotheses: 1. Bacterial amyloid proteins cause crossseeding of amyloid misfolding in neuronal proteins in the gut (including the mouth and nose) which are transmitted via the autonomic nervous system to the brain in a prion-like manner Friedland, Journal of Alzheimer's Disease, 2015 Two hypotheses: 2. The innate immune system recognizes bacterial amyloids utilizing TLR2 and related pathways causing priming of immune cells which enhance the response to the amyloid proteins deposited in the brain with aging, enhancing neuroinflammation. Friedland, Journal of Alzheimer's Disease, 2015 Exposure to the Functional Bacterial Amyloid Protein Curli Enhances Alpha-Synuclein Aggregation in Aged Fischer 344 Rats and Caenorhabditis elegans Shu G. Chen, Vilius Stribinskis, Madhavi J. Rane, Donald R. Demuth, Evelyne Gozal, Andrew M. Roberts, Rekha Jagadapillai, Ruolan Liu, Kyonghwan Choe, Bhooma Shivakumar, Francheska Son, Shunying Jin, Richard Kerber, Anthony Adame, Eliezer Masliah & Robert P. Friedland Scientific Reports| 6:34477 | DOI: 10.1038/srep34477 1 2016 The effect of bacterial amyloid on AS misfolding in the rat and C elegans • Aged Fischer 344 rats (males, >24m) • N=9-12 • Oral administration of • E coli making curli (wild type) • E coli mutant lacking the ability to make curli • Vehicle alone C elegans expressing AS-YFP Chen et al , Scientific Reports, 2016 AS expression Microgliosis AS expression Curli exposure enhanced AS aggregation PK- PK+ PK = Proteinase K digestion Toll like receptor 2 (TLR2) expression was increased in hippocampus and striatum TLR2 C elegans expressing AS-YFP Worms fed on bacteria that produce amyloid (left) developed more and larger α-synuclein inclusions (green) than did worms eating control bacteria (right). C elegans expressing AS-YFP Congo Red AS-YFP Merge Congo Red stained deposits (arrowheads) in the head region of C. elegans expressing AS-YFP in the body wall muscles colocalized with AS-YFP aggregates. S. Chen and colleagues at CWRU Summary Exposure to the bacterial amyloid protein curli • enhanced Alpha synuclein (AS) expression in the gut and brain and AS aggregation in the brain (rats) • enchanced neuroinflammation as assessed by microgliosis, astrogliosis and TLR2, IL6, TNF expression (rats) • enhanced AS aggregation in muscle (transgenic C elegans) Chen et al , Scientific Reports, 2016 Further considerations • Is it surprising to see that the microbiota influence inflammation in the brain? An illustration of “immume tolerance”? Immune system Microbiota Immunological tolerance - the failure to mount an immune response to an antigen. Immune tolerance: why? • “The potential for detrimental inflammation suggests that the responses of T cells and B cells to the gut microbiota must be tightly regulated” Honada and Littman, 2016 • “The innate immune system might therefore function to promote the growth of beneficial members of the microbiota and to contribute to the maintenance of a stable community of microorganisms” Thaiss et al, 2016 Olfactory receptor neurons are in direct contact with the environment (Urban and Tripathy, 2012; Hommet et al, 2012) “Adapting Koch’s Postulates” • Byrd, Segre Science, 2016: “Criteria for disease causation must take microbial interactions into account” Commensal organisms can protect the host against pathogens “colonization resistance” (Commensal organism absence can enhance pathogenicity) Robert Koch, 1890 “Adapting Koch’s Postulates” • Byrd, Segre Science, 2016: “Criteria for disease causation must take microbial interactions into account” • Commensal organisms can protect the host against pathogens “colonization resistance” (Commensal organism absence can enhance pathogenicity) The new understanding: • Disease can be caused by bacterial communities, not only single players • Disease can be caused by microorganisms without infection Recent developments • The microbiota are altered in Parkinson’s disease Scheperjans et al 2015, 2016 • Gut bacteria are involved in amyloid pathology in AD model transgenic mice Minter et al 2106 • Gut bacteria are involved in AS pathology in PD model transgenic mice Harach et al 2017 “Gut Microbiota Regulate Motor Deficits and ” Neuroinflammation in a Model of Parkinson's Disease ASO Tg mouse ASO Tg mouse-germ free Frontal cortex AS aggregates (red), phoshorylated AS (green) Sampson et al, Cell, 2016 Based on what we know about the microbiota what can we do now? Gene therapy and epidemiology of dementia • It is difficult to change the human genome, but the metagenome can be adjusted through diet • This can be done in as little as 2 weeks, with consequences for inflammation, oxidative toxicity and cancer in humans Greer and O’Keefe 2011 Ou et al 2012 Therapeutic options: Gene therapy • • • • • • • • • • Dietary fiber Dietary diversity Dental care Prebiotics Probiotics Antibiotics (pro and con, not from food) Fecal transplant Vaccine Phages Amyloid inhibitors (e.g, quorum sensing, green tea polyphenols) Oli et al 2012; Syed, Boles 2014, Wang et al, 2014 Further implications 1 • ALS, C9ORF72 and macrophages (O’Rourke et al, 2106) • Stroke and TLR2 • Idiopathic Creutzfeldt Jakob disease • Fungi and Alzheimer’s disease Further implications 2 • Strains • • • • • • • diversity of conformational states, multiple forms of disease incubation periods host specificity Deposition patterns in the brain proteolysis digestion patterns Stability Best demonstrated for Prions, also reported for neurodegenerative disease proteins Hypothesis: Strains of bacterial amyloids induce cross-seeding in a strain specific manner, thereby initiating CJD, AD, PD, ALS, PSP and their complex phenotypes. Cohen et al, 2016, Macedo et al 2016, Chen et al 2016 The story • Why do people get sporadic Parkinson’s, Alzheimer’s or other related neurodegenerations? • The microbiota (i.e., gut bacteria) have been shown to have important roles in human metabolism and immunity • A wider perspective on the role of bacteria and other of our partner organisms is needed, beyond the considerations of Koch’s postulates and infection Conclusions • We have more nucleotide sequences in our gut than in our own cells • We evolved with these organisms – they are not there “by accident” • Evolutionary conserved proteins are widespread and can lead to diseases via molecular mimicry through similarity of tertiary structures • Gut bacteria may be involved in neurodegenerative disease • Gene therapy: we can change the population of organisms in the gut through diet & other measures and influence disease risk and course Outstanding questions • • • • • • • Which bacteria and which amyloid? Oxidative toxicity? Why not aggregated in gut? Distribution in the brain (olfactory lobe)? Effects on phenotype? How to prevent/treat? Is this why AD/PD is more common in developed than in developing countries? Thank you