Enteric and Dietary Short Chain Fatty Acids Possible Triggers of Autism Associated Epilepsy and Movement Disorder
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Enteric and Dietary Short Chain Fatty Acids
b
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, methylationMethyl 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
hyperreligiousit y
-hypergraphia -number fixation - preoccupation with detail
?hyperinhibition
Temporal lobectomy for intractable seizure
Activated Microglia in Epilepsy
Kainate mouse hippocampus
Human epileptic focusmesial temporal sclerosis
Found in experimental and human epilepsy and brain tumour- ?epileptogenic factors Similar to autism neuropathology
Nonepileptic tissue from same 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 Increased Neuronal Density Altered Cell Migration Seizure Disorder Hormonal Sex Hormones Oxytocin Vasopressin
Systemic Changes Immune System Gastrointestinal System Metabolic Disorder Detoxification Systems (glutathione)
AUTISM
Genetic Factors Neurotransmitter Growth Factors Cell-cell Interaction Sex Linked (Fragile X) White Matter Disorder Glial/microglial Changes Neuroinflammation (Impaired Neurodevelopment and Cortico-cortical processing)
Environment Metals Hydrocarbons Infectious Drug (valproate) Diet- Wheat Casein Allergy 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
Mean Number of Snake Postures
15
Snake Posture
8
Mean Retropulsion Bouts
12
*
6
Baseline Treatment
9
*
+
4
6
2
3
0 Low Propionate High Propionate Propanol Sodium Acetate PBS
0 Low Propionate High Propionate Propanol Sodium Acetate PBS
50
Turning
40
Mean Turning
#
30
#
20
Legend * = Significantly different from all control groups. # = Significantly different from low PA, propanol, PBS. + = Significantly different from propanol and PBS
10
0 Low Propionate High Propionate Propanol Sodium Acetate PBS
Propionic acid causes movement disorder with caudate spiking
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
500
Number of Movements
6000
SAL PPAH
Mean Number of Movements
400
Mean Distance (cm)
5000
SAL PPAH
*
*
4000
300
*
200
*
*
3000
*
2000
* *
* *
100
1000
0 BL T1 T2 T3 T4 T5 T6 T7 D8
0 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 infectionsLong 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 PPA • Subcortical spiking with movement • 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
Enteric and Dietary Short Chain Fatty Acids
b
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, methylationMethyl 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
hyperreligiousit y
-hypergraphia -number fixation - preoccupation with detail
?hyperinhibition
Temporal lobectomy for intractable seizure
Activated Microglia in Epilepsy
Kainate mouse hippocampus
Human epileptic focusmesial temporal sclerosis
Found in experimental and human epilepsy and brain tumour- ?epileptogenic factors Similar to autism neuropathology
Nonepileptic tissue from same 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 Increased Neuronal Density Altered Cell Migration Seizure Disorder Hormonal Sex Hormones Oxytocin Vasopressin
Systemic Changes Immune System Gastrointestinal System Metabolic Disorder Detoxification Systems (glutathione)
AUTISM
Genetic Factors Neurotransmitter Growth Factors Cell-cell Interaction Sex Linked (Fragile X) White Matter Disorder Glial/microglial Changes Neuroinflammation (Impaired Neurodevelopment and Cortico-cortical processing)
Environment Metals Hydrocarbons Infectious Drug (valproate) Diet- Wheat Casein Allergy 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
Mean Number of Snake Postures
15
Snake Posture
8
Mean Retropulsion Bouts
12
*
6
Baseline Treatment
9
*
+
4
6
2
3
0 Low Propionate High Propionate Propanol Sodium Acetate PBS
0 Low Propionate High Propionate Propanol Sodium Acetate PBS
50
Turning
40
Mean Turning
#
30
#
20
Legend * = Significantly different from all control groups. # = Significantly different from low PA, propanol, PBS. + = Significantly different from propanol and PBS
10
0 Low Propionate High Propionate Propanol Sodium Acetate PBS
Propionic acid causes movement disorder with caudate spiking
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
500
Number of Movements
6000
SAL PPAH
Mean Number of Movements
400
Mean Distance (cm)
5000
SAL PPAH
*
*
4000
300
*
200
*
*
3000
*
2000
* *
* *
100
1000
0 BL T1 T2 T3 T4 T5 T6 T7 D8
0 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 infectionsLong 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 PPA • Subcortical spiking with movement • 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
