Acetylcholine as a Neurotransmitter (A*)

Question 1

Describe the process of acetylcholine release, reuptake and recycling.

Answer 1

1 - Ca2+ influx triggers exocytosis of acetylcholine-containing vesicles in the presynaptic terminal by activating the Ca2+ sensor synaptotagmin.

2 - The vesicle fuses with the membrane of the presynaptic terminal, releasing ACh into the synaptic cleft.

3 - ACh binds to ACh receptors, inducing a receptor potential.

4 - Acetylcholinesterase breaks down the ACh-receptor complex to produce choline and acetic acid.

5 - Choline reuptake back into the presynaptic terminal occurs via the Na+/Choline cotransporter.

6 - Choline Acetyltransferase catalyses the conversion of choline and acetyl CoA into acetylcholine.

7 - ACh transporters load ACh into vesicles.

Question 2

How does butyrylcholinesterase differ in function from acetylcholinesterase?

Answer 2

Butyrylcholinesterase differs from acetylcholinesterase in that:

1 - It is a nonspecific cholinesterase.

2 - It is located in the presynaptic terminal rather than in the synaptic cleft.

Question 3

List the categories of acetylcholine receptors.

Are these receptors ionotropic or metabotropic?

For each category, give an example of an agonist (not including acetylcholine) and an antagonist.

Answer 3

Acetylcholine receptors include:

1 - Nicotinic receptors.

• These are ionotropic receptors.
• Nicotine is an agonist of nicotinic receptors.
• Curare is an antagonist for nicotinic receptors.

2 - Muscarinic receptors.

• These are metabotropic receptors.
• Muscarine is an agonist for muscarinic receptors.
• Atropine is an antagonist for muscarinic receptors.

Question 4

Describe the structure of nicotinic acetylcholine receptors.

Answer 4

• Nicotinic AChRs are LGICs.
• Nicotinic AChRs have a pentameric structure (have 5 subunits).
• Receptor subtypes are on the basis of subunit composition:
• Subunits can be a combination of alpha, beta, gamma, delta and epsilon, and there are variants of each of these (e.g. alpha 1-9).
• The current naming system focuses on the alpha subunit variant.

Question 5

Which acetylcholine receptor subtype is used in the neuromuscular junction?

Answer 5

The neuromuscular junction uses alpha 1 nicotinic receptors (not to be confused with alpha 1 adrenoceptors!).

Question 6

What is the difference in the speed and duration of receptor potentials induced in nicotinic and muscarinic receptors?

Answer 6

• Nicotinic receptors mediate fast, short-lasting transmission of action potentials.
• Muscarinic receptors mediate slow, long-lasting transmission of action potentials.

Question 7

List the nicotinic receptor subtypes that are mostly located on presynaptic terminals.

What quality of these receptors make them ideal for being presynaptically located?

List 3 cellular functions of presynaptic nicotinic receptors.

Answer 7

• Alpha 7 and 9 nicotinic receptors are mostly located on presynaptic terminals.
• They have a high calcium permeability, making them ideal presynaptic receptors since they are able to induce Ca2+-mediated cell signalling pathways.

Their cellular functions include:

1 - Modulation of gene expression.

2 - Modulation of neurotransmitter release.

3 - Desensitisation.

Question 8

Describe the signalling cascades that occur in response to Ca2+ influx from presynaptic nicotinic acetylcholine receptors.

What cellular responses might result from these signalling cascades?

Answer 8

1 - The presynaptic nAChR opens, causing Ca2+ influx.

2 - Ca2+ influx results in:

• PKA activation, both directly and through cAMP activation.
• CaMK activation.
• PI3K activation, both directly and via JAK2 activation.

4 - PKA and CaMK activate ERK.

• ERK stimulates exocytosis and contributes to memory and addiction.

5 - PI3K activation results in:

• Increase in expression of cell surface NGF receptors. NGF receptors promote cell survival when bound to NGF.
• Activation of PKB. PKB promotes survival by upregulating antiapoptotic enzymes and downregulating proapoptotic enzymes. In the absence of this signalling mechanism, the cell dies -‘use it or lose it’. This depends on forming strong connections with other neuronal networks.

Question 9

Describe the ‘shortcut pathway’ of muscarinic acetylcholine receptor signalling.

What proportion of muscarinic signalling is accounted for by this pathway?