Future directions TOPIC Non UofL collaborators Metagenomic study of PD in humans Ireland Curli biochemistry and exposure studies Cleveland, Poland C elegans studies Cleveland Fungi and AD Ireland TMA metabolite studies Japan, UK Metaproteomics Japan Strep mutans exposure +/_ bacterial amyloid protein SpaP C9ORF72 mice/cells Stool transplant in ALS Polyphenol treatment of bacterial amyloid Hydrogen sulfide and the gut RED =underway issues • • • • • • • • • Stool does not sample mucisal organsims Fungi Phages small intestine?metaproeomics Microbial products (metabolites) Hos tgenetics Mouse a samodel Choice of controls Functional metagenmoics • Ignores nose and mouth Further issues • • • • Metaproteomics Fungi Hos tgenetics Fundctional metagenmics Therapies • • • • • • • • • • • • • • • Synbiotics Prebiotics P[robuotics Curds FMT Phages Diet Dental care Antibiotics Combinations Vaccine Medical foods (personalized nutritional prescriptiom) Low hanging fruit Amyloid inhibitors MyD88 inhinbiiton (TLRs) • S salivarius (tongue) M18 kills strep mutans makes dextranses, all ser • Slide of lo ahnging fruit?otypes (blis technology) Challenges to these hypotheses 2 • The disease may influence the metagenome (causes or consequences?) • Studies looking at the association of one bacterial amyloid to disease may be negative because that particular one is not not involved • There are lots of bacterial amyloids • Which disease? (strains) Conclusions: Proposals • Bacterial and other amyloids may • seed self propagating amyloid prion-like misfolding of neuronal proteins • Induce inflammation to enhance neurodegenerative and cerebrovascular diseases The oral/nasal microbiota may have critical influences on the brain and brain vessels, just as the colonic microbiota have been shown to have profound influences on systemic metabolism and inflammation Friedland, 2015 C9ORF72 • Hexanucelotide repeat • GGGGCC • Major cause of FTLD, familial and sporadic as well as familial and sporadic ALS; may have early onset psychiatric manifestations • May have insoluble aggregates, affecting protein expression Open reading frame: part of a reading frame that contains no stop codons (or termination codon) (a nucleotide triplet within messenger RNA that signals a termination of translation). Van Rheenun et al, 2012; Friedland et al, 2013; Arighi et al, 2012; Mori et al 2013 Stroke and vascular cognitive impairment Multi-infarct dementia Vascular dementia Vascular cognitive impairment Microscopic infarctions Binswanger’s disease Microglia and macrophages respond to systemic inflammation (circulating cytokines) and produce inflammatory mediators that damage axons Moreno et al, 2011 MRI of Binswanger’s disease A Model of the Diet, Gut Microbiota, Atherosclerosis, and the Risk of Thrombosis Phosphatidly choline rich diet ATHEROGENESIS TMA FM03 TMAO HYPERACTIVE PLATELETS Choline MI, STROKE TMA Tilg NEJM 2016 Gut bacteria and cardiovascular disease • Choline, phosphatidylcholine [lecithin] and carnitine can be converted by gut bacteria to trimethylamine [TMA] • Which is oxidized in the liver by flavine monooxygenases (FMOs) to trimethylamineoxide [TMAO] • TMAO is pro-atherogenic (promotes vascular inflammation, platelet hyperactivity, clotting and leukocyte endothelial adhesion). • Microbiota are related to MI size in rats (Lam et al, 2012) • Suppression of TMAO/TMAO production inhibits atherogenesis. Hazen group , CCF 2011-present Procedures/choices • Mice and/or rats • Exposure to bacteria and/or purified curli • Oral/nasal exposure routes • What immune markers (B, T cells, cytokines, TLRs) to study • How to test behavior • Immunohistochemistry and oxidative stress “Theories are not true or false, they are fertile or sterile” Claude Bernard (1813-1878) Fat, fibre and cancer risk in African Americans and rural Africans • “….the food changes resulted in remarkable reciprocal changes in mucosal biomarkers of cancer risk and in aspects of the microbiota and metabolome known to affect cancer risk, best illustrated by increased saccharolytic fermentation and butyrogenesis, and suppressed secondary bile acid synthesis in the African Americans” O’Keefe et al Nat Commun 2015 Polio Cases in the USA CDC, 2012 The gut microbiome in Parkinson’s disease “Anti-inflammatory" butyrate-producing bacteria from the genera Blautia, Coprococcus, and Roseburia were significantly more abundant in feces of controls than PD patients.” Keshavarzian et al 2015 Who does a cow get then nutrients it needs? We need research diversity • We need a diversity of approaches to Parkinson's and related diseases • Parkinson’s can be cured, and prevented. We need to learn how. Dr. Louis Pasteur H2S and Als Davoli et al 2015 Bovine Spongiform Encephalopathy (BSE) • • • • First recognized among cattle in the United Kingdom in 1986 Cases may have occurred in the early 1980s Peaked in 1992-1993: 36,682 cases reported in 1992 alone As of June 1, 2005, 180,800 total cases in Great Britain Prion diseases (Spongioform encephalopathies) • • • • • Creutzfeldt-Jakob disease Kuru Fatal familial insomnia Scrapie Bovine spongiform encephalopathy (BSE) Gut has about 300 million neurons Furness 2012 Molecular mimicry • Where a harmless species imitates the warning signals of a harmful species directed at a common predator H.Bates (1825-1892) • “…natural selection can lead to the mimicking of antigenic determinants of hosts by their parasites” R. Damian, 1965 May involve adaptive changes, reflect evolutionary conservation or occur by chance Microbiota, microbiome, metagenome • The greatest opportunity for humans to be exposed to foreign antigens is through the microbiota (the microbe population in and on our bodies) • Microbiome or Metagenome: The combined genetic material of the microorganisms in a particular environment • Fungi, bacteria, Archaea, animal and plant viruses, and other larger life-forms Chief questions 1. What initiates misfolding in the proteinopathies (AD, PD, ALS, FTLD, CBD, PSP, CJD)? • Is it stochastic? Prusiner, 2013 • Or induced by environmental or endogenous amyloids? 