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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
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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
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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
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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
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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
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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
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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
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Metaproteomics
Fungi
Hos tgenetics
Fundctional metagenmics
Therapies
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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)
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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)
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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,
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• Dietary diversity
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• Avoid red meat (especially organs)
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• Prebiotics (consider fiber supplements)
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• Probiotics
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• Dental care (flossing and cleaning)
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• 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
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