Answer 9

The shortcut pathway of muscarinic acetylcholine receptor signalling:

1 - An agonist binds to the muscarinic receptor.

2 - GTP phosphorylates the alpha subunit.

3 - The alpha subunit separates from the beta and gamma subunits.

4 - The beta and gamma subunits activate a nearby K+ channel, causing K+ influx.

• This pathway only accounts for the minority of muscarinic acetylcholine receptor signalling. Most muscarinic signalling is mediated by cascades of downstream signalling messengers.

Question 10

Describe the structure of muscarinic acetylcholine receptors.

Answer 10

• Muscarinic AChRs are metabotropic.
• Muscarinic AChRs have 7 transmembrane domains (like all GPCRs).
• Receptor subtypes include M1-5.
• All receptors have an intracellular loop between transmembrane segments 5 and 6, distinguishing them from other GPCRs
• This loop is involved in regulation of the receptor function.
• Different subtypes are coupled to different second messenger systems.

Question 11

What property of muscarinic receptor agonists makes studying muscarinic pharmacology particularly difficult?

Answer 11

• Studying muscarinic pharmacology is made notoriously difficult by the fact that there are few selective muscarinic receptor agonists.
• This is due to the similarity in structure / high sequence homology among the muscarinic receptor subtypes.
• This also means that muscarinic drugs have many side effects.

Question 12

List 2 recent approaches to studying muscarinic receptor pharmacology.

Answer 12

Recent approaches to studying muscarinic receptor pharmacology include:

1 - The use of (more selective) allosteric modulators of muscarinic AChRs.

2 - Using receptor knockouts.

Question 13

Which muscarinic receptor subtypes are involved in postsynaptic transmission?

List their effects on the postsynaptic cell.

Answer 13

• Postsynaptic excitatory: mostly M1 and M3 (but M1 is mixed pre- and postsynaptic):

1 - Generation of non-selective cation currents.

2 - Amplification of the postsynaptic cell’s responsiveness to other coincident excitatory inputs. This is known as heterosynaptic facilitation, and is particularly evident in some glutamatergic synapses.

3 - Promoting ‘high-frequency gamma oscillations’.

• Postsynaptic inhibitory: mostly M2 (but M2 is mixed pre- and postsynaptic).

Question 14

List 3 presynaptic muscarinic receptor subtypes.

List the regions of the brain in which these receptors are highly expressed.

Are these receptors facilitatory or inhibitory?

Answer 14

Presynaptic muscarinic receptor subtypes:

1 - Inhibitory M2 in the cortex (but M2 is mixed pre-and postsynaptic).

2 - Facilitatory M4 receptors in the cortex and on glutamatergic terminals in the hippocampus are an example of presynaptic muscarinic receptors.

3 - Facilitatory M1 receptors on dopaminergic neurones of the nigrostriatal pathway (but M1 are generally postsynaptic).

*Neither M2 nor M4 are exclusively presynaptic.

Question 15

Describe the distribution of nicotinic and muscarinic acetylcholine receptors in the brain.

Answer 15

• Both nicotinic and muscarinic acetylcholine receptors are widespread throughout the brain, however some areas are predominated by a particular type:
• Muscarinic receptors predominate in the brainstem (medulla, pons and midbrain).
• Nicotinic receptors predominate in the substantia nigra, locus coeruleus and septum.

Question 16

List 4 general functions of cholinergic transmission in the CNS.

Answer 16

Cholinergic transmission in the CNS contributes to:

1 - Arousal (observed as EEG synchronisation).

2 - Sleep-wake cycle.

3 - Learning and memory (as well as glutamatergic transmission, as discussed in the y2 memory lecture).

4 - Motor functions.

Question 17

How does cholinergic transmission contribute to REM sleep?

Answer 17

• Pontine cholinergic neurones increase firing prior to onset of REM sleep.
• These are ‘REM-on’ cells.

Question 18

How does cholinergic transmission contribute to arousal?

Answer 18

Cholinergic agonists increase arousal, whereas cholinergic antagonists decrease arousal.

Question 19

List the diseases that are the main therapeutic targets of cholinergic therapy.

Answer 19

The main therapeutic targets of cholinergic therapy are:

1 - Diseases that affect cognitive function, such as Alzheimer’s disease (and more broadly, dementia).

2 - Diseases that affect motor function, such as Parkinson’s disease.

Question 20

Give an example of a cholinergic therapy for Parkinson’s disease.

How does it work?