2. Is the inflammatory response to amyloids in the brain and to vessel pathology with aging enhanced by priming of the innate immune system by the microbiota? Background • AS aggregation starts in olfactory bulb and gut neurons, both exposed to the microbiota Rey et al, 2013; Attems et al, 2014 • PRPsc begins in DMN of the vagus in scrapie (similar to AS in PD) Ryder et al, 2001 • Cross-seeding of amyloid misfolding is documented (AS, Tau; PRPsc, Abeta, as well as curli) Guo et al, 2011, Hartman et al 2013 Is there a precedent for an oral factor to induce an amyloid misfolding disease? Is there a precedent for an oral factor to induce an amyloid misfolding disease? • Bovine spongiform encephalopathy (BSE) in cattle • Scrapie in sheep • New variant Creutzfeldt Jakob Disease (nvCJD) in humans Amyloid misfolding can be triggered by food: “Amyloidogenic potential of foie gras” “These data provide evidence that that an amyloidcontaining food product hastened the development of amyloid protein A amyloidosis in a susceptible population” Solomon et al, PNAS 2007 The gut is the most important organ of the immune system • 70% of lymphocytes are in the gut--dendritic and lymphoid cells Mabbott et al 2006 • B cells develop in bone marrow and and also gut, stimulated by microbes (receptor editing to prevent recognition of self) Wesemann et al, 2013 • Differentiation of Th17 cells requires gut bacteria and germ free mice have poor immune systems • M cells sample the gut content over Peyer's patches, and enteroendocrine cells of the gut epithelium are in direct contact with mucosal neurons via a cytoplasmic process Bohórquez et al, 2015 Hypotheses • Bacterial amyloid in the nose and gut trigger, through molecular mimicry, misfolding of NGD proteins (AS, Tau, AB, others) in brain and gut neurons and initiate autocatalytic selfpropagating templated nucleation dependent amyloid creation as well as inflammation and oxidative stress [through TLR2/1, NFKb, CD14 and iNOS] • Similar mechanisms produce inflammation involved in cerebrovascular disease (stroke, CAA, VCI) Summary: Bacterial amyloid and inflammation • Toll Like Receptors (TLRs) are the principal sensors of the innate immune system • Bacterial amyloid is recognized by the innate immune system as conserved molecular structures (pathogen associated molecular patterns, PAMPs) and activates TLR2/1, CD14 and other immune modulators • TLR2/1 and CD 14 is an important “trimolecular complex” and activates microglia, produces NFkB and iNOS, COX2, thus oxidative toxicity Madan, Amar 2008, Liu et al, 2012, Tukel et al, 2010; Kim et al 2013 Rapinski et al, 2013 Summary: Bacterial amyloid and inflammation • TLR2 is a primary receptor for AD Aβ peptide and PD alpha synuclein to trigger neuroinflammatory activation (activation of Cytokine and inteferon genes) • TLR2 is involved in atherosclerosis, according to studies in APOE KO mice , promotes lipid accumulation Madan, Amar 2008, Liu et al, 2012, Tukel et al, 2010; Kim et al 2013, Rapinski et al, 2013 Oxidative toxicity • Bacterial amyloid activates the innate immune system and increases iNOS • HNE increases cell to cell transmission of aggregates of AS Bae et al 2013 • Liposomal dysfunction increases seed production (less autophagy) • Free radicals are involved in all neurodegenerations and vascular disorders Alpha synuclein (AS) • Intestinal myenteric neurons have aggregated AS in PD and in aged Fischer 344 rats Innervation of the myenteric plexus by AS fibers and varicosities in the stomach (A) and proximal duodenum (B) of aged control F344 rats Phillips et al 2009 Eliezer Masliah, MD MD, National Autonomous University of Mexico Pathology, National Institutes of Health, Mexico City Postdoctoral studies, UCSD Full Professor, Director of the Lab of Experimental Neuropathology, Neurosciences Department, UCSD Challenges to these hypotheses 1 • Why is it so slow? -the prion disorders can have a 20-30 year incubation period (lag phase) -Amyloid propagation is not linear -Bacterial amyloid may shorten the lag phase • What about inherited cases? - the mutations may enhance sensitivity to a ubiquitous pathogenic mechanism (i.e., PRP Polymorphism -129 MV or VV is protective against BSE) • Ankylosing spondylitis is related to genotype (HLA B27) and Klebsiella overgrowth in the colon Rashid et al, 2012 Challenges to these hypotheses 2 • The disease may influence the metagenome (causes or consequences?) • Studies looking at the association of one bacterial amyloid to disease may be negative because that particular one is not not involved Bacteria, inflammation and vascular disease • “Atherosclerosis is a chronic inflammatory disease involving activation of innate and adaptive immunity” [Tabas, 2014] • Monocytes and T lymphocytes respond to oxidized LDL • Oxidized LDL signals through TLRs • Ruptured plaques have more inflammatory cells • Incr. CRP is a RF for stoke and CAD • Periodontitis, the most common oral bacterial disease, is linked to stroke and CAD • Strep and other oral bacterial DNA have been found in ruptured cerebral aneurysms Pyysalo et al, 2013; Jashari ey al, 2012 Lusis, 2000; Shalhoub et al, 2011; Lee et al, 2013 CVD, Aneurysms and the microbiota • Oral bacterial DNA has been found in atherosclerotic plaques and aneurysms • In ruptured cerebral and abdominal aneurysms DNA from oral bacteria were detected with intense CD14 and TLR2 staining Fernandez et al, 2014, Pyysalo et al, 2013 The collagen-binding protein of Streptococcus mutans is involved in haemorrhagic stroke Controls (N=35) Cerebral hem Ss (N=74) Odds ratio 9% CNM pos. S mutans 27% CNM pos S mutans 3.95 (1.09-14.4) • “Strains isolated from haemorrhagic stroke patients aggravate haemorrhage in a mouse model, indicating that they are haemorrhagic stroke-associated” Nakano et al, Science Comm 2013 TLR and atherosclerosis • TLR2/4 activation of the endothelium promotes lipid accumulation and leukocyte accumulation (proatherogenic) Curtiss et al 2009 How can the microbiota influence stroke? • Inflammation and priming of innate immunity Curtiss et al 2009 • Medin and amyloid formation Westermark et al, 2011 • TMAO and dietary choline/phosphatidylcholine and carnitine metabolism Koeth at al, 2013 • Collagen binding proteins (e.g., Strep mutans) Nakano et al, 2013 • Vitamin K LeBlanc et al, 2012 • Salicylate production Carr, 2014 • Aneurysm creation and/or rupture Pyssalo et al, 2013 Based on what we know about the microbiota what can we do now? • Diet: fruit, vegetables, beans, whole grains, fiber, fish, PUFAs, antioxidants, • • Dietary diversity • • Avoid red meat (especially organs) • • Prebiotics (consider fiber supplements) • • Probiotics • • Dental care (flossing and cleaning) • • Avoid antibiotics as treatment (unless necessary) or in food What can we do now with patients and others? ???? fiber 20g of fiber per cup, cooked What can we do now with patients and others? 0g of fiber 20g of fiber per cup, cooked Based on what we know about the microbiota what can we do now? • Hydration (thirst changes with aging) • Avoid high total plasma homocysteine (folate, B12, B6) • Avoid obesity and diabetes • Avoid excess alcohol • Monitor vitamin D levels (modulates the immune system) • Head trauma [“No injury to the head is too trifling to be despised” Hippocrates] • Exercise Future directions • This is the first study of the influence of bacterial amyloid on the nervous system • Studies of curli protein exposure (C elegans, mice) S. Chen, CWRU) • Metagenomic study of PD in humans (K. LaFaver, A. Gadre, W. Winstead, P. O’Toole, Cork, Ireland) • Gut TLRs and iron transport molecules (R.RahaChowdhury, Cambridge) • Metaproteomics of the microbiota (largely unexplored) Patho- and Saluto-genesis • Pathogenesis the origination and development of a disease Princeton Univ. • Salutogenesis (lat. salus = health, genere = to create) a shift from the disease-metaphor to the vision of wellbeing WorldNet, Antonovsky, A. Health, Stress and Coping. 