Neurotoxicity:

Toxicology of the Nervous System

John J Woodward, PhDDepartment of NeurosciencesIOP471Nwoodward@musc.edu

www.people.musc.edu/~woodward

1930’s – Ginger-Jake Syndrome• During prohibition, an alcohol beverage was

contaminated with TOCP (triortho cresyl phosphate) causing paralysis in 5,000 with 20,000 to 100,000 affected.

1950’s – Mercury poisoning• Methylmercury in fish in Japan cause death and

severe nervous system damage in infants and adults (Minimata disease).

Historical Events

Central Nervous System (CNS)• Brain & Spinal Cord

Peripheral Nervous System (PNS)• Afferent (sensory) Nerves –

Carry sensory information to the CNS

• Efferent (motor) Nerves – Transmit information to muscles or glands

Cells of the Nervous System

Neurons• Signal integration/generation; direct control

of skeletal muscle (motor axons)

Supporting Cells (Glia cells)• Astrocytes (CNS – blood brain barrier)

• Oligodendrocytes (CNS – myelination)

• Schwann cells (PNS – myelination)

• Microglia (activated astrocytes)

Cellular Events in Neurodevelopment

Events: Division Migration Differentiation Neurogenesis Formation of synapses Myelination Apoptosis

Active throughout childhood & adolescence

Underlying Cellular Biology

6

Development of GABA and Glutamate Synapses in Primate Hippocampus

•GABA synapses develop on contact

•Glutamate synapses develop but require a developed spine to become active

•GDPs dominate early developmental neuronal activity and disappear prior to birth (primates) or during early neonatal life (rodents)

Neurons are post-mitotic cells

High dependence on oxygen

• Little anaerobic capacity

• Brief hypoxia/anoxia-neuron cell death

Dependence on glucose

• Sole energy source (no glycolysis)

• Brief disruption of blood flow-cell death

High metabolic rate

Many substances go directly to the brain via

inhalation

Why is the Brain Particularly Vulnerable to Injury?

Blood Supply to the Brain

Blood-brain Barrier

Anatomical Characteristics• Capillary endothelial cells are tightly joined – no pores

between cells• Capillaries in CNS surrounded by astrocytes• Active ATP-dependent transporter – moves chemicals

into the blood

Not an absolute barrier• Caffeine (small), nicotine• Methylmercury cysteine complex• Lipids (barbiturate drugs and alcohol)• Susceptible to various damages

BBB can be broken down by:

• Hypertension: high blood pressure opens the BBB

• Hyperosmolarity: high concentration of solutes can open the BBB.

• Infection: exposure to infectious agents can open the BBB.

• Trauma, Ischemia, Inflammation, Pressure: injury to the brain can open the BBB.

• Development: the BBB is not fully formed at birth.

What causes neurotoxicity?

Wide range of causes

Chemical

Physical

• Inhalation (e.g. solvents, nicotine, nerve gases)

• Ingestions (e.g. lead, alcohol, drugs such as MPTP)

• Skin (e.g. pesticides, nicotine)• Physical (e.g. load noise, trauma)

Toxicants and Exposure

NEURONS

CELL MEMBRANE AND MEMBRANE PROTEINSIon Channels

•Important for establishing resting membrane potetial

•Synaptic transmission/nerve conduction

Sodium channel

•Voltage-sensitive •Ligand-gated

Types of Neurotoxic Injury

Neuron

Normal

Neuronopathy

Axonopathy

Myelinopathy

Transmission

Axon

Synapse

Myelin

Types Of Neurotoxicity

Neuronopathy• Cell Death. Irreversible – cells not replaced.• MPTP, Trimethyltin

Axonopathy • Degeneration of axon. May be reversible.• Hexane, Acrylamide, physical trauma

Myelinopathy• Damage to myelin (e.g. Schwann cells)• Lead, Hexachlorophene

Transmission Toxicity• Disruption of neurotransmission, toxins, heavy

metals, organophosphate pesticides, DDT, drugs (eg., cocaine, amphetamine, alcohol)

