Nutritional Factors and Epigenetics in

Brain Development and Autism

Richard Deth, PhD

College of Pharmacy

Nova Southeastern University

FINANCIAL DISCLOSURE:

1. Member of the scientific advisory board of Immunotec Inc.

2. Research grant support from A2 Milk Corporation

3. Focus for Health advisory board member

AUTISM:

• Complex neurodevelopmental

disorder usually appearing at

1-3 yrs of age.

• Deficits in social interaction

and communication

• Repetitive stereotyped

behavior and abnormal

preoccupations

• Complex etiology involving a

combination of environmental

risk factors and genetic

predisposition

The prevalence of autism in the US has

increased >100-fold in the last 50 years

This large increase is not explained by genetic factors,

but implies a role for environmental factors

OUTLINE:

1. Development of the Brain, the Mind and Neural Networks

2. Redox/Methylation Metabolism and Autism

3. DNA Methylation and Epigenetic Regulation of Gene Expression

4. Metabolic roles of Folate, Vit B12, Vit B6, cysteine and lithium

5. D4 Dopamine Receptors and Attention

6. Default mode network dysfunction in autism

Development of the Brain,

the Mind and Neural Networks

Normal Development of the Brain

Development of the Mind

Higher level cognitive abilities (e.g. language and

social skills) require connected networks of neurons

Individual experience programs your brain

to be a personalized version of the worldwide web

Starting at birth, specialized network-forming neurons

(PVIs) migrate from stem cell incubator regions into the

cortex and hippocampus

The networked brain

HOW DO NETWORKS FORM?

Parvalbumin-expressing

interneurons (PVIs)

WERNICKE’SAREA

SPEECH

When neuronal networks fire at the same frequency,

they work together and their information combines

ON

OFF

BROCA’SAREA

Broca’s

areaWernicke’s

area

Visual Information

Auditory Information

COMPLEXPERCEPTIONS

Dopamine promotes attention by increasing

the frequency of network firing

ON

OFF

DOPAMINE

50 Hz (gamma)

ATTENDED

INFORMATION

PVI NEURONSD4R

5 Hz

Csibra et al. Science 290 1582-1585 (2000)

Increased gamma frequency activity in response to a

visual task at eight months of age vs. six months

ON

OFF

DOPAMINE

50 Hz (gamma)

ATTENDED

INFORMATION

PVI NEURONSD4R

5 Hz

↑ Energy Demand

↑ ATP Production

↑ Oxidative Metabolism↑ Reactive Oxygen Species

High Antioxidant Demand

High firing rates create high antioxidant demand

Glutathione: GSH

The primary antioxidant in cells

GLYCINEGLUTAMATE

CYSTEINE

Reducing Thiol

Environmental toxins cause their effects

by depleting cellular antioxidant levels

GSH Synthesis and Methylation Metabolic Pathways

MethionineSynthase

HCY

MET

SAH

SAM

>200Methylation

Reactions

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

D4HCY

D4SAM

D4SAH

D4MET

ATPPP+Pi

MethylTHF

THF

PhospholipidMethylation

Adenosine

Dopamine

Cysteine

( - )

EAAT3

AstrocytesCysteinylglycine GSH

GSSG

REDOX

SENSITIVE

ACTIVITY NEURON

D4 Dopamine Receptor

PL Methylation Cycle

Methionine

Methylation

Cycle

GSH Synthesis

Neurotrophic

Growth Factors (+)

REDOX: - The balance between reduced vs. oxidized states

- The balance between antioxidant supply and demande.g. the antioxidant glutathione (GSH)

GSH (reduced form)GSSG (oxidized form)

METHYLATION:- Addition of a carbon atom (CH3- methyl group)

e.g. DNA methylation

- S-Adenosylmethionine (SAM) is the universal donorSAM (methyl donorSAH (methylation inhibitor)

REDOXSTATUS

METHYLATIONSTATUS

Restriction of transsulfuration makes growth factors more crucial

as a source of cysteine to make the antioxidant glutathione

MethionineSynthase

HCY

MET

SAH

SAM

>200Methylation

Reactions

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

D4HCY

D4SAM

D4SAH

D4MET

ATPPP+Pi

MethylTHF

THF

PhospholipidMethylation

Adenosine

Dopamine

Cysteine

( - )

EAAT3

AstrocytesCysteinylglycine GSH

GSSG

REDOX

SENSITIVE

ACTIVITY NEURON

GSH Synthesis

Neurotrophic

Growth Factors (+)

