Enteric and Dietary Short Chain Fatty Acids

Possible Triggers of Autism Associated

Epilepsy and Movement Disorder
Derrick F. MacFabe M.D. (Assist. Professor & Director)
The “Kilee Patchell-Evans” Autism Research Group
Depts. of Psychology and Psychiatry (Div. Develop. Disabilities)
Schulich School of Medicine and Dentistry (U. of Western Ontario)
 OVERVIEW

Clinical Presentation of Autism Spectrum Disorders- A systemic disorder?

History of Seizure and/or Seizure Disorder in Autism- Are they linked?

Dietary Exacerbation of Autism- GI Factors Link to Antibiotic Associated Diarrhea –

Clostridia

Enteric Short Chain Fatty Acids- A Common Link?

Biological Effects- Brain, Gut, Immune, lipid metabolism, oxidative stress,

glutathione, gene induction, cell- cell communication- seizure and ASDs

Kilee Patchell-Evans Autism Research Group-multi-disciplinary

Using Animal Models to Study ASD’s- rational study of environmental factors

-hyperactive,repetitive/anti-social behavior
-brain electrical activity- seizure and ticks (ie movement disorder)
-neuroinflammation and mitochrondrial dysfunction
-Common infections, chronic antibiotics, clostridia, carnitine and
convulsions- “THE PERFECT STORM”
 DISCLAIMER

Research depicts studies using rodents with EEG electrodes/drug ports to measure
brain activity and behaviour, with studies based on extensive biochemical and tissue
culture research

Similar to human patients undergoing workup for surgical treatment of epilepsies

Research ethics in strict accordance with Canadian Council on Animal Care and
University of Western Ontario Animal Use Committee

This basic science research in no way is intended to support any treatment

claims by any groups not medically sanctioned by experienced physicians
practicing evidenced based medicine in autism spectrum and related disorders.
Autism – A Brain Disorder of Movement, Sensory
Sensitivity and Impaired Socialization
Affects 1 in 150 persons (males>females)

Abnormal Social Interaction

Speech and Language Difficulties

Repetitive Stereotyped Movements

Self-Injurious Impulsive Behavior

Sensitivity to Sensory Input

Seizure Disorders/ cortical dysplasia

Savant Syndrome (rare)- Regression?

Immune Abnormalities CNS/GI*

Genetic/environmental interaction
 Where is the Lesion? What is the Lesion?

Primary Brain Disorder?

Systemic Disorder Which Affects The Brain?

Does Autism and Epilepsy Share Common Factors?

 The Genetics of Autism
- Identical twin studies- 50-80% concordance - genetics and environment
- Multiple Chromosomes- 2,3,7, 15, 16, 17 X- chromosome mapping
-Multiple genes- brain development, neurotransmitters, language centres
-Intercellular connections- Neurexins
-Disorder of Gene expression (methylation, acetylation of histones)
-Met Receptor Tyrosine Kinase &
-Protein Kinase C beta 1 (brain, gut, immune)

-Oversimplistic to say one specific genetic cause

-Epigenetics- interaction with genes/environment
-“Spontaneous (?)” genetic mutation
- Other genetic disorders where autism is associated
Fragile X**
Angelman/Prader Willi Syndrome**
Epilepsy- “tuberous sclerosis”*
Rett Syndrome
Mitochondrial genetic disorder?
 Enlarged White Matter in ASD patients (Herbert)

Grey matter atrophy white matter hypertrophy

 microglia

Autism- A White Matter Disorder? Brain Interconnectedness

Neurodevelopment- “Lets Build a Brain”
- Complex development
timing important
- Many neurons die

Genetic (instruction)
Environment

Insults:
Infection (virus)/inflammatory (IL-6)
toxins (alcohol)/metals/drugs(valproate)
(germ cell-fetus-neonate)

Cell to Cell Communication is

Important in the organization of the
developing nervous system

(programmed cell death and ordered cell migration)

Neuropathology of Human Autism– Neuroinflammation/mild cell loss

NRL AUT MG

MG RG

RG

RG MG
Autism- A Disorder of Energy Utilization and Toxin Elimination

Oxidative Stress:
Inflammation, impaired metabolism
Process similar to memory!!!
Antioxidants- glutathione
Facilitators of mitochondrial
function-carnitine, methylation-
Methyl B12 (accessibility to CNS?)
A mitochondrial disorder?
(Mitochondrial DNA mutations- risk)
 Role of Vaccine MMR- Controversial

-Epidemiological evidence not conclusive (sensitive subgroup?)

-Continued increase in ASD despite reduction in thimersol
- Danger of tunnel vision – Many other factors occurring at that
time period (paediatric infections, antibiotics, pathogen spread )
- Lack of immunization = “home for viruses” and mutation
multiple children with developmental delay
i.e. congenital rubella syndrome
- Ongoing epidemiology- Queens prospective study (Dr. Holden)
Seizure Disorders and Autism Spectrum Disorders
 Review of Epilepsy/Movement Disorder in Autism

High association with Autism- over 40% difficult to examine and assess

Previous history of seizure in infancy or seizure disorder

Risk factors for seizure or movement disorder often similar for autism

Difficult to determine relation of seizure to autism behaviour

i.e inattention, repetitive behaviour, ticks, aggression, hyperactivity

Autism as a movement disorder- ticks, repetitive behaviours- sub cortical

very difficult to measure

Gather information from patients with surgical treatment of epilepsy and movement
disorder (deep brain electrographic changes)

Empiric dietary treatment shares some similarities- low carbohydrate

Common mechanisms?
 Epilepsy
Prolonged neuronal
discharge

limbic areas very

sensitive
Hippocampus

Many seizures not

convulsive, and many
“convulsive” behaviours
not seizures!

Many common triggers

with ASDs
 Autism and The Limbic System: the Biology of Social
Behaviour

Limbic System - Primitive Brain Areas- emotions and complex behavior

- Response to environment, social cues, drive, fear, learning, memory

- Feeding, sexuality, reward, feelings of significance
-Important: complex movement, motivation, socialization, impulsivity
-Meaning of facial expression
- Epilepsy, OCD/anxiety/mood disorder, schizophrenia, drug addiction
AUTISM
 Limbic Seizures

mainly originate- hippocampus and amygdala

complex partial seizures:
complex motor activities
complex visual, auditory hallucinations, social withdrawal
emotional responses
Sensory Changes :
odours
pain and pleasure
visceral sensations from
abdomen
complex visual scenes
feelings of familiarity
(déjà vu), unfamiliarity
inappropriate significance
Heart rate, blood pressure, respiratory changes
GI changes (gut motility, acid secretion)
Innattention, repetitive movements picking, grabbing
Post seizure hyperactivity, mood swing
-very similar to autism!
?Interictal Temporal Lobe Personality (Geschiwind’75)

humourlessness
Post-ictal psychosis

-hypergraphia
-number fixation
- preoccupation
hyperreligiousit with detail ?hyperinhibition
y
Temporal lobectomy for intractable seizure
 Activated Microglia in Epilepsy

Human epileptic focus-

mesial temporal sclerosis
Kainate mouse hippocampus

Found in experimental and

human epilepsy and brain
tumour- ?epileptogenic factors Nonepileptic tissue from same
Similar to autism neuropathology patient
Autism and Basal Ganglia - The Neurology of Habit

Deep Brain structures:

Appropriately sequencing
movement
Learned movements

Disorders of Movement:
Ticks, impulsive actions
and twirling

Repetitive Stereotypic
Movements in Autism
 Enlarged Brain Size Systemic Changes
Increased Neuronal Density Immune System
Altered Cell Migration Gastrointestinal System
Seizure Disorder Metabolic Disorder
Detoxification Systems
Hormonal (glutathione)
Sex Hormones
Oxytocin
Vasopressin
AUTISM

Genetic Factors
Neurotransmitter
Growth Factors
Cell-cell Interaction
Environment
Sex Linked (Fragile X)
Metals
Hydrocarbons
White Matter Disorder Infectious
Glial/microglial Changes Drug (valproate)
Neuroinflammation Diet- Wheat
(Impaired Neurodevelopment and Casein Allergy
Cortico-cortical processing) Carbohydrate?
 Autism- The Blind Men and the Elephant

ASDs

Some common underlying cause involving behaviour, brain changes,

seizures, ticks, GI/dietary symptoms, immunology, genetics,
oxidative stress, environment, increase?????
The Kilee Patchell-Evans Autism
Research Group

University of Western Ontario

The “Kilee Patchell-Evans Autism Research Group”
David Patchell-Evans- CEO GoodLife Fitness
“GRAIFs” Gut Related Autism Inducing Factors
Microbiome (100x host cells)

Bacterial metabolites- symbiosis/dysbiosis

Opportunistic Infections- key risk factor

i.e clostridia, yeast (chronic antibiotics)

Cell wall- LPS, beta glucan- innate immunity

Fermentation products of dietary carbohydrate

- Short chain fatty acids*

Barriers, variable metabolism

Acquired/genetic (met receptor tyrosine

kinase)
Digestive Tract Pathology in Autism- Lymphoid Nodular Hyperplasia
(Wakefield, Horvath)

Intestinal pathology on a subset of autistic patients

Associated with regressive onset and GI symptoms
Moderate inflammatory process (nonspecific?)
Cause????
Can Enteric Bacteria Affect Brain Development/Behaviour?

Psssst…
Dr. MacFabe
Feed Us and
Spread Us
Around!!

Clinical- Food Craving/Symptom Worsening

Gut changes (gluten/casein) poorly studied (antigenic mimicry)
Early gut colonizers- alteration with antibiotics (increased incidence)
“Leaky” or malabsorbtive digestive tract (impairment of barriers)
Production of bacterial metabolites (fuel for brain)
Effect on Brain development, physiology, behaviour, immune function
 Clostridium Difficile – Epidemic

Genetics ARE important- Genetic mutations of infectious processes too!

“Wash Yer Hands Eh?”
 Propionic Acid- Neuroactive properties

Weak organic acid: lipid/water soluable

Uptake passive active (monocarboxylate receptors)
Intracellular concentration (intracellular acidification)
Unique CNS/GI immunological properties
 Short Chain Fatty Acids – Propionic Acid
Propionate:

Byproduct of bacterial metabolism

Clostridium, propionibacteria (gut/acne)
(butyrate, acetate)- short chain fatty acids

Common preservative of wheat and

dairy products

Increased by ethanol, B12/biotin deficiency

Variable metabolism of propionate in population – Multiple
mechanisms and multiple clinical presentation
shares similarities with autism including seizures- underreported???

Role of diet, gut bacteria/barriers and “sickness” in propionate levels

(other short chain fatty acids and metabolites)
 Automated Behavioural Monitoring (Ethovision):
- Computerized long term quantification of movement
- Combined with drug administration, brain electrical activity
- Repetitive behaviour

Vehicle

Propionate Autism Model

 Intraventricular Propionate- “ritual”

Hippocampal EEG- Repetitive motor loop

Normal EEG
Propionate induced kindling in Cortex,Hippocampus, Caudate
No effects seen with control treatments (PBS, propanol)
Brain “remembers” exposure
Intraventricular Propionate/Acetate Induces Seizure- role of pH?
 10 Retropulsion 15 Snake Posture

Mean Number of Snake Postures

8 12
Mean Retropulsion Bouts

Baseline
* Treatment
6 9 *
+
4 6

2 3

0 0
Propanol PBS PBS
Low High Sodium Propanol
Low High Sodium
Propionate Propionate Acetate
Propionate Propionate Acetate

50
Turning

40

#
#
Legend
Mean Turning

30

* = Significantly different from all control groups.

20
# = Significantly different from low PA, propanol, PBS.
+ = Significantly different from propanol and PBS

10

Propionic acid causes movement

0

Low Propanol PBS

disorder with caudate spiking
High Sodium
Propionate Propionate Acetate
Caudate Spiking and Limb Dystonia Only Caused by Propionate

Movement disorder effects of PA

Most sensitive
 Long Term Propionate (2xday for 7days)- Increases Activity

7000
Distance Moved Number of Movements
500
SAL
6000 PPAH
400 SAL

Mean Number of Movements

*
5000 PPAH
Mean Distance (cm)

*
4000 300
* * * *
3000
* *
200
*
2000 *
100
1000

0 0
BL T1 T2 T3 T4 T5 T6 T7 D8 BL T1 T2 T3 T4 T5 T6 T7 D8

Day Day

Marked increase in hyperactivity/ preservation after PPA withdrawal

-tissue for – Immunohistochemical/biochemical study
 Social Behaviour (Ignoring/Mean Distance Apart)
(Shultz et al. Neuropharmacology, 2008 in press)

vehicle Propionate

Effect apparent after one dose, reversible post metabolism

Reduced play behaviour
Long term effects- Ethovision- stereotypies/ object fixation
 Neuropathology of Propionate in Rodent Model:

Similarities to metabolic/autism spectrum disorders

Innate neuroinflammation, oxidative stress, BBB
Altered gene expression
Altered lipid metabolism
Anti Nitrotyrosine Immunoreactivity- oxidative stress

Saline High Dose Propionate

Propionate causes increase anti Nitro-tyrosine immunoreactivity

in hippocampal formation
increases “oxidative stress”
Reduction of glutathione
Electron Microscopy- Mitochondrial Dysfunction

PBS PPA
 Diabetes Autism

Type 1 Type II
Can’t metabolize glucose Can’t metabolize SCFAs?
Multi- system involvement
Multiple Causes (Genes/diet/environment)
Present with Metabolic Crisis (i.e infection)
Treatment-Carbohydrate restriction (direct/indirect)
Treatment-Insulin/glyburide Carnitine/bacterial
eradication/probiotics/MB12?
Multi- system approach
Common Infections, Chronic Antibiotics, Clostridia and Carnitine=
Convulsions “The Perfect Storm”

Carnitine

Carnitine- Shuttle for mitochondrial fatty acid beta oxidation

Routine pre- peri or post natal infections-
Long term antibiotics (beta lactams)- deplete carnitine transport
“Barren Gut”->Growth of clostridials- increased SCFA production
Further sequestration of carnitine
Impaired fatty acid metabolism- mitochondrial encephalopathy
Sooo…Is our PPA rodent model like human autism?
• Hyperactivity
• Complex Movement
• Object fixation
• Intermittent seizure
• Subcortical spiking with movement PPA
• Kindling/ Neuroplasticity
• Social Impairment
• No gross neurotoxicity
• Astrogliosis/microglia/ Neuroinflammation
• White matter damage (lipoperoxidation, edema, cholesterol)
• Oxidative stress/ impaired glutathione (broad spectrum detoxifier)
• induce catecholamine/CREB expression (epigenetics)

•Propionate is known to cause:

• Neutrophil/monocyte migration (specific SCFA receptors)
• Mitochondrial uncoupling (fatty acids), increases in odd chain FAs, low chol.
• Neuronal structural changes (cytoskeleton)/gene expression)
• Intracellular acidification - Dopamine/glutamate/5HT release – gene induction
• Impairment in cell-cell signal transduction (gap junctions, cytokines)
Cautious Optimism- need for further rational study!
Short Chain Fatty Acids not “Good” or “Bad”
Timing/Amount/Genetic Sensitivity Critical
 Summary
Autism is a complex problem needing a multi-disciplinary approach
Considerable overlap between autism and seizure disorder!

with modern brain research techniques, much is available to rationally

examine autism as a defined brain disorder

detailed study of behaviour/ EEG in animal models necessary to

determine cause and develop rational clinical studies and treatment

Factors in brain development- neural migration, embryonic cell death

toxic environmental compounds (dietary and enteric fatty acids)
role of diet and gut bacteria (antibiotic exposure)

Gut metabolites can alter brain electrical activity, behaviour,

Pathology, gene inductions and cellular metabolism

Variable exposure/breakdown in humans/microflorae/antibiotics

 Acknowledgements- Kilee Patchell- Evans Autism Research Group

Klaus-Peter Ossenkopp, Ph.D., Donald Peter Cain, Ph.D., Martin Kavaliers, Ph.D.,
(Department of Psychology (Neuroscience)- University of Western Ontario)
Fred Possmayer PhD. (Biochemistry, Obstetrics/Gynecology)
Lisa Tichenoff, Kelly Foley MSc., Roy Taylor, Francis Boon, Soelaha Shams ,Melissa
Gordon, Andrew Franklin MSc., Jennifer Hoffman MSc., Jennifer Martins MSc., Karina
Rodriguez-Capote, PhD, Yalda Mohammad-Asef

Heng Yong-Yi Ph.D and Leo Lau Ph.D (Surface Science Western- The University of
Western Ontario)

Jeanette Holden, Ph.D (Queens University)

Martha Herbert, M.D. Ph.D, (Harvard University)

Edmund F. La Gamma, M.D. , Bistra Nankova, Ph.D

(Westchester Medical Center - New York Medical College)

Valerie Hu, Ph.D (George Washington University)

Tim Delorey, Ph.D (Mountainview Research Institute)

Sidney Finegold, M.D. (UCLA)

AUTISM- HOPE FOR THE FUTURE