1979 Collaborators University of Louisville • M. Brown, MD • D. Demuth, PhD • A. Gadre, MD • E. Gozal, PhD • R. Jagadapillai, PhD • B. Jenson, MD • S. Jin, PhD • H. Kaplan, MD • R. Kerber, PhD • R. Liu, PhD • E. O’Brien, PhD • M. Rane, PhD • A. Roberts, PhD • K. Sharp, PhD • V. Stribinskis, PhD • W. Winsted, MD • Z. Wong, MD Neurology Oral Health Surgery (ENT) Pediatrics Pediatrics Vaccinology Medicine Ophthalmology Public Health Neurology Public Health Medicine Physiology Mechanical Engineering Biochemistry Surgery (ENT) Neurology Others • J. Carr, PhD • T, Heiman-Patterson MD • E. Masliah, MD • P O’Toole, PhD • R. Raha-Chowdhury, PhD Univ. of Cambridge, UK Drexel Univ., Phila. UCSD, San Diego Univ. College, Cork, Ireland Univ. of Cambridge, UK Lab of Neurogeriatrics and Benjamin Franklin, 2000 Bacterial amyloid and inflammation • Toll Like Receptors (TLRs) are the principal sensors of the innate immune system • Bacterial amyloid is recognized by the innate immune system and activates TLR2/1, CD14 and other immune modulators • TLR2/1 and CD 14 is an important “trimolecular complex” and activates microglia, produces NFkB and iNOS, thus oxidative toxicity • TLR2 is a primary receptor for AD Aβ peptide and PD alpha synuclein to trigger neuroinflammatory activation Madan, Amar 2008, Liu et al, 2012, Tukel et al, 2010; Kim et al 2013 Rapinski et al, 2013 Limitations • Which amyloid is important? • Can the potential for cross-seeding be predicted in silico? • How to distinguish the effects of infection from that of the bacterial amyloid? • What about amyloids in food? [milk, meat, fungi, maize, berries, as well as various other plants] Cui et al, 2008; Miglio et al 2013 Specific Aim 2 • To evaluate the effects of oral administration of amyloid producing bacteria on gut colonization, gingivitis, periodontitis, serum inflammatory and free radical markers, and development of aggregated AS in gut and brain • Rats will be assessed for gut bacteria in feces, serum markers of immune response, oxidative toxicity, curli levels and antibodies, at monthly intervals for 9 months. Periodontitis • Very common, often asymmetrical • Linked to heart disease and stroke through unknown mechanisms • Associated with CD68 upregulation (marker of microglial activation) Allam et al, 20111; Fan et al 2011 Investigations • Underway in vitro and in vivo (human immune markers, yeast, aged rats, Tg mice) • Proposed: Metagenomic studies of AD, PD, CVD, delirium including the gut as well as nasal cavities & ear • Translational potential ? Challenges to these hypotheses 1 • Why is it so slow? -the prion disorders can have a 20-30 year incubation period (lag phase) -Amyloid propagation is not linear -Bacterial amyloid may shorten the lag phase • What about inherited cases? - the mutations may enhance sensitivity to a ubiquitous pathogenic mechanism (i.e., PRP Polymorphism -129 mv is protective against BSE) • Ankylosing spondylitis is related to genotype (HLA B27) and Klebsiella overgrowth in the colon Rashid et al, 2012 Challenges to these hypotheses 2 • The disease may influence the metagenome (causes or consequences?) • Studies looking at the association of one bacterial amyloid to disease may be negative because that particular one is not not involved • J Alzheimers Dis. 2010;21(1):81-6. doi: 10.3233/JAD-2010100075. • Cerebrospinal Fluid from Alzheimer's disease patients promotes amyloid beta-protein oligomerization. • Ikeda T, Ono K, Elashoff D, Condron MM, Noguchi-Shinohara M, Yoshita M, Teplow DB, Yamada M. Could the initiating agent be ubiquitous? • TTR mutant mice and protected in a germ free environment • Ankylosing spondylitis is related to genotype (HLA B27) and also Klebsiella overgrowth in the colon Noguchi et al, 2002; Rashid et al, 2012 Challenges, cont. • How to explain the diversity of the diseases? (early or late onset, asymmetry of onset, severity of progression) Strains ! [as well known for prions] Collaborators University of Louisville • R. Friedland, MD • D. Demuth, PhD • R. Liu, PhD • J. Rebolledo-Mendez • A. Roberts, PhD • A Bhatnagar PhD • V. Stribinskis, PhD • H. Kaplan, MD • A. Gadre, MD • W. Winstead, MD • K. LaFaver Neurology Oral Health Neurology ASNB Physiology Cardiology Biochemistry Ophthalmology ENT ENT Neurology Others • M. Chapman, PhD • R. Kalaria, PhD • M. Ihara, PhD • E. Masliah MD • R. Raha Chowdhury PhD • E. Mukaekova-Ladinska, MD • R. Vaishnav, PhD Univ. of Michigan Newcastle Univ., UK Kyoto Univ., Japan UCSD, San Diego Univ. of Cambridge, UK Newcastle Univ. UK Baroda, India Collaborators University of Louisville • R. Friedland, MD • D. Demuth, PhD • R. Liu, PhD • J. Rebolledo-Mendez • A. Roberts, PhD • A Bhatnagar PhD • V. Stribinskis, PhD • H. Kaplan, MD • A. Gadre, MD • W. Winstead, MD • K. LaFaver Others • M. Chapman, PhD • R. Kalaria, PhD • M. Ihara, MD, PhD • E. Masliah MD • R. Raha Chowdhury PhD • E. Mukaekova-Ladinska, MD • R. Vaishnav, PhD Neurology Oral Health Neurology ASNB Physiology Cardiology Biochemistry Ophthalmology ENT ENT Neurology Univ. of Michigan Newcastle Univ., UK Kyoto Univ., Japan UCSD, San Diego Univ. of Cambridge, UK Newcastle Univ. UK Baroda, India Does the microbial metagenome influence neurodegeneration and cerebrovascular disease through molecular mimicry? • Molecular mimicry: pathogenic or beneficial • Mechanisms of molecular mimicry: • Primary structure: Potato virus Y Tobacco mosaic virus Neuromyelitis optica • Epigenetic: mRNA Friedland et al, JBC 2008 Liu et al, PLOS One, 2013 Vaishnav et al, JNI, 2013 Rebolledo –Mendez et al, CIB 2013 • Tertiary Structure: Protein misfolding and neurodegeneration Inflammation and oxidative stress Does the microbial metagenome influence neurodegeneration through molecular mimicry? Robert P. Friedland MD [email protected] University College Cork, Ireland September 13, 2013 Neurodegenerative disorders • • • • • • Disease Alzheimer disease Frontotemporal dementia Creutzfeldt-Jacob Parkinson’s disease Dementia with Lewy Bodies Huntington’s disease Motor neuron disease Protein ABeta Tau, TDP-43 PrP a-synuclein a-synuclein Huntingtin TDP-43, FUS Bacteria that make amyloid proteins • • • • • • • • • Bacillus subtilis Escherichia coli Klebsiella pneumoniae Mycobacterium tuberculosis Salmonella enterica Salmonella typhimurium Staphlococcus aureus Streptococcus mutans, coelicolor Xanthomonas axonopodis The list is incomplete. Genes for producing amyloids are also found in these phylum of