TRANSSULFURATION ISPARTIALLY BLOCKED IN NEURONAL CELLS

Certain Nutritional Factors Support Redox/Methylation

MethionineSynthase

HCY

MET

SAH

SAM

>200Methylation

Reactions

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

D4HCY

D4SAM

D4SAH

D4MET

ATPPP+Pi

MethylTHF

THF

PhospholipidMethylation

Adenosine

Dopamine

Cysteine

( - )

EAAT3

AstrocytesCysteinylglycine GSH

GSSG

NEURON

Vit B6

Methyl B12

Blood-Brain

Barrier

Lithium(+)B12

DNA Methylation and

Epigenetic Regulation

of Gene Expression

EPIGENETICS: -Regulation of gene expression by methyl groups on DNA

and histone proteins

-Transmitted across generations

-The primary driving force for development

- Oxidative stress interferes with epigenetic regulation

Methyl Group

Oxidative Stress

Exposure to environmental toxins alters

developmental trajectory by interfering

with epigenetic regulation

Toxic

Exposure

Normal

Developmental

Trajectory

Abnormal

Developmental

Trajectory

Rate of DNA methylation change is

70-fold lower in children 0-10 yrs

than during fetal development.

Rate of change in adults > 10 yrs

is 600-fold lower than during fetal

development.

Brain DNA methylation changes are 600-fold more dynamic

during fetal development, 20-fold higher in children vs. adult

Numata S et al. Am J Hum Genet. 2012 Feb 10;90(2):260-72.

Redox/Methylation Metabolism

and Autism

A number of studies have reported abnormal

DNA methylation in autism and other

neurodevelopmental disorders.

DNA Methylation, Hydroxymethylation and Formylation

in Human Frontal Cortex of Autistic and Schizophrenic subjectsY. Zhang, N. Hodgson, H. Abdolmaleky, R. Deth, M.S. Trivedi,

Department of Pharmaceutical Sciences, Northeastern University, Boston, MA

and Department of Pharmaceutical Sciences, Nova Southeastern University, Fort Lauderdale, FL

CpG methylation of DRD4 gene

ASD and SCZ subjects

Individual CpG methylation in DRD4 gene measured using bisulfite conversion followed by pyrosequencing in postmortem frontal

cortex samples from age matched Controls (N=9) with Autism (N=12); Age matched Controls (N=9) with Schizophrenia (N=9). *indicates a significant difference between groups (* p<0.05). Site-4 was not detected in all samples although we got good signal incontrols.

D4 dopamine receptor gene is hypermethylated

In both autism and schizophrenia

Trivedi et al.

Starting in 2004, >50 studies link ASD to oxidative stress, low levels of the antioxidant glutathione (GSH)

and impaired methylation

38%

28%

36%

2006:

James SJ et al. Am J Med Genet B Neuropsychiatr

Genet. 2006 Dec 5;141B(8):947-56.

2007: A Redox/Methylation Hypothesis of Autism

Deth R et al. Neurotoxicology. 2008 Jan;29(1):190-201.

97% correct

classification

2017: Measuring Redox/Methylation Metabolites

Correctly Identifies Autistic Subjects

Methionine synthase expression

decreases with age

Methionine synthase expression

is decreased in autism

2013: Human brain expression of folate and B12-

dependent methionine synthase decreases

across the lifespan and is 50% lower in ASD

Muratore CR et al. PLoS One. 2013;8(2):e56927

Methionine synthase expression in cortex is lower

in autistic subjects vs. age-matched controls

The ASD decrease is greatest for youngest subjects,

representing an abnormal acceleration of the

usual age-dependent decrease.

This acceleration may represent a lost opportunity

for learning and development.

Brain levels of redox and methylation

metabolites normally change with age, but

change earlier in autistic subjects

Zhang Y, et al. PLoS One. 2016 Jan 22;11(1):e0146797.

