Amyotrophic Lateral Sclerosis

see also Motor Neuron Disease

• ALS (also called Lou Gehrig’s disease) a rapidly progressive and fatal neurodegenerative disease.
• Most common type of motor neuron (MN) disease.
• Characterised by gradual degeneration and death of MNs.
• Most people who develop ALS are 40-70 years old.
• The average age for the onset of ALS is 55.
• ALS occurs throughout the world with no racial, ethnic, or socioeconomic boundaries.

ALS can be classified as

• Sporadic: most common form.  Random and no known cause
• Familial: Inherited form (rarer occuring in ~5%)
• Guamanian: An extremely high incidence of ALS was observed in Guam in the 1950s

Symptoms

Usually begins with a weakness of one limb, which progresses to two/more limbs.  Can also present as dysarthria/dysphagia.

As it progresses, symptoms can include twitching, cramping, partial/complete paralysis, falls/instability, lack of fine motor control/coordination, persistent fatigue etc.  Eventually, ALS will effect the respiratory system and cause difficulty breathing.  Combined with swallowing difficulties, pneumoniae/choking is the most common cause of death.

Pathology

The main feature of ALS is degeneration of the upper and lower motor neurons in the motor cortex, brain stem and spinal cord.  Proteinous inclusions are found in both motor neurons and astrocytes in human ALS.

Familial ALS

Molecular Genetics of Huntington’s Disease

Huntington’s disease is caused by an autosomal dominant mutation in the gene encoding for huntingtin protein (htt).

NB KO htt is inviable- so it is of some importance, but we still do not know what this is.

The genetic defect is an expansion of a CAG repeat pattern found in the first exon of the gene.  The normal gene carries around 30 CAG repeats.  Individuals with HD typically have around 40 (the threshold is usually 36/7, but can range to >100).

The age of onset is typically affected by the number of CAG repeats.  Those with 36/7 will usually develop HD in their old age, whereas those with 100 can develop HD in their twenties.

Note that not all indivuals with 37 CAG repeats will develop HD, and patients with 35 can.  This, combined with age, results in a picture of age-dependent, incomplete penetrance.

Expanded CAG repeats are unstable

In normal alleles, CAG repeat changes occur in less than 1% of offspring (i.e. Mendelian inheritance).  In HD alleles, CAG repeat changes occur in 70% of offspring, 73% of which are expansions.  This causes anticipation, that is for HD to occur at an earlier age in offspring (it is technically possible for one generation to be diagnosed before the parent).  Large expansions (>7 CAG) are more common in paternal transmission.

HD mutations are probably gain of function/dominant-negative

Dominant – negative mutations are those in which the mutated protein interferes with the normal WT protein to cause >50% loss of function.

This is probably the case given that transgenic mice which have two WT alleles and a HD allele (i.e. triploid) display features of HD.  Also, HD/- mice (monoploid) are normal.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a gene in which the number of CAG repeats can be manipulated and has been used as an alternative model for HD.  In mice with polyQ (CAG repeats) HPRT, they show neurological deficit(despite HPRT not known to be involved with any neurological CAG disorders).

Pathology and Neurobiology of Huntington’s Disease

The largest changes in the brain of HD patients are seen in the basal ganglia, particularly the striatal medium spiny projection neurons.

Striatonigral Pathway and Dopamine

Dopamine at D2 receptors disinhibit the indirect pathway and promote choreic symptoms.  Suppressed by drugs that deplete dopamine.

Dopamine at D1 receptors disinhibit the direct pathway, promoting dystonia and rigidity.

In huntingtons, dopamine receptors eventually deplete (D2 before D1).

ALS Therapies

Riluzole

The only approved drug for ALS, riluzole extends survival by 2-3 months but does not have any lasting protection and is certainly by no means curative.  It’s acute effects (animal models) include:

• decreasing persistent V-gated Na currents
• potentiation of calcium-dependent K currents
• inhibiting glutamate release

which all act to decrease excitotoxicity in motor neurons.