bacteria: Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteoabacter, Thermodesulfobacteria [Bednarska et al , 2013; Hartman et al, 2013] Lab of Neurogeriatrics and BF 2000 Every neurodegenerative disease is associated with a protein folding problem Protein misfolding disorders • • • • • • Disease Alzheimer disease Frontotemporal dementia Creutzfeldt-Jacob Parkinson’s disease Dementia with Lewy Bodies Huntington’s disease Motor neuron disease Protein ABeta Tau, TDP-43 PrP a-synuclein a-synuclein Huntingtin TDP-43, FUS Proteins acquiring -sheet conformation are toxic Protein misfolding disorders • • • • • • Disease Alzheimer disease Frontotemporal dementia Creutzfeldt-Jacob Parkinson’s disease Dementia with Lewy Bodies Huntington’s disease Motor neuron disease Protein ABeta Tau, TDP-43 PrP a-synuclein a-synuclein Huntingtin TDP-43, FUS Amyloid • Beta pleated sheet • Misfolded (or purposefully folded in a Beta-pleated sheet) • Pathogenic or functional (in all forms of life from bacteria to humans) • Resistant to inactivation by proteases, heat, denaturation, time • Fiber formation is not energy dependent • Extracellular or intracellular • CNS or systemic Does the microbial metagenome influence neurodegeneration through molecular mimicry? Robert P. Friedland MD [email protected] University College Cork, Ireland September 13, 2013 Autoimmunity and molecular mimicry The immune system protects us from invasion, but: “ ….unfortunately, this set of tools, known as the immune system, sometimes goes awry and attacks the body itself.” Johns Hopkins Autoimmune Research Center What role does autoimmunity have in tissue remodeling and repair, which are especially important in aging? Molecular mimicry • “…natural selection can lead to the mimicking of antigenic determinants of hosts by their parasites” Damian, 1965 • Where a harmless species imitates the warning signals of a harmful species directed at a common predator Henry Bates (1825-1892) Molecular mimicry • “…natural selection can lead to the mimicking of antigenic determinants of hosts by their parasites” Damian, 1965 • Where a harmless species imitates the warning signals of a harmful species directed at a common predator Henry Bates (1825-1892) May involve adaptive changes, reflect evolutionary conservation or occur by chance Molecular mimicry Antigenic similarity between macromolecules, which plays a role in the pathogenesis of disease May be pathogenic or beneficial Edward Jenner (1749-1823) Vaccinia Poxvirus (in green) and its host cell (in red) Jason Mercer/Swiss Federal Institute of Technology Does the microbial metagenome influence neurodegeneration and cerebrovascular disease through molecular mimicry? • Molecular mimicry: pathogenic or beneficial • Mechanisms of molecular mimicry: • Primary sequence: Potato virus Y Tobacco mosaic virus Neuromyelitis optica • Epigenetic: mRNA Friedland et al, JBC 2008 Liu et al, PLOS One, 2013 Vaishnav, JNI, 2013 (Rebolledo –Mendez et al, CIB, 2013) • Tertiary Sequence: • Protein misfolding and neurodegeneration • Inflammation and oxidative stress A question concerning Alzheimer's disease: 2001: • Since anti Ab vaccination has a potent biological effect on Ab deposition, could molecular mimicry explain why people remain unaffected at later ages? Ab and the potato virus Y nuclear inclusion b (Nib) protein The N terminal region of Ab is therapeutic in Tg mice and most highly immunogenic for B cells (Petrushina et al, 2007) Friedland et al, Journal of Biological Chemistry, 2008 Potato Virus Y infects: • • • • • • • • • • • • • Capsicum annuum Capsicum frutescens Chenopodium amaranticolor Chenopodium quinoa Lycium Lycopersicon esculentum Nicotiana glutinosa Nicotiana tabacum Physalis floridana Solanum chacoense Solanum demissum Solanum tuberosum Tinantia erecta chili pepper chili pepper goosefoot goosefoot (quinoa- cereal) wolfberry tomatoes tobacco tobacco gooseberry potato potato potato widow’s tears J Biological Chemistry, 2008 Cited three times since 2008 Plant viruses • Do not replicate in humans • Are not thought to influence human health or disease • Eating peas infected with Cow pea mosaic virus causes immunity to measles • Tabasco sauce contains live pepper viruses • 97% of viral RNA or DNA in the gut are from plant viruses • 67% of fecal samples from humans have pepper mild mottle virus (Olszewska et al, 2003; Zhang et al 2006; Colson et al, 2010) Does the microbial metagenome influence neurodegeneration and cerebrovascular disease through molecular mimicry? • Molecular mimicry: pathogenic or beneficial • Mechanisms of molecular mimicry: • Primary sequence: Potato virus Y Tobacco mosaic virus Neuromyelitis optica • Epigenetic: mRNA Friedland et al, JBC 2008 Liu et al, PLOS One, 2013 Vaishnav, JNI, 2013 (Rebolledo –Mendez et al, CIB, 2013) • Tertiary Sequence: • Protein misfolding and neurodegeneration • Inflammation and oxidative stress Does the microbiome influence neurodegeneration and CVD through molecular mimicry? • Molecular mimicry: pathogenic or beneficial • Mechanisms of molecular mimicry: • Primary sequence: Potato Virus Y Tobacco mosaic virus Neuromyelitis optica (Vaishnav et al, J Neuroimmunology, 2013) • Epigenetic: mRNA (Rebolledo –Mendez et al, 2013) • Tertiary Sequence: • Protein misfolding and neurodegeneration • Inflammation and oxidative stress Prions Shortened form of Proteinaceous infectious particles They are abnormal variants of normal proteins Prions have the ability to convert the normal forms that they come into contact with into abnormal (misfolded) forms. Specific strains are transmissible! Prusiner, 2013 “A unifying role for prions in neurodegenerative diseases” AD, CJD, PD, ALS 80% are sporadic, Late onset “…some event occurs with aging that renders a disease-specific protein pathogenic” “…stochastic (random) refolding of the etiologic protein into a misfolded infectious state known as a prion” Prusiner, 2012, Science Transmissibility • While the prion disorders, including kuru, scrapie, CJD, bovine spongiform encephalopathy (mad cow disease) are well known to be transmissible, transmission of AD from human subjects to nonhuman primates had not been well documented Goudsmit, J. et al. Evidence for and against the transmissibility of Alzheimer disease. Neurology (1980) Intracerebral Inoculation of Alzheimer Brain Extracts Elicits Amyloid B Pathology in Non-human Primates Dmitry Goldgaber Peter Davies Pierluigi Gambetti Lary C. Walker Lon R. White Pedro Piccardo David M. Asher Robert P. Friedland ICAD 2010 Chimp 62. Abeta 4G8 Transmissibility of ABeta • Recent findings show that that ABeta pathology may be transmitted from humans and transgenic mice bearing human AD mutations to animals containing the human APP protein via intracerebral or IP inoculation Jucker, Meyers-Leuhman et al, 2006; Eisele et al, 2010, Walker, 2013 • Synthetic ABeta can also cause seeding; after IP injection AB seeds may go through macrophages Walker et al, 2013 • Seeded nucleation dependent aggregation Brundin et al 2010 How is the misfolding initiated in sporadic cases? Dorsal motor nucleus of the vagus in PD and LBD • Alpha synuclein, ubiquitin and TAU • James Parkinson reported “torpid bowels” in 1817 • Lewy described DMN Vagus pathology (1930s) • AS pathology spreads along interconnected pathways to the CNS from the periphery 10-20 yrs before motor signs Huang et al 2002; Braak et al, 2003, 2006; (Braak and del Tedici 2008) Kalaitzakais et al, 2008; Miller et al, 2009 Alpha synuclein (AS) • Presynaptic signaling protein membrane trafficking protein found in Lewy bodies, Lewy neurites in PD, LBD & in neuritic plaques in AD as misfolded aggregates • Intestinal myenteric neurons have aggregated AS in PD innervation of the myenteric plexus by AS fibers and varicosities in the stomach (A) and proximal duodenum (B) of control rats. Phillips et al 2009 Pathogenesis of BSE in sheep and CWD in Elk • PrP(Sc) found first in the brain in the dorsal motor nucleus of the vagus, after involving the enteric nervous system and spleen • Dendritic cells take prions from gut to lymphoid cells van Keulen et al, 2008 “Amyloidogenic potential of foie gras” “These data provide evidence that that an amyloidcontaining food product hastened the development of amyloid protein A amyloidosis in a susceptible population” Solomon et al, 2007 Systemic amyloidoses and cross-seeding • AS, protein A amyloid, apolipoprotein AII, prion protein, amyloid β, tau and others • Studies of the systemic amyloidoses have documented the cross-seeding of amyloidoses by amyloids with similar structure but differing sequence following delivery by inoculation or feeding or drinking Lundmark et al, 2005; Yan et al. 2007. Solomon et al, 2007, Ono et al 2012 “Distinct a-Synuclein Strains Differentially Promote Tau Inclusions in Neurons” Guo et al 2013 Cross-seeding effect of Abeta and AS • Ono et al 2012 • “Aβ and αS acted as seeds and affected each other's aggregation pathways in vitro” • AS strains promote tau inclusions in neurons (Guo et al 2013 What might be the triggers of misfolding? • Amyloid in Food Beef “..food products made from aged cattle posses amyloid-enhancing potential” Yoshida et al, 2009 Milk and amyloid AA Zing et al 2001 Amyloidogenic potential of foie gras Solomon et al 2007 Fish • Feces Amyloid AA in Cheetah • Other amyloids ? Friedland and Petersen, 2008 Zhang et al 2008 Microbiome, metagenome Shall we ruminate on that? Olfactory receptor neurons are in direct contact with the environment (Urban and Tripathy, 2012; Hommet et al, 2012) Olfactory receptor neurons are in direct contact with the environment (Urban and Tripathy, 2012; Hommet et al, 2012) Bacterial amyloid • Extracellular secreted proteins, biofilms, resistant to attack, adaptive • E. coli fibers called curli • Also, Pseudomonas, Staph, Strep, Proteobacter, Salmonella, Enterobacter, Fungi, yeast others • In gut and elsewhere Wang et al, 2008; Larsen et al, 2007; Chapman et al, 2008; Nishimori et al, 2010 E coli with curli fibers CsgA polymerized Courtesy of Chapman Lab, U. Michigan Amyloid adhesins are abundant in natural biofilms “…amyloid (has) almost a prion-like ability to get through the gut and act as a seed for amyloid growth and disease induction. This would raise the possibility that one variable in classical amyloid disease is exposure to environmental amyloids –” Larsen et al, 2007 The gut is the most important organ of the immune system • 70% of lymphocytes are in the gut • Gut to neuron -- dendritic and lymphoid cells involved Mabbott et al 2006 • B cells develop in bone marrow and and also gut, stimulated by microbes (receptor editing to prevent recognition of self) Wesemann et al, 2013 • Differentiation of Th17 cells requires bacteria • Germ free mice have poor immune systems • M cells sample the gut content over Peyer's patches Inflammation in AD • Microglial activation by sAPP • Cytokines, IL 1Beta • Oxidative toxicity • Increased expressions of TLR2 and TLR4 on peripheral blood mononuclear cells from patients with Alzheimer's disease Zhang et al, 2011, Griffin, 2013 Innate immune system is involved in neurodegeneration • - TREM2 -- regulates phagocytosis and activation of monocytes, linked to AD via QWAS Guerreiro, 2013, Neumann et al 2013 • HLA-DRB5 and DRB1 -- major histocompatibility complex II, presents antigens on the surface of lymphocytes and macrophages-- influence susceptibility to multiple sclerosis as well as Parkinson’s disease Sawcer et al, 2011; Nalls et al, 2011 Oxidative toxicity • Bacterial amyloid activates the innate immune system and increases iNOS • HNE increases cell to cell transmission of aggregates of AS (Bae et al 2013) • Liposomal dysfunction increases seed production (less autophagy) • Free radicals are involved in all neurodegenerations and vascular disorders Bacterial amyloid and inflammation • Toll Like Receptors (TLRs) are the principal sensors of the innate immune system • Bacterial amyloid is recognized by the innate immune system as conserved molecualr structures (pathogen associated molecular patterns, PAMPs) and activates TLR2/1, CD14 and other immune modulators • TLR2/1 and CD 14 is an important “trimolecular complex” and activates microglia, produces NFkB and iNOS, COX2, thus oxidative toxicity • TLR2 is a primary receptor for AD Aβ peptide and PD alpha synuclein to trigger neuroinflammatory activation (activation of Cytokine and inteferon genes) • TLR2 is involved in atherosclerosis according to studies in APOE KO mice , promotes lipid accumulation Madan, Amar 2008, Liu et al, 2012, Tukel et al, 2010; Kim et al 2013 Rapinski et al, 2013 Bacterial amyloid and inflammation • Toll Like Receptors (TLRs) are the principal sensors of the innate immune system • Bacterial amyloid is recognized by the innate immune system and activates TLR2/1, CD14 and other immune modulators • TLR2/1 and CD 14 is an important “trimolecular complex” and activates microglia, produces NFkB and iNOS, thus oxidative toxicity • TLR2 is a primary receptor for AD Aβ peptide and PD alpha synuclein to trigger neuroinflammatory activation • TLR2 is involved in atherosclerosis according to studies in APOE KO mice , promotes lipid accumulation Madan, Amar 2008, Liu et al, 2012, Tukel et al, 2010; Kim et al 2013 Rapinski et al, 2013 periodontitis • Strep mutans makes amyloid • P gingivalis uses TLR2 TLR2 and Abeta • Fibrillar amyloid-beta peptides activate microglia via TLR2 • “These studies delineate a novel role of TLR2 signaling pathway in mediating fibrillar Abeta peptide-induced activation of microglia” • Abeta enhances TLR2 via NALP3 (an innate receptor), enhances Th17 cells and IL17A production and may be enhanced by curli fibers Jana et al,. 