Answer 20

• Benztropine is a cholinergic therapy for Parkinson’s disease.
• Benztropine is a muscarinic antagonist (probably acting at both M1 and M3 receptors) that decreases overactive striatal cholinergic transmission resulting from a decrease in dopaminergic inhibition (see y2 Parkinson’s lecture).
• Benztropine can be used to delay L-DOPA therapy in early Parkinson’s disease, or allow for a lower L-DOPA dose.

Question 21

Give an example of a cholinergic therapy for Alzheimer’s disease.

Answer 21

AChE inhibitors such as tacrine and donepezil can be used as a symptomatic treatment for Alzheimer’s disease.

*Evidence for efficaciousness of donepezil is poor.

Question 22

List 2 factors that affect the efficaciousness of AChE inhibitors such as tacrine and donepezil for the treatment of Alzheimer’s disease.

Answer 22

Factors that affect the efficaciousness of AChE inhibitors such as tacrine and donepezil for the treatment of dementia include:

1 - Sex.

2 - ApoE genotype, a protein involved in metabolism of fats.

Question 23

For Alzheimer’s disease, Schizophrenia, Parkinson’s disease and drug addiction, name the subtype of muscarinic receptors that are targeted by the drugs used to treat those conditions.

Are these agonists or antagonists?

Answer 23

• Alzheimer’s disease: M1 and M5 agonists.
• Schizophrenia: M1 and M4 agonists.
• Parkinson’s disease: M1 and M5 antagonists.
• Drug addiction: M5 antagonists.

Question 24

A*:

Describe the cholinergic-adrenergic hypothesis of mood disorders.

On which research was this hypothesis founded?

Answer 24

• The cholinergic hypothesis of mood disorders (Janowsky et al., 1972) states that:
• Hyperactivity of adrenergic neurotransmission from sympathetic neurones and hypoactivity of cholinergic neurotransmission from parasympathetic neurones is responsible for the manic symptoms of mood disorders.
• Hypoactivity of adrenergic neurotransmission from sympathetic neurones and hyperactivity of cholinergic neurotransmission for parasympathetic neurones is responsible for the depressive symptoms of mood disorders.
• This hypothesis was founded on two pieces of research:

1 - Increases in arousal mediated by adrenergic transmission is inhibited by cholinergic transmission.

2 - Administration of cholinesterase inhibitors reduced manic symptoms in patients suffering from mania, and induced depressive symptoms in healthy patients.

*In short, high ACh = depression, high NA = mania.

Question 25

A*:

What might underlie the physiological changes described in the cholinergic-adrenergic hypothesis of mood disorders?

Give an example of a potential drug based on the cholinergic hypothesis of mood disorders.

Give an example of a limitation of this drug, and a solution for how this can be resolved.

Answer 25

• Hypercholinergic transmission in depression is thought to be caused by reduced expression of M2 receptors.
• M2 receptors are inhibitory autoreceptors, therefore reduced M2 receptor activity leads to an increase in cholinergic transmission.
• Hyoscine is a nonselective muscarinic antagonist that causes an antidepressant effect by inhibiting facilitatory postsynaptic cholinergic transmission.
• The limitation of directly stimulating / antagonising the orthosteric site of muscarinic receptors is that receptor internalisation, desensitisation and receptor downregulation will occur with drug exposure. This would require an increasing dose over time (Jeon et al.,2015).
• This could be overcome by developing specific positive allosteric modulators for M2 receptors.

Question 26

A*:

List the GPCR alpha subunits with which each muscarinic receptor is associated.

Answer 26

GPCR alpha subunits with which each muscarinic receptor is associated:

• M1 = Gq.
• M2 = Gi/o.
• M3 = Gq.
• M4 = Gi/o.
• M5 = Gq.

Question 27

A*:

Describe the role of acetylcholine in cognitive disorders.

Give an example of a drug class used to treat the cognitive symptoms of neuropsychiatric disorders.

Give an example of an emerging drug class for treating the cognitive symptoms of neuropsychiatric disorders.