Novel Targets

1. Glutamate Transport

Ceftrixone is an antibiotic that increases glial mediated glutamate transport by stimulating expression of EAAT2.  In animal models, it prolongs life and increases EAAT2 mRNA levels.  It is currently in trials for therapeutic use.

2. Protein Misfolding and Aggregation

Heat shock proteins are involved in protein folding and degradation.  Abnormalities in HSPs promote motor neuron degeneration in ALS.  Arimoclomol is an oral drug that increases expression of HSPs involved with neuroprotective mechanisms, and delays progression and extends life in mouse models (SOD1 mutants).  It is also thought to be able to penetrate the BBB.  Clinical trials are also underway.

3. Mitochondrial Targets

Agents that improve mitochondral function (creatins) have beneficial effects in SOD1 mutant mice, but have been disappointing in trials.

Olesoxime is a mitochondrial pore modulator which also delays disease progression and prolongs survival in models.  Trials are underway.

Dexpramipexole lowers oxidative stress and maintains mitochondrial function (also extends life in models).  While it is known to be safe in humans, trials specific for ALS are underway.

Stem Cell Therapy

In theory, stem cells could be implanted to replace dying motor neurons.  However, because of the rapid progression of ALS, even if this does work, it might not work in time.

A more likely approach is to use stem cells to differentiate into non-neuronal cells that will release neuroprotective factors.

Muscle Targets

ACE-031 is a protein that inhibits negative regulators (e.g. myostatin) of muscle growth, promoting lean muscle growth and strength.  Subcutaneous treatment with ACE-031 is well tolerated in humans.

CK-2017357 activates fast skeletal muscle troponin complex by increasing its sensitivity to calcium, and thus increasing muscle force.  It is being tested in clinical trials (where it has been shown to be both effective and safe at increasing pulmonary function so far).

RNA targets

Using antisense oligonucleotides and small inhibitory RNAs to inhibit mutant SOD1 in familial ALS has therapeutic potential.  It has been shown to slow disease progress and increase survival in mouse models, and clinical trials are underway.  However, this approach will NOT help the majority of patients with the sporadic form of ALS.

Molecular mechanisms in ALS

Oxidative Damage

Mutations in SOD1 cause structural changes in SOD1 that expose the Copper site to aberrant (different from normal) substrates, particularly hydrogen peroxide (to form hydroxyl radicals), and will also promote the copper-mediated catalysis of peroxynitrite damage to intracellular proteins (via nitration of tyrosine residues of target proteins- signalling cell death).

Protein Aggregation

Abnormal Protein aggregation is a common feature of many neurodegenerative disorders including ALS.  In ALS, aggregates are primarily found in motor neurons and glial cells.  Several studies show SOD1 as a component of these aggregates and it has been shown that SOD1 has an increased propensity for aggregation.

Mitochondrial Dysfunction

Mitochondria show signs of swelling and dysfunction in early ALS.  In sporadic ALS, defects of the electron transport chain have been found.  Mutant SOD1 has also been found to directly disrupt mitochondrila function (SOD1 aggregates are found in the mitochondria too), possibly through disruption of the association between cytochrome C and the inner mitochondrial membrane.  Mitochondrial dysfunction ultimately leads to the production of ROS and cell damage/DNA damage.

Excitotoxicity

In motor neurons, most glutamate is cleared from the synapse by the glial glutamate transporter, EAAT2 (also known as GLT1).  In sporadic ALS, glutamate levels are increased in the CSF, suggesting problems with glutamate handling.  Glutamate transport is also markedly reduced, due to loss of EAAT2 (shown by reduced EAAT2 protein levels in mutant SOD1 mouse models) and selective inactivation of EAAT2 by mutant SOD1.

GluR2 and ALS

In most cells, AMPARs are tetramers of at least one GluR2 subunit and have low Ca permeability.  The levels of mRNA for GluR2 in motor neurons is lower than that of other neurons.  Lack of GluR2 accelerates motor neuron degeration and life span in SOD1 mutant mice and replacement of GluR2 will increase life span.