Changes in autism are indicative of a premature

decrease in methylation and increased

transsulfuration pathway activity

MethionineSynthase

HCY

MET

SAH

SAM

>200Methylation

Reactions

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

( - )

( - )

PARTIALLY BLOCKED IN NEURONAL CELLS

EAAT3

HealthyGlial Cells

(Astrocytes)Cysteinylglycine GSH

GSSG GSCbl

MeCbl

SAMOHCbl

TNF-alpha( + )

( - )Inflammation

Vaccination

CBS

CGL

The pro-inflammatory cytokine TNF-alpha decreases

methionine synthase expression, similar to autism brain

Muratore CR et al. PLoS One. 2013;8(2):e56927

Vitamin B12 (Cobalamin) in the Human Frontal Cortex

MethionineSynthase

HCY

MET

SAH

SAM

>150Methylation

Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

D4HCY

D4SAM

D4SAH

D4MET

ATPPP+Pi

MethylTHF

THF

PhospholipidMethylation

Adenosine

Dopamine

Cysteine

( - )

PI3-kinase

( + )

PARTIALLY BLOCKED IN NEURONAL CELLS

EAAT3

HealthyGlial Cells

(Astrocytes)Cysteinylglycine GSH

GSSG

Growth

Factors

GSCbl

MeCbl(MethylB12)

SAMOHCbl

Vitamin B12 status in postmortem human brain

during aging and autism

Stabler SP N Engl J Med. 2013

Jan 10;368(2):149-60.

Levels of MethylB12 (MeCbl) and total Cbl

decrease with age in frontal cortex and levels are

prematurely decreased in autism

Zhang Y, et al. PLoS One. 2016 Jan 22;11(1):e0146797.

Ave age = 8 yrs

Thus in autism something has accelerated the

normal age-dependent decline in brain MeCbl

D4 Dopamine Receptors

and Attention

CH3

DOPAMINE

DOPAMINE –STIMULATED PHOSPHOLIPID METHYLATION

MethylfolateMethionine

Synthase

Sharma A et al. Mol Psychiatry. 1999 May;4(3):235-46.

Structural features of the dopamine D4 receptor

Seven repeats are

associated with

increased risk of

ADHD

D4 receptors are located in the post-synaptic density (PSD),

where dopamine-stimulated phospholipid methylation (PLM)

can affect neighboring proteins, “tuning” the neural

network firing rate to gamma frequency.

PSD-95

PLM

The number of D4 receptor repeats

affects the availability of phospholipids

for methylation.

DOPAMINE

7-repeats

2 or 4-repeats

Gamma frequency responses are stronger for 7-repeat D4 receptor

or 10-repeat dopamine transporter

Demiralp T et al. Cereb Cortex. 2007 May;17(5):1007-19.

Perineuronal Net

Neuropsin

mNRG-1

NRG-1Ligand

Erb-B4

PI3K

Akt

EAAT3

ApoER2

Cysteine

SelP

Selenoproteins

Parvalbumin-expressingGABAergicInterneuron

Cysteine

GSH GSSG

Dopamine

D4 Receptor

↑ROS

↑ATPDemand

GammaSynchrony

( - )

PLM ?SO3-2

Gamma synchronization during attention involves

redox-sensitive parvalbumin-expressing interneurons

and parvalbumin expression is decreased in autism

Soghomonian JJ et al. Autism Res. 2017 Nov;10(11):1787-1796.

Default mode network

dysfunction in autism

The Default Mode Network is crucial for self-identity,

social interactions, future anticipation and other

domains that are dysfunctional in autism

Synchronized Activity

of Neural Networks

Creates an

Electromagnetic

Field,

Allowing Information

To Freely Interact

Instantaneously

A speculation about

the mind:

Gamma ActivityMIND

FIELD

100-fold lower coherence in ASD

Activity of the Default Mode Network correlates

with social measures in autistic subjects

D4 Receptors

DOPAMINE

↓ Gamma

Synchrony

DNA

Methylation

Phospholipid

Methylation

↓ Attention/Self-identity

∆ Epigenetic

Effects

↓ Learning

Oxidative Stress

Environmental

Factors

AUTISM

Brain Samples:

Autism Tissue Program

Harvard Brain Tissue Resource Center

Tissue Resource Center (Australia)

Stanley Medical Research Foundation

and donor families.

Collaborators:

Antonio Persico

Suzanne De la Monte

Hamid Abdolmaleky

Yahya Al-Farsi

Steve Walker

Bernat Kocsis

Grant Support:

Autism Research Institute

SafeMinds

NIH

National Autism Association

Autism Speaks

A2 Milk Corporation

ACKNOWLEDGMENTS

Current and Former

Grad Students:

Christina Muratore

Nate Hodgson

Alok Sharma

Malav Trivedi

Yiting Zhang

Matt Schrier

Mostafa Waly

TALK ABOUT CURING AUTISM

https://www.facebook.com/tacaflorida

https://tacanow.org/local-chapters/southeast/florida/

For further information from autism moms: