Chemical-Induced Brain Injury

1. Chemical-Induced Brain Injury Mohamed B. Abou-Donia, Ph.D. Department of Pharmacology and Cancer Biology Duke University Medical Center Durham, North Carolina 27710 donia@duke.edu

2. Brain Injury • Little is known about the etiology of many brain diseases, such as Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis (ALS), or Autism. • Chemicals can cause brain injuries that resemble brain diseases, for example: a. Manganese causes brain injury similar to that of Parkinson’ disease b. Organophosphate-Induced Delayed Neurotoxicity (OPIDN) has been misdiagnosed as Multiple Sclerosis.

3. The Brain The brain contains approximately 200 billion cells (neurons) and a trillion supporting cells. Brain neurons are all formed before birth, and no new neuronal cells are born after birth.

4. The Brain The brain requires one fifth of the oxygen and energy (glucose) consumed by the body to maintain its function. The hourly flow of blood through the brain is approximately 13 gallons, which accounts for one fifth of the blood pumped by the heart.

5. Brain Regions The brain is divided into three sections: a.Forebrain: cerebrum, limbic system (amygdala,hippocampus, septum), thalamus, hypothalamus. b. Midbrain c.Hindbrain: Pons, medulla oblongata, cerebellum

6. Cerebral Cortex • Language • Vision • Higher-order processing • Movement

7. Cerebral Cortex II • Corpus striatum • Degenerates in Parkinson’s • Paralysis • Loss of sensory input • Loss of reasoning, judgment, memory, etc

8. Limbic System • Emotion • Memory • Consolidation • Storage • Working memory • Movement

9. Limbic System • Emotion • Memory • Consolidation • Storage • Working memory • Movement

10. Cerebellum • Motor learning • Posture • Gait

11. Brain Supporting Cells Supporting cells continue to divide throughout life. A. Glial Cells: 1. Astocytes or astroglia: provide structural (Blood Brain Barrier) and nutritive support and form “glial” scar after injury. 2. Oligodendrocytes or oligodedroglia: form myelin sheath of axons in the CNS. 3. Microglia: Activated after injury and act as scavengers taking up debris. B. Endothelial Cells: Form the walls of brain capillaries and the BBB.

12. Blood brain barrier Blood Vessel Blood brain barrier

13. Fenestra General capillary Brain capillary Intercellularclef Endothelialcell Pinocytotic vesicles Mitochondria Endothelialcell Astroglial process Pericyte Basement membrane Tight junction

14. The Blood Brain Barrier (BBB) Is formed by brain capillary walls endothelial cells: 1. Tight junctions 2. No fenestras 3. Few pinocytotic vesicles in the cytoplasm (BChE) 4. Increased mitochondria (active transport) 5. Basement membrane (AChE) 6. Astrocytic feet ensheathe 95% of endothelial cells 7.Pericytes with smooth muscle-like properties 8. P-glycoprotein (P-gp) to remove undesired substances.

15. Nerve Cell Processes

16. Nervous System-Specific Proteins 1. Axons a. Neurofilament proteins (NFP) b. Tau proteins c. Tubulin 2. Dendrites Microtubule Associated Proteins-2 (MAP-2) 3. Myelin Myelin basic protein (MBP) 4. Astrocytes a. Glial Fibrillary Acidic Proteins (GFAP) b. S-100 protein

17. The Cytoskeleton • The cytoskeleton in the neuron consists of straight and parallel: a. 25-nm microtubules (α- and β-tublin) b. 10-nm neurofilaments c. Microtubule associated proteins (MAP-2 and Tau) that function as cross-bridges to link microtubules and neurofilaments. 2. The cytoskeleton gives the cell its shape and transport material within and outside the cell.

18. Neurofilament Proteins Neurofilaments consist of 3 polypeptides: • A 200-k Da, outer or high molecular weight protein (NFH), • A 160-k Da, middle or medium molecular weight protein (NFM), and • A 70-k Da, core and low-molecular weight protein (NFL). NFH is peripherally located, and especially vulnerable to injury and is an early marker to neuronal damage.

19. Tau Proteins Tau Proteins are: • Present almost exclusively in the axon. • Involved in microtubule assembly and stabilization.

20. Tubulin • Tubulin is present in all cells 2. It is present at high level in the brain where it comprise approximately 10% of brain protein. 3. Testes have high contents of tubulin

21. MAP-2 Microtubule Associated Proteins-2 (MAP-2): • Are found exclusively in the somato-dendrites of the neurons • They promote polymerization and stabilization of microtubules.

22. Myelin Basic Protein (MBP) MBP is a major constituent of the myelin that is formed by: 1.The supporting cells, oligodendrocytes in the central nervous system and 2. Schwann cells in the peripheral system.

23. Astrocytic Proteins Astrocytes form the following proteins: 1. Glial Fibrillary Acidic Protein (GFAP) is secreted following axonal injury to form gliotic scar. 2. S-100 is a calcium binding proteins that is formed in response to acute brain injury such as brain infarction and has been used to assess ischemic brain damage.

24. Autoantibodies against Brain Specific Proteins • Normally, small amounts of brain-specific proteins may leak into the circulation, where they react with B lymphocytes to form autoantibodies that are reactive against these proreins. • Autoantibodies are increased with age. • Damage to neuronal and glial cells in the brain or of the blood brain barrier (BBB), causes more leakage of these proteins into blood stream, with subsequent increased formation of the autoantibodies against them.

