EVALUATING THE POTENTIAL OF PROBIOTIC AND PREBIOTICS FOR INFLUENCING BRAIN FUNCTION -

FROM FETUS TO ADULTS

Mark Lyte, Ph.D., M.S., MT(ASCP) Texas Tech University Health Sciences Center

ISAPP - Potential to employ probiotics/prebiotics for fetus and infants to improve well-being

May 19-21, 2015

As of 6/15: Veterinary Medicine, Iowa State University

Structure of Talk

Brief overview of selected aspects of gut-brain communication pathways.

Introduction of neurochemical-mediated mechanisms within the microbiota-gut-brain axis.

Discussion of these neurochemical or microbial endocrinology-based mechanisms as basis for probiotics/prebiotics to influence and infant and adult brain function and behavior.

What role can probiotics and prebiotics play in modulating the complex interactions between host, diet and microbiota that may ultimately influence adult and infant behavior? Will employ a Socratic-based format throughout this discussion

Where we have come from…

Emergence of human microbiome and what it means for behavior in a microbiota-gut-brain axis Not really new – In 1917, Kellogg and use of

colectomy to improve human temperament and health: ”Should the colon be sacrificed or reformed…I have labored constantly and earnestly to devise and perfect methods for changing the intestinal flora…” Kellogg, JAMA, 1917(68:1957)

How far things have gone…

What do we know from published studies?

Isolation from previously believed sterile areas Translocation from mother’s gut to placenta via bloodstream

Jimenez et al., Curr. Microbiol. 51:270, 2005 Lactobacillus and Bifidobacterium DNA in placenta – no viable cells

Satorki et al., Lett. Appl. Microbiol. 48:8, 2009 Placental membrane has microbiome signature similar to oral cavity

Aagaard et al., Sci. Transl. Med. 6:237ra65, 2014

Alteration of microbiota can result in later neurodevelopmental, brain and behavioral issues Studies involved germ-free mice

Diaz Heijtz et al., PNAS, 108:3047, 2011 Clarke et al., Mol. Psychiatry, 18:666, 2013

A word about timing…

Neurodevelopment and microbiome undergoing massive changes – at the same time Presents both opportunities and problems at the

same time

Developing scientific headwinds…

Nature, 512:247-8, 2014

Issue is: Correlation and

Causation

Pressing need to identify mechanisms Entering Microbiome Version 2

Can probiotics and prebiotics influence brain and behavior?

There are numerous studies which have shown that probiotics, and prebiotics, can influence brain and behavior. Studies include both humans and lab animals Range of behaviors as well as neurodevelopmental aspects have

been reported Feeding of Saccharomyces boulardii can alter neurochemistry

of enteric neurons by unknown mechanism. Kamm et al., Neurogastroenterol. Motil. 16:53, 2004

From these studies has emerged the concept of the microbiota-gut-brain axis.

Furness et al., Adv. Exp. Med. and Biol. 817: 39-72, 2014

How extensive is the innervation in the ENS where bacteria are in close proximity?

Vagal villus afferents

Powley et al. Journal of Comparative Neurology 519:644-60, 2011

Question is where does information flow and possible bi-directionality

Presence of afferents in vagal crypts

Powley et al. Journal of Comparative Neurology 519:644-60, 2011

Vagal villus and vagal crypt afferents are independent of each other.

Gut vagal afferents modulate behavioral responses

Take home message: “Emphasizes an important role of afferent visceral signals in the regulation of emotional behavior”.

What is consequence of this “bottom-up” information flow from gut-to-brain?

Klarer et al. performed sub-diaphragmatic deafferentation Klarer et al. Journal of Neuroscience 34:7067-76, 2014

Anxiety-like behavior

Thinking about prebiotics and probiotics from a different perspective Neurochemical production by both host and probiotic, or prebiotic-dependent, is a proposed mechanistic linkage. Probiotics have long been recognized to produce neurochemicals

Report dating from 1930’s of acetylcholine production in probiotics

Food production microbes well known to produce neurochemicals Cheese and meat starter cultures utilizing Enterococcus spp. to produce tyramine

Host interaction with food-borne neurochemicals also long recognized Need for MAO-compliant diets for individuals receiving antipsychotic drugs

Neurochemicals as a mechanistic linkage between microbe and host is the field of

Microbial endocrinology Use of probiotics according to a microbial endocrinology-based

approach has been referred to as “psychobiotics”. - Dinan et al., Biol. Psychiatry 74:720-6, 2013

Uptake into portal circulation

Enteric nervous system (ENS)

MICROBIOTA

PROBIOTICS

Food-derived substrates and

neurochemicals PREBIOTICS

BRAIN

1 6

3 4

5 2

Behavior and cognition

7

Correlation and causation

Lyte, Gut Microbes, 5:381-9, 2014

Presence of neurochemicals in food

Bananas 700 µg/g dopamine and 70 µg/g NE Division between peel and pulp Waalkes et al., Science 127:648, 1958.

Tribal pulses 8 g of L-Dopa per 100 g of flour Resistant to destruction by autoclaving and boiling

Other common foodstuffs Tomatoes - dopamine, tyramine Cheese - tyramine

Presence of hormones in the microbial world

Bacteria Insulin-like material - present in all strains examined GABA – Clinical bacterial pathogens Somatostatin - Bacillus subtilis Catecholamines – E. coli Specific receptors have been demonstrated - 100% homology of E. coli EnvY

gene for high affinity opioid binding site. Probiotics produce neurochemicals such as GABA Tryptamine – Human microbiota

Protozoa Catecholamines - Crithidia fasciculata, Paramecium Serotonin - Tetrahymena pyriformis

Fungi Sex pheromone - Truffles (Androsterol)

Do neurochemicals affect bacteria?