2008; Nishimori et al 2012 • “Since TLR2 recognizes both curli fibrils and host amyloid fibrils, including amyloid-beta and SAA, it is possible that T cells and antigen-presenting cells become exposed to various amyloids through out their life span, which could generate a memory response. However, to support this hypothesis and determine the involvement of TLR2 in such immune responses, further investigation is needed. “ Nishimori et al 2012 Stroke and vascular cognitive impairment Multi-infarct dementia Vascular dementia Vascular cognitive impairment Microscopic infarctions Binswanger’s disease Microglia and macrophages respond to systemic inflammation (circulating cytokines) and produce inflammatory mediators that damage axons Moreno et al, 2011 MRI of Binswanger’s disease Bacteria, inflammation and vascular disease • • • • • • Atherosclerosis and arteriolosclerosis Monocytes and T lymphocytes respond to oxidized LDL Oxidized LDL signals through TLRs Ruptured plaques have more inflammatory cells Incr. CRP is a RF for stoke and CAD Periodontitis, the most common oral bacterial disease, is linked to stroke and CAD • Strep and other oral bacterial DNA have been found in ruptured cerebral aneurysms Pyysalo et al, 2013; Jashari ey al, 2012 Lusis, 2000; Shalhoub et al, 2011; Lee et al, 2013 Bacteria, inflammation and vascular disease • • • • • • Atherosclerosis and arteriolosclerosis Monocytes and T lymphocytes respond to oxidized LDL Oxidized LDL signals through TLRs Ruptured plaques have more inflammatory cells Incr. CRP is a RF for stoke and CAD Periodontitis, the most common oral bacterial disease, is linked to stroke and CAD • Strep and other oral bacterial DNA have been found in ruptured cerebral aneurysms with intense CD14 and TLR 2 staining Pyysalo et al, 2013; Jashari ey al, 2012 Lusis, 2000; Shalhoub et al, 2011 TLR and atherosclerosis • TLR2/4 activation of the endothelium promotes lipid accumulation and leukocyte accumulation (proatherogenic) Curtiss et al 2009 Periodontitis • Very common, often asymmetrical • Linked to heart disease and stroke through unknown mechanisms • Associated with CD68 upregulation (marker of microglial activation) Allam et al, 20111; Fan et al 2011 Diet “…diet shapes the microbiota, which then affects health in older people” Claesson et al, 2012 A Modest Proposal for a Longitudinal Study of Dementia Prevention (with apologies to Jonathan Swift, 1729) Friedland, Nandi 2013 Hypotheses 1 • Neurodegenerative disorders are caused by exposure to environmental proteins which cause [through molecular mimicry] the autocatalytic self propagating templated nucleation dependent misfolding of a host protein. The protein conformation in linked to the strain of misfolding • Neurodegenerative disorders and stroke are initiated and/or propagated by inflammation linked to bacterial amyloid (TLR2/1, CD14) Hypotheses 2 • Amyloid plaques are common with aging (Savva et al, 2009). AD and PD are caused by the immune response to the brain Abeta/AS through TLR2/1, CD14, induced by bacterial amyloid in the metagenome. • This immune response to metagenome bacterial amyloid is caused by the failure to develop tolerance to our commensal microbiome in early life. • Oral bacterial disease has been shown to be related to vascular disease – the mechanism is a bacterial amyloid induced inflammatory state mediated by TLR2/1 and CD14 Challenges to these hypotheses 1 • Why is it so slow? -the prion disorders can have a 20-30 year incubation period (lag phase) -Amyloid propagation is not linear -Bacterial amyloid may shorten the lag phase • What about inherited cases? - the mutations may enhance sensitivity to a ubiquitous pathogenic mechanism (i.e., PRP Polymorphism -129 mv is protective against BSE) • Ankylosing spondylitis is related to genotype (HLA B27) and Klebsiella overgrowth in the colon Rashid et al, 2012 Challenges to these hypotheses 2 • The disease may influence the metagenome (causes or consequences?) • Studies looking at the association of one bacterial amyloid to disease may be negative because that particular one is not not involved Could the initiating agent be ubiquitous? • TTR mutant mice and protected in a germ free environment • Ankylosing spondylitis is related to genotype (HLA B27) and also Klebsiella overgrowth in the colon Noguchi et al, 2002; Rashid et al, 2012 Challenges, cont. • How to explain the diversity of the diseases? (early or late onset, asymmetry of onset, severity of progression) Strains ! [as well known for prions] Investigations • Underway in vitro and in vivo (human immune markers, yeast, aged rats, Tg mice) • Proposed: Metagenomic studies of AD, PD, CVD, delirium including the gut as well as nasal cavities & ear • Translational potential ? Population pyramid of Japan: 2005 and 2030. Muramatsu N , and Akiyama H The Gerontologist 2011;51:425-432 . Conclusions • Molecular mimicry may involve patho- or salutogenic mechanisms • Plant viruses, in addition to animal viruses, bacteria and food may be involved • Homology of primary or tertiary structures may lead to molecular mimicry • Mechanisms may be immunological or proteomic (protein misfolding through cross-seeding) • These processes may be involved in neurodegenerative diseases, autoimmune conditions and cardiac-cerebrovascular diseases “Everything Should Be Made as Simple as Possible, But Not Simpler” • Albert Einstein ? • William Whewell (1794-1866) “Everything Should Be Made as Simple as Possible, But Not Simpler” • Albert Einstein ? • William Whewell (1794-1866) • “Gut microbiome studies are inherently interdisciplinary, drawing on approaches and technical skill sets from the biomedical sciences, ecology, and computational biology.” Kostic et al 2013 Specific aims 1. To evaluate the influence of bacterial amyloids on Abeta and alpha synuclein aggregation in vitro and in silico 2. To examine the influence of gut bacteria on aggregation of alpha synuclein in the gut wall of the aging rat 3. To examine the gut flora of Tg mice and cases of AD and PD as compared to Wt mice and healthy age and gender matched controls Misao Okawa, world's oldest woman, 115th birthday celebrations March 2013, Osaka, Japan Buddhika Weerasinghe / Getty Images • Goudsmit, J. et al. Evidence for and against the transmissibility of Alzheimer disease. Neurology 30, 945950 (1980). Human Transmissable Spongifom Encephalopathies (TSE) - Prionoses • • • • • Kuru Creutzfeldt-Jakob Disease (CJD) Variant Creutzfeldt-Jakob Disease (vCJD) Gerstmann-Sträussler Scheinker Syndrome (GSS) Fatal Familial Insomnia (FFI) They are transmissible to animals but not to PrP knockout mice Gajdusek, Gibbs et al Amyloid deposits are numerous in chimp 145: all types are present: diffuse, dense core and neuritic Transmissable amyloids • • • • Prions (Kuru, CJD, BSE, CWD) Alzheimer’s disease (A Beta & Tau) Parkinson’s disease (Alpha synuclein) Lewy body disease (Alpha synuclein) Iatrogenic CJD • • • • • Corneal transplant HGH Surgical instruments, electrodes Dura matter grafts Transfusions Implications • • • • • Diet Supplements Transfusions Surgery and instruments Organ donation Holmes and Diamond (Annals of Neurology, in press) Hypotheses 3. The laterality of disease is related to laterality of bacterial involvement or asymmetry of the autonomic nervous system 4. AD or PD cases have more amyloidogenic bacteria than healthy age matched controls Hypotheses 5. If the amyloidogenic bacteria in the gut can be removed protein misfolding disorders may be prevented, delayed or ameliorated, even in familial cases bearing pathogenic mutations (eg; TTR mutant mice are protected in a germ free environment) How could exposure to plant viruses influence human disease? 