25. Brain-Specific Protein Autoantibodies: Biomarkers for Neurological Diseases Brain-Protein protein autoantibodies have been detected in the sera of patients with: • Alzheimer’s Disease • Parkinson’s Disease • Myasthenia Gravis • Multiple Sclerosis • Kuru Disease • Creutzfeldt-Jacob Disease, and • Picks Disease • Down Syndrome

26. Chemical-Induced Axonal Degeneration and Gliosis in Animals Axonal and gliosis are induced in animals by: • Organophosphates (Abou-Donia, 1982) • n-Hexane, MBK (Lapadula et al, 1988) • Carbon disulfide (Wilmarth et al., 1993) • Acrylamide (Reagan et al., 1994) • Glycidamide (Reagan et al., 1995)

27. Mechanisms of Neurotoxicity • Nonspecific: lack of oxygen (hypoxia) • Selective: Chemicals can target: • Nucleus • Axon • Myelin • Synapse

28. Hypoxia • Anoxic • Respiratory paralysis • Failure of blood to carry oxygen hemoglobin • Ischemic • Cardiac arrest • Hypotension (vasodilation) • Hemorrhage/thrombosis • Carbon monoxide • Cytotoxic • Cytochrome oxidase inhibition • Metabolic inhibition • Repeated hypoxia (e.g., TIA)

29. Selective neurotoxins • Cell body • Synaptic • Axon • Central-peripheral proximal axonopathy • Central-peripheral distal axonopathy • Neurofilamentous • Tubulovesicular • Conduction

30. Chemical-Induced Neurodegeneration The following chemicals caused neuronal degeneration in the cerebral cortex, hippocampus, and cerebellum of brain of exposed rats. 1. Organophosphorus Compounds: Sarin, Malathion, Chlorpyrifos, TOCP, TmCP, TpCP. 2. Pyrethroids: Permetrhrin

31. Increased Autoantibodies Against Brain-Specific Proteins 1. Axonal Degeneration Neurofilament proteins, Tau, tubulin 2. Demyelination Myelin Basic Protein (MBP) 3. Dendrite Degeneration MAP-2 4. Astrogliosis GFAP 5. Acute Brain injury S-100

32. Consequences of Axonal Degeneration Increased autoantibodies against neurofilaments, tau, tubulin or/and MBP indicate axonal degeneration. Degeneration in the cerebral cortex leads to: 1. Motor and sensory abnormalities, 2. Ataxia, 3. Deficit in posture, locomotion, and skilled movements 4. Fine motor movements (fingers, speech, facial expression, etc., 5. Weakness

33. Consequences of Axonal Degeneration Axonal degeneration of the limbic system including the hippocampus leads to: • Learning and memory deficits 2. Neurobehavioral (mood, emotion and judgment) abnormalities

34. Consequences of Axonal Degeneration Increased autoantibodies against MAP-2 suggests damage to the dendrite-rich Purkinje cells in the cerebellum resulting in: 1. Gait and coordination abnormalities 2. Staggering gate 3. Ataxia

35. Consequences of Gliosis Increased autoantibodies against GFAP suggests: • Axonal injury (forms scar). • Neuropsychiatric disorder.

36. Consequences of Gliosis Increased autoantibodies against S-100 suggest: 1. Traumatic brain damage 2. Acute phases of brain injury such as brain infarction, and 3. It as been used to assess ischemic brain damage. 4. Can help to differentiate between acute and chronic brain injury.

37. Hypothesis: Increased Autoantibodies Against Brain Specific Protein Hypothesis: • Following neuronal injury, neuron- and glia-specific proteins are released into circulation. • Released necrotic neuronal and glial elements accumulate and stimulate B lymphocytes to produce autoantibodies that are reactive against these proteins. • Increased autoantibodies against brain-specific proteins in the serum are indicative of neuronal damage.

38. Significance Antibody test that could detect ongoing or at-risk status of neurodegenerative diseases would be desirable, because: 1. Antibodies are extremely sensitive and specific measure 2. They can amplify the signal of an altered biological environment.

39. Specific Aim To correlate neurological deficits in persons following chemical exposure with sera levels of autoantibodies against brain neuronal and glial specific proteins.

40. Methods • Patients. Individuals exposed to pesticides, industrial chemicals, and flight cabin fumes. 2. Sera from the patients and healthy controls were obtained. 3. Western Blotting. Standard brain-specific proteins are separated on SDS –PAGE. 4. Proteins were transferred into PVDF membranes. 5. Membranes were incubated with serum samples at 1:50 dilution. 6. After washings, the membranes were incubated with horseradish peroxidase-conjugated goat anti-human IgG. 7. The membranes were developed by enhanced chemiluminescence. 8. The signal intensity was quantified. 9. The results were normalized to sera albumin.

41. Results • We have confirmed the strong association between levels of autoantibodies against brain-specific proteins and chemical-induced neurological deficits. • Increased autoantibodies were more frequent among the patients than the controls. • The results indicate axonal and dendrite degeneration followed by demyelination of brain neurons.

42. Conclusions In the absence of neurological diseases, while not diagnostic for a specific illness, the presence of increased circulating autoantibodies against neuronal and glial proteins is consistent with, and can be used as further confirmation for chemical-induced injury.