Answer 27

• It is thought that hypofunction of cholinergic neurones projecting from the cortex to the hippocampus underlies the cognitive deficits in neuropsychiatric disorders such as Alzheimer’s disease and schizophrenia (Langmead et al., 2008).
• E. g. the cholinergic hypothesis of memory dysfunction states that cholinergic hypoactivity underlies the memory deficits in healthy individuals and in neuropsychiatric disease (Bartus et al., 1982).
• Hence, the use of acetylcholinesterase inhibitors such as neostigmine and rivastigmine improve cognitive function (see card 21 for other examples).
• An emerging drug class for treating the cognitive symptoms of neuropsychiatric disorders are muscarinic AChR agonists.
• Xanomeline is a muscarinic agonist that acts preferentially at M1 and M4 receptors. The principal site of action for treating cognitive symptoms is M1 receptors. It was shown to improve cognitive symptoms in AD patients (Bymaster et al., 1997).

Question 28

A*:

Describe 3 mechanisms of muscarinic drugs for the treatment of Alzheimer’s disease.

List 2 specific drugs that do this.

Answer 28

1 - Muscarinic drugs restore activity in hypoactive cholinergic pathways.

2 - An increase in alpha secretase expression. This alleviates symptoms and slows progression in AD by diverting away from the gamma secretase pathway that leads to the formation of amyloid plaques.

3 - An increase in activation of PKC, which inhibits the kinases responsible for phosphorylation of tau.

• This can be achieved by talsaclidine, a selective M1 agonist, and xanomeline, an M1/M4 agonist.

Question 29

A*:

List 2 limitations of muscarinic therapies for Alzheimer’s disease.

Answer 29

Limitations of muscarinic therapies for Alzheimer’s disease:

1 - Targeting the orthosteric binding site of muscarinic receptors causes many side effects. This is due to the similarity in structure between muscarinic receptor types, which makes it difficult to develop selective drugs. Sequence homology is less preserved in allosteric sites.

2 - In AD, M1 receptors have been been shown to exhibit impaired G-protein-receptor coupling (Flynn et al., 1991). Simply activating M1 receptors by way of muscarinic agonists may therefore have limited clinical effect, since ligand binding to the receptor might not trigger the downstream cascades.

Question 30

A*:

What is biased agonism and probe dependence?

How do these properties influence drug design?

Answer 30

From Davie et al., 2013:

• Biased agonism is the notion that different allosteric modulators, even though acting the same receptor, activate different downstream cascades to different extents.
• Probe dependence is the notion that the modulatory effect of an allosteric drug is dependent on the pharmacological profile of the drug acting at the orthosteric site.
• Therefore, to account for probe dependence when developing an allosteric modulator, it is important to investigate the action of the drug in combination with endogenous orthosteric ligands that are involved in the disease pathology.

Example:

• Chan et al., 2008, investigated an M4 PAM as a treatment for schizophrenia, and concluded that the ligand exhibited a high selectivity for the M4 receptor and showed a high degree of positive modulation.
• Investigations by other groups found that the drug exhibits:

1 - Variation in the degree of allosteric modulation on different downstream cascades (biased agonism).

2 - No positive modulatory effect when acting at a receptor that isnt bound to an agonist at the orthosteric site (probe dependence).

• Probe dependence is a consideration unique to allosteric modulators that must be considered in drug development.

Question 31

A*:

Give an example of an emerging PAM that is being developed for treating the cognitive symptoms of neuropsychiatric disorders.

How is it likely to be administered?

Answer 31

• PQCA is an emerging PAM that has shown efficacy for improving cognition in animal models of Alzheimer’s disease.
• Human trials have not yet occurred.
• The efficacy of PQCA has been shown to be improved with coadministration of AChE inhibitors such as donepezil, therefore PQCA is a potential adjunct therapy to AChE inhibitors.

Question 32

A*:

What are gamma oscillations?

What are the functions of gamma oscillations?

How might gamma oscillations be involved in schizophrenia?

Answer 32

• Gamma oscillations are high-frequency fluctuations of 30-200Hz in extracellular potentials known as local field potentials.
• They are due to the combined effect of excitatory glutamatergic and inhibitory GABAergic activity of surrounding neurones.
• The functions of gamma oscillations are thought to include:

1 - Synchronising the electrical activity of adjacent populations of neurones.

2 - Communicating extrasynaptically across brain regions.

• In schizophrenia, gamma oscillations show both a reduction in frequency and amplitude (Williams and Boksa, 2010).
• It is thought that these changes to gamma oscillations in schizophrenia is due to impaired GABA transmission. In a study by Lewis et al. (2008), the frequency and amplitude of gamma oscillations were enhanced in Schizophrenia patients following administration of a benzodiazepine.
• This finding was paired with improvement of working memory.