Interactive microbial endocrinology environment – From Host to Bacteria

Lyte, BioEssays, 33:574-581, 2011

MICROBIOTA/ PROBIOTICS

First observation: Yersinia enterocolitia

Lyte & Ernst, Life Sci. 50:203-12, 1992

What is the spectrum of neurochemicals that

microbiota/probiotics make that may affect host?

Probiotics can also be influenced by stress-related neurochemicals such as norepinephrine. L. acidophilus ASF360 and S. thermophilus NCIMB 41856 increased growth rate >5-fold. --Bailey et al. PLOS One 6:e26507, 2011

What in vivo evidence exists?

Sudo and colleagues measured the role of the microbiota in generation of luminal catecholamines

Lasano et al., Am J Physiol Gastrointestin Liver Physiol 303:G1288-1295, 2012

What do probiotics make?

Lyte, BioEssays, 33:574-581, 2011

Neurochemical levels produced by probiotics are physiologically relevant – often in the mg/ml level

GABA in various probiotic strains

Interactive microbial endocrinology environment – From Bacteria to Host

Lyte, BioEssays, 33:574-581, 2011

MICROBIOTA/ PROBIOTICS

Probiotics as neurochemical delivery system to influence behavior:

Probiotics can be used to influence behavior Numerous studies by groups have amply shown

the ability of probiotics to influence anxiety-like conditions in humans and animals, as well as other behavioral states.

Difference is mechanism responsible for the ability of probiotics to influence behavior Immunological mechanisms among those considered What if considered from the lens of microbial

endocrinology? Lyte, BioEssays, 33:574-581, 2011

Pathways by which probiotic produced neurochemicals may reach target

Concept that there are multiple host targets Brain – CNS Uptake into portal circulation

ENS Receptors in the gut epithelium

Immune system components within the gut Peripheral sites (organ systems) that then influence behavior Above suggests gut-derived neurochemicals do not have to

reach brain to have host effect Also other microbiota targets

Direct effect of probiotics on prevalence and function of other microbiota

Further on routes by which probiotics influence brain and behavior

Uptake into portal circulation Will 108-1010 probiotic microorganisms be enough? Need a way of measuring Could it survive transit and make it all the way to the brain?

Question of real estate? Does location of probiotics influence where it may

interact with components of the ENS? How quickly does a probiotic get “up to speed”

once ingested? Going from a low temperature to 37°C may take too

long?

Evidence from functional foods that this approach may work

Lactobacillus brevis FPA 3709 produced high levels of GABA in black soybean milk (Ko et al. Process Biochem. 48:559-68, 2013)

Strain located after screening of 15 different fish species intestines

Black soybean milk made from homogenized beans

Rats were administered GABA-rich soybean milk initially freeze-dried, homogenized and added as powder

GABA-rich soybean milk as effective as fluoxetine as antidepressant in rat forced swimming model Ko et al. Process Biochem. 48:559-68, 2013

Prebiotics – view and role

Current definition of dietary prebiotic as “…a selectively fermented ingredient…”. Gibson et al., Food Sci. and Technol. Bull: Functional Foods 7:1-19, 2010.

Bindels et al. have published “Towards a more comprehensive concept for prebiotics”. Bindels et al., Nat. Rev. Gastroenterol. Hepatol.

doi:10.1038/nrgastro.2015.47, 2015 Argue for a “…more rational basis for the identification of

prebiotic compounds, and a framework by which the therapeutic potential of modulating the gut microbiota could be more fully materialized”.

Critical as to consideration of microbial endocrinology and probiotics as drug delivery vehicles.

Prebiotics and microbial endocrinology

Diet contains large and varied amount of neurochemical precursors that may serve as prebiotics to increase ability of probiotics to produce neurochemicals of interest.

Use of prebiotics is being examined with resistant starch RS is portion of starch that resists digestion by pancreatic

amylase in small intestine thereby available to microbiota in large intestine

Behavior often compared to soluble, fermentable fibers such as guar gum.

Resistant starch as dietary means to alter microbiome

PLOS ONE, under revision

CLASS PHYLUM

Resistant starch-induced behavioral changes

Submitted, under revision

Elevated plus maze

Detection of neurochemicals in stool

NCS HA-7

CAVEAT: Correlation and causation

Use of neuroimaging to detect microbiota-induced changes in structure

Maternal and Infant Microbiome Determinants of Brain and Behavioral Development

PROBIOTICS

NIH R21/R33 Phased Innovation Award Multi-PI: Coe (UW-Madison), Lyte (TTUHSC) and Phillips

(ISU) First large scale characterization of infant primate

microbiome and will demonstrate that bacteria acquired from the mother affect behavioral and neural development.

Specific Aim #1: Infant microbiota acquired from mother will affect behavioral and neural functioning.

Specific Aim #2: To demonstrate that the infant microbiome is associated with brain structure and maturation.

Specific Aim #3: To interrogate the microbiome with novel methods and discover new mediating pathways.

Maternal and Infant Microbiome Determinants of Brain and Behavioral Development

Speculation (possibilities) and concern…

Could probiotics influence ongoing neuro-degenerative diseases If a probiotic that produced l-dopa was consumed, to what extent might it

delay onset or reduce effects of Parkinson's?

Do probiotics interact with clinical psychoactive drugs? Body of literature shows receptors so the answer is “yes”

If a probiotic or prebiotic (induction by bacteria) can make a make neurochemical that may influence brain function, what will be consequence for other microbiota – good, bad?

Neurochemical production needed for other bacteria – probiotic consequences

Strandwitz et al., RISE 2014-Northeastern University, Abstract ID#: 417

Neurochemicals produced by probiotic may influence growth of other microbes in gut in addition to desired host effect. Does this suggest that probiotic may be acting indirectly

through other microbes to affect host?