Ion Channels are Targets for a Variety of Toxins, Chemicals and Therapeutic Compounds

Natural ToxinsSnake, insect,plant toxins

(cobra venom, scorpion, curare)

Environmental ChemicalsHeavy metals, industrial solvents

(lead, benzene, aromatic hydrocarbons)

Therapeutic DrugsAnesthetics, Benzodiazepines(lidocaine, halothane, valium)

Drugs of abuse(Ketamine, alcohol, inhalants)

Neurotoxicology

Heavy Metals

Lead – environmental exposure (paint, fuels)

Mercury – exposure via diet (bioaccumulation in fish)

Historical Sources of Lead Exposure

Ancient/Premodern History

• Lead oxide as a sweetening agent

• Lead pipes (“plumbing”)

• Ceramics• Smelting and

foundries

Modern History• Gasoline (leaded)• Ceramics• Crystal glass• Soldering

– pipes– “tin” cans– car radiators

• House paint

Nervous Systems Effects Developmental Neurotoxicity Reduced IQ Impaired learning and memory Life-long effects Related to effects on calcium permeable

channels (NMDA, Ca++ channels)

Lead Neurotoxicity

Mechanisms of Damage to the Nervous System by Lead

Central• Cerebral edema• Apoptosis of neuronal cells• Necrosis of brain tissue• Glial proliferation around blood vessels

Peripheral• Demyelination• Reversible changes in nerve conduction velocity (NCV)• Irreversible axonal degeneration

• Natural Degassing of the earth• Combustion of fossil fuel• Industrial Discharges and Wastes• Incineration & Crematories• Dental amalgams• CF bulbs

Environmental Sources of Mercury

Toxicity of Mercury

• Different chemical forms – inorganic, metallic, organic (

• Organic mercury (methylmercury) is the form in fish; bioaccumulates to high levels

• Organic mercury from fish is the most significant source of human exposure

• Brain and nervous system toxicity– High fetal exposures: mental retardation, seizures, blindness– Low fetal exposures: memory, attention, language

disturbances

Hg0 Hg2+ CH3Hg+)

MeHg Consumption Limits

US EPA – 0.1 ug/kg-day

US FDA – 1 ppm (mg/kg) in tuna

Consuming large species such as tuna and swordfish even once a week may be linked to fatigue, headaches, inability to concentrate and hair loss, all symptoms of low-level mercury poisoning. In a study of 123 fish-loving subjects, the researchers found that 89% had blood levels of methylmercury that exceeded the EPA standard by as much as 10 times.

How Much Tuna Can You Eat Each Week? A safe level would be approximately 1oz for every 20lb of body weight. So for a 125lb (57kg) person, 1 can of tuna a week maximum.

Excitotoxicity-Glutamate Mediated Cell DeathExcitotoxicity-Glutamate Mediated Cell Death

Experimental ObservationsExperimental Observations

Glutamate induces a delayed cell death in neuronsGlutamate induces a delayed cell death in neurons

This cell death requires extracellular calcium and is blocked by This cell death requires extracellular calcium and is blocked by antagonists of NMDA receptorsantagonists of NMDA receptors

Hypothesis: Prolonged or inappropriate activation of NMDA Hypothesis: Prolonged or inappropriate activation of NMDA receptors underlies glutamate excitotoxicity of neurons receptors underlies glutamate excitotoxicity of neurons

Glutamate Synapses

Excitatory synapse of brain

Required to generate action potentials

Both AMPA and NMDA receptors are critical for normal brain function

NMDA-hi Ca++ permeabilityGlutamate synapse

Overview of Glutamate and ExcitotoxicityOverview of Glutamate and Excitotoxicity

Glutamate activates two types of ion channels (AMPA and NMDA)