1. Molecular mimicry and immune mechanisms (These mechanisms may be Pathogenic or Salutogenic) 2. Epigenetic interactions may be found with miRNAs and other factors (Vaishnav et al, 2012) TAU • Tau aggregates induce Tau misfolding, blocked by immunodpletion of tau • Method: worms were fixed by incubation in 4% paraformaldehyde in DPBS for 15 h at 4 oC, and were permealized at 37 oC for 15 h in a solution of 1% Triton X-100, 5% beta-mercaptoethanol, 125 mM Tris-HCl, pH 7.5. Worms were then mounted on glass slide and stained with 0.5 mg/ml Congo red in 50% ethanol for 1 min. Destaining was immediately carried out using several rinses of 50% ethanol until solution became colorless, and then one rinse each with 75% ethanol, 50% ethanol, and water. A drop of Fluoromount was applied and stained worms were visualized for red fluorescence using a Leica fluorescence microscope. Hayashi et al, 2010 • Pathogen-Mediated Inflammatory Atherosclerosis Is Mediated in Part via Toll-Like Receptor 2-Induced Inflammatory Responses • “Human and mouse studies support a role for the oral pathogen Porphyromonas gingivalis in atherosclerosis, although the mechanisms by which this pathogen stimulates inflammatory atherosclero- sis via innate immune system activation is not known” Transmissibility • Recent findings show that that ABeta pathology may be transmitted from humans and transgenic mice bearing human AD mutations to animals containing the human APP protein via intracerebral or IP inoculation (Meyers-Leuhman et al, 2006; Eisele et al, 2010). Anatomical considerations • Nose and mouth and cerebral vessels • Includes those with amyloid • Includes proteobacteria l(Branton et al, 2013) Deep sequencing found bacterial RNA in 70% of brain samples Transmission studies-results • In brains of chimpanzees, mangabey and capuchins inoculated with brain biopsy tissue of patients with early AD at the time when animals were young we detected amyloid deposits before animals aged. • No amyloid depositions we detected in animals inoculated with non-AD tissue. • Neurofibrillary tangles were not observed. Hypotheses The development of disease is related to a. exposure b. protective mechanisms (intestinal barrier, chaperones, aggresomes, proteostasis, antioxidants) c. Age (length of exposure) d. Genetics Challenges to these hypotheses • Why do some people get AD/PD and others not? Genetics (eg; Apo E; PrP 129 polymorphism) Chaperones (ApoE) Other factors (brain clearance, oxidative toxicity, free radicals, trauma, proteosome function proteostasis, mitochondrial dysfunction, metals, pesticides, aggresome production, immunological responses) Inflamm caused by the gut bacteria may cause AD by decreasing Apoe expression • Apo e expression is downregulated by activated macrophages, proinflammatory cytokines and IFN gamma and TNFalpha • Antininflam stimuli like TGF beta and estrogen protoe Apo E synthesis • Mediators of Inflammation Volume 2011, Article ID 949072, 10 pages doi:10.1155/2011/949072 • Review Article • Cross-Talk between Apolipoprotein E and Cytokines Hongliang Zhang,1, 2 Li-Min Wu,1 and Jiang Wu1 • Peridontic bacteria have been found in coronary arteries • Marcelinio et al 2010 Primate studies: Conclusions • Brain tissue obtained from human subjects with early onset AD inoculated in the brains of young nonhuman primates caused the development of cerebral ABeta deposits after an incubation d • d of many years but before such deposits would be expected to develop as a feature of healthy aging. Parkinson’s disease • Alpha synuclein misfolding in Lewy bodies in the substantia nigra pars compacta and also in the colonic submucosa (aggregates) • Fetal dopaminergic neurons transplanted into PD brain develop Lewy bodies • Alpha synuclein Tg mice have accelerated deposits after intracerebral inoculation of material form the same Tg mice Li et al, 2008, Kordower et al, 2008, Luk et al 2012 Parkinson’s disease and Lewy Bodies and Lewy neurites • • • • Misfolded alpha-synuclein Found in colonic biopsies Dorsal motor nucleus of the vagus Salivary glands, Anterior olfactory nucleus and bulb Cersosimo et al, 2011 • There is no evidence of person to person transmission of any neurodegeneratice disease, including CJD Serum anti-TMV-IgG1 * Scatter plot: Median Bar graph: Mean S.E. Hypotheses • bacterial amyloid impairs perivacualr drainage of AB • Can we do matagenomic analyaiss loking for bacterial amyloid genees? microbiome • Related to sex hormones in early live and risk of DM 1 • Autoantibody production • Involved in production of proatherogenic compounds (trimethylamine-N-oxide< TMAO) • Markle et al, 2013; Koeth et al, 2013 Metabolic Function • • • • Fermentation of non-digestible substrates Bile acid bio-transformation Vitamin production Energy extraction Protective Function Colonization resistance Innate and adaptive immunity Structural and Histological Function • Epithelial cell growth and differentiation • Intestinal villi and crypt development Courtesy of I. Kirpich • Oxidized LDL signal through TLR • Activates macrophages increase cytokine and chemokine production biases toward M! and Th1 proinflamm pathway Shalhoub et al, 2011 Infectivity of alpha-synuclein CD36, another innate immunity receptor • Membrane glycoprotein that aids FA uptake, receptor for apoptotic cells, LDL, fibrillar beta amyloid, components of bacterial cell walls, promotes vascular amyloid deposition (CAA) • Febbraio, Silverstein, 2007; Park et al, 2013 Immune System homeostasis and gut bacteria “Peripheral education of the immune system by colonic commensal microbiota” Lathrop et al, Nature, 2011 “Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota” Atarashi et al, Nature, 2013 Conclusions: Medline search – 7/4/2013 • Genome-Brain • Metagenome –brain • Metagenome -eye Conclusions: Medline search –2013-2015 July 2013 • Genome-Brain Sept. 2015 37,299 42,964 • Metagenome –brain 41 60 • Metagenome-nose ---- 15