Growth Factor Deficiency

This is one of the possible non-SOD1 related pathophysiological mechanisms behind ALS.  Targeted deletion of part of the VEGF gene causes motor pathology and features of ALS pathology (possible role of VEGF in ALS).  This is reinforced by the fact that progression of motor neuron disease is delayed in SOD1 mutant mice after:

• Overexpression of VEGF
• ICV administration of VEGF
• IM delivery of VEGF-expressing lentiviral vectors

Other growth factors (IGF-1 and GDNF (Glial cell derived neurotrophic factor)) also have similar effects.

Role of Glial Cells

Glial cells seem to contribute to disease progression.  Astrocytic inclusions are an early indicator of SOD1 mutant toxicity.  Also, astrocytes expressing mutant SOD1 secrete factors that are toxic to motor neurons, e.g. proinflammatory cytokines and chemokines (e.g. TNF-alpha).

Defective Axonal Transport

Neurofilaments are the most common cytoskeletal protein in motor neurons and play a key role in axonal growth.  Abnormal accumulation of neurofilaments in the soma and axons are hallmarks of ALS.  Transgenic mice with mutations or overexpression of neurofilaments display motor neuron dysfunction.  Mutant SOD1 mice have defective axonal transport.

Why is this selective for motor neurons?

One possibility is that motor neurons are more susceptible to excitotoxicity.

• Spinal motor neurons receive very strong glutaminergic input
• They express Ca(2+) permeable AMPA receptors
• They have low Ca(2+) buffering capacity

Familial ALS

Accounts for 5-10% of cases of ALS.

20% of familial cases are caused by a dominantly inherited mutation in the protein Cu/Zn Superoxide dismutase (SOD1).  The ala-to-val substitution (A4V) is the most common mutation and results in aggressive disease (mean survival 1yr post-diagnosis).  Mutant SOD1 is also a key component of protein aggregates in ALS.

SOD1 converts superoxide radicals to hydrogen peroxide and oxygen.

SOD1 is a metalloprotein (proteins with a metal ion co-factor) and is a key enzyme involved in anti-oxidant defence mechanisms.  It is found in the cytosol, nucleus and mitochondrial membrane.

More than 140 mutations in the SOD1 gene have been identified (confirmed in transgenic animal models- which have strengthened our understanding of SOD1 and ALS).  Early research has shown that the mechanism of mutated SOD1 is NOT via a loss of function/enzyme activity (SOD1 KO mice do not develop ALS).  Therefore, mutations cause SOD1 to gain a toxic property.

NMDAR activation and cell death

Mitochondrial Dysfunction

The mitochondrial membrane is depolarised by Ca(2+) uptake, which inhibits ATP production, and can even deplete cytosolic ATP levels due to reversal of the mitochondrial ATPase.  Loss of ATP reduces the cell’s ability to regulate intracellular calcium levels further, worsening the situation.  Crucially, mitochondrial calcium promotes the production of ROS, causing further mitochondrial damage and cellular damage.

Calpain Activation

Excessive calcium influx also impairs calcium efflux mechanisms via PMCA (plasma membrane calcium ATPase) and NCXs (Na/Ca exchangers).

Calpains are calcium dependent proteases that are activated by excessive NMDAr calcium influx.  They cleave the major NCX3 isoform, impairing its function, and play a role in inactivating PMCA following excitotoxic insult.

Stress-activated Protein Kinases

P38 MAPK and JNK (SAPK family members) contribute to cell death via NMDAR activation too.

• In cerebellar and cortical neurons, NMDAR dependent P38 MAPK activation involves nNOS (neuronal nitric oxide synthase)

Neuroprotective mechanism of NMDAR activation

PI3-Kinase Cascade

The PI3-Kinase/Akt pathway is activated by NMDARs in many neurons.

Akt goes onto inhibit/downregulate glycogen synthase Kinase 3 beta (GSK3-beta) (which plays a role in apoptotic signalling pathways), Bcl-2 (Beta-cell lymphoma-2) associated death promotor (BAD) (also involved in apoptosis) and p53, which is known to be involved in a number of pro-apoptotic pathways including Bcl-2 associated X-protein (BAX).