Cell Death is associated with excessive calcium entry through NMDA receptors

Both Native and Recombinant NMDA Receptors Can Both Native and Recombinant NMDA Receptors Can Cause Excitotoxicity Cause Excitotoxicity

NeuronsNeurons

Transfected CHO cellsTransfected CHO cells

NMDA-induced Excitotoxicity is NR2 Subunit Dependent in NMDA-induced Excitotoxicity is NR2 Subunit Dependent in Recombinant Expression SystemsRecombinant Expression Systems

NMDARS require two NR1 subunits and two NR2 subunits

-NR2 family-NR2A, 2B, 2C, 2D

-NR2A, NR2B high excitotoxicity potential

-NR2C, NR2D lower excitotoxicity potential

Calcium and ExcitotoxicityCalcium and Excitotoxicity

Glutamate-mediated apotosis in spinal motor neurons Glutamate-mediated apotosis in spinal motor neurons is blocked by calpain inhibitorsis blocked by calpain inhibitors

Expose cells to 10 µM Glu in absence or presence of calpeptinExpose cells to 10 µM Glu in absence or presence of calpeptin

Monitor apoptosis (left panel) or membrane potential (right panel)Monitor apoptosis (left panel) or membrane potential (right panel)

The Calcium That Triggers Excitotoxicity is Source-DependentThe Calcium That Triggers Excitotoxicity is Source-Dependent

Calcium entry via NMDA Calcium entry via NMDA receptors can trigger neuronal receptors can trigger neuronal cell deathcell death

Calcium entry through other Calcium entry through other channels (eg. VSCC) does notchannels (eg. VSCC) does not

Location of NMDA receptors is Location of NMDA receptors is also important, synaptic versus also important, synaptic versus extrasynapticextrasynaptic

Synaptic and non-synaptic Synaptic and non-synaptic NMDA Receptors Increase NMDA Receptors Increase CalciumCalcium

L-type calcium channel L-type calcium channel increase calciumincrease calcium

Synaptic NMDA receptors Synaptic NMDA receptors and L-type channels do no and L-type channels do no affect mitochondrial affect mitochondrial functionfunction

Extrasynaptic NMDA Extrasynaptic NMDA receptors disrupt receptors disrupt mitochondrial function and mitochondrial function and are linked to excitotoxicityare linked to excitotoxicity

Mitochondrial Dysfunction Mitochondrial Dysfunction Resulting from Calcium Resulting from Calcium

Overload is Source-SpecificOverload is Source-Specific

Calcium Mito Vm

Glutamate Excitoxicity in OligodendrocytesGlutamate Excitoxicity in Oligodendrocytes

Historically, oligos were thought to lack Historically, oligos were thought to lack NMDA receptorsNMDA receptors

More recent studies demonstrate More recent studies demonstrate NMDA and non-NMDA currents in NMDA and non-NMDA currents in oligosoligos

These receptors may be activated by These receptors may be activated by injury or ischemic conditions that result injury or ischemic conditions that result in the release of glutamatein the release of glutamate

Loss of oligo processes may underlie Loss of oligo processes may underlie myelin degeneration associated with myelin degeneration associated with many diseases such as cerebral palsy, many diseases such as cerebral palsy, spinal cord injury and multiple sclerosisspinal cord injury and multiple sclerosis

Glutamate Excitoxicity in OligodendrocytesGlutamate Excitoxicity in Oligodendrocytes

Oxygen-glucose Oxygen-glucose deprivation (OGD)-deprivation (OGD)-model of ischemic model of ischemic damagedamage

Leads to loss of oligo Leads to loss of oligo processesprocesses

This is prevented by This is prevented by blockers of NMDA blockers of NMDA receptors (MK801)receptors (MK801)

Glutamate and Human Brain TraumaGlutamate and Human Brain Trauma

Glutamate in Human Brain Following StrokeGlutamate in Human Brain Following Stroke

Glutamate

Threonine

Glutamate levels remain high after stroke

Threonine, a structural amino acid, is measured as a control