• In laymens, it prevents the combining of BAD/BAX with other proteins so they cannot form the active complexes.

Antioxidant defences

The balance of reactive oxygen species (ROS) production and neutralisation is important to protect against cellular damage and death.  NMDAR activity seems to play a role in the regulation of a cell’s vulnerability to oxidative stress: neurons with higher NMDAR activity withstand oxidative damage more than electrically quiet neurons.  Conversely, if NMDAR activity is blocked, cells become highly vulnerable to oxidative damage.

The proposed mechanism behind this is that synaptic activity exerts changes in the thioredoxin-peroxiredoxin system. (Thioredoxin reduces (chemically) hyperoxidised peroxiredoxins (antioxidant enzymes)- freeing them to reduce ROS.)  Synaptic activity also promotes a series of gene-expression changes that boost anti-oxidant defenses e.g. inhibition of thioredoxin inhibitor TXNIP (a FOXO target gene).

Mechanisms of Neuronal Death

Apoptosis

Mild excitotoxic insult allow NMDAR activation by ambient concentrations of glutamate. This leads to increased mitochondrial [Ca2+] and free radical production, yet relatively preserved ATP generation. Consequently, the mitochondria can release cytochrome c (Cytc), caspase 9, apoptosis inducing factor (AIF), and other mediators that lead to apoptosis.

Necrosis

After severe insults e.g. ischaemia, NMDAR activation is enhanced resulting in increased intracellular calcium levels.  This activates nitric oxide synthase (NOS), which increases mitochondrial Ca(2+) and superoxide generation followed by formation of peroxynitrite (ONOO-).  This free radical causes DNA and cellular damage, leasing to the activation of poly-ADP-ribose polymerase (PARS).  Mitochondrial calcium accumulation and oxidative damage lead to activation of the permeability transition pore (PTP) that is linked to excitotoxic cell death.

NMDAR Neuroprotection vs Excitotoxicity

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2837198/

NMDAR blockade and over excitation both seem to cause cell death.

• NMDAR blockade in vivo reduces neuron viability and elimination causes apoptosis of developing neurons (all post-natally)
• In adult CNS, NMDAR blockade enhances neuronal loss
• Physiological levels of NMDAR activity are required for neuronal survival, synaptic plasticity and neuronal development.

The response to NMDAR activity can be shown like this

I.e.

• Pro-survival actions of NMDAR activity are mediated by synaptic NMDARs
• When extracellular glutamate levels are chronically elevated, extrasynaptic NMDAR (and to a lesser extent, synaptic NMDAR) activation is coupled with pro-death signalling pathways that shut down CREB (cAMP response element binding protein) function and triggers mitochondrial depolarisation (i.e. loss of proton gradient and no ATP production)

Conclusion- What determines if NMDAR activity is neuroprotective or excitotoxic?

• Stimulus Intensity
  • The magnitude of NMDAR activation (i.e. intensity or duration)
    • Modest/physiological NMDAR activity promotes neuroprotection
    • Too little or too much promotes cell death
  • The implication is that pro-survival signalling requires lower calcium levels than cell death pathways.
• NMDAR Locus
  • Synaptic
    • Ca influx via sNMDARs is well tolerated
    • Activates ERK/MAPK pathway (cell survival)
    • Activates CREB-dependent gene expression
    • activates the pro-survival PI3-kinase/Akt pathway
  • Extrasynaptic
    • Ca influx via eNMDARs triggers cell death
    • Induces ERK inactivation
    • causes CREB dephosphorylation
    • No activation of PI3-kinase/Akt pathway
• Subunit composition
  • NMDAR consists of one NR1, one or more NR2 (A/B/C/D) with or without one NR3 subunit
    • In the hippocampus, NR2A and NR2B predominate
    • 2A seems to be mainly synaptic and 2B extrasynaptic
    • However, whether 2B is more likely to cause cell death is still debated
    • http://www.jneurosci.org/content/27/11/2846.short (2A promotes survival)
    • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2635533/ (opposite)