Neuropeptides (A*)

Question 1

What is a peptide?

What is a neuropeptide?

Answer 1

• A peptide is a chain of at least 2 amino acids joined by a peptide bond.
• Neuropeptides are a class of peptide signalling molecules released and synthesised by neurones that target GPCRs to produce a neuromodulatory effect when coreleased with classical neurotransmitters. They also serve a neuroendocrine function, as many are secreted into the circulation to travel to distal targets.

Question 2

How do neuropeptides travel to their targets in the body from their site of synthesis?

Answer 2

Neuropeptides travel to their targets through the blood (they travel as hormones, e.g. oxytocin to the mammary gland and myometrium, or vasopressin to the distal convoluted tubule and vasculature).

*Neuropeptides can also be confined to synapses.

Question 3

List 2 generic roles of neuropeptides.

Answer 3

Generic roles of neuropeptides include:

1 - Neuromodulation of classical neurotransmitters (usually has no postsynaptic effect without classical neurotransmitters).

2 - Trophic roles (e.g. influencing DNA synthesis and stimulating growth of smooth muscle).

Question 4

Describe the process of neuropeptide synthesis.

Give an example of a specific neuropeptide synthesis pathway.

Answer 4

Neuropeptide synthesis is similar to classic protein synthesis. In summary, different neuropeptides are derived from the differential cleavage of precursor proteins, which in turn are derived from differential splicing of mRNA:

1 - Alternative splicing of primary mRNA produces an alpha, beta or gamma spliced mRNA.

2 - Spliced mRNA is translated to produce a prepropeptide.

3 - Prepropeptides are cleaved by signal peptidase enzymes to produce propeptides.

• Propeptides often contain multiple copies of neurotransmitters, e.g. TRH propeptide contains 5 copies of TRH. Other propeptides contain multiple different peptides.

4 - Convertase enzymes convert propeptides into peptides.

Example:

Splicing of the preprotachykinin A (PPT) gene:

• Alpha mRNA produced from splicing of the preprotachykinin A gene produces substance P.
• Beta mRNA produced from splicing of the preprotachykinin A gene produces substance P + neurokinin A + neuropeptide K.
• Gamma mRNA produced from splicing of the preprotachykinin A gene produces substance P + neuropeptide gamma.

Question 5

List 9 differences between the mode of release, action and reuptake of neuropeptides and classical neurotransmitter release.

Answer 5

1 - The peptide, once synthesised, is packaged into a vesicle at the golgi apparatus in the soma (whereas most classical neurotransmitters are made at the presynaptic terminal).

2 - The neuropeptide travels down the axon in high concentrations in large dense core vesicles (in comparison to classical neurotransmitters which are stored in small synaptic vesicles and are already present in the presynaptic terminal).

3 - At the presynaptic terminal, large dense core vesicles do not cluster at the active zone (the zone where most small synaptic vesicles cluster), and instead are dispersed throughout the terminal.

4 - Specific proteins induce exocytosis of large dense core vesicles at extrasynaptic domains by a mechanism that requires lower Ca2+ than normal exocytosis.

• Low Ca2+ is required because most voltage-gated Ca2+ channels are clustered at the active zone, so Ca2+ concentration is low at extrasynaptic domains in the presynaptic terminal.

5 - Extrasynaptic release of neuropeptides into the extracellular space enables them to engage in volume transmission, sometimes over relatively long distances, or in paracrine signalling. Neuropeptides may also act as autocrine molecules, binding to autoreceptors on the same neurone from which the neuropeptide was released. On the other hand, most classical neurotransmitters simply engage in synaptic transmission.

6 - Most neuropeptides are unable to produce a postsynaptic potential on their own. Some are able to produce a slow postsynaptic potential (as opposed to small neurotransmitters such as glutamate that produce fast postsynaptic potentials), however most neuropeptides modulate the postsynaptic potential induced by other primary neurotransmitters, e.g. amplifying the EPSP induced by glutamate.

7 - There is no reuptake mechanism for neuropeptides. Instead, their action is terminated by inactivating ectoenzymes. This means neuropeptides cannot be reused / recycled.

8 - Some neuropeptides released from the soma are inactive, and must be activated either by enzymes contained within their vesicles or in the extracellular space by activating enzymes.

9 - Neuropeptides, since they are produced at the soma, cannot be quickly replenished, since they must be synthesised and transported down the axon to the presynaptic terminal (slow vesicle recycling in neuropeptides vs fast vesicle recycling in neurotransmitters).

Question 6

Why must neuropeptide receptors have a high affinity for neuropeptides (relative to the affinity of classical neurotransmitter receptors for their ligands)?

Answer 6

Neuropeptide receptors must have a high affinity for neuropeptides because neuropeptides are involved in volume transmission, so their concentration at the receptor site is relatively low (micromolar - compared to the millimolar concentrations of classical neurotransmitters at synapses).

Question 7

At which type of receptors do neuropeptides act?

Answer 7

Neuropeptides act at GPCRs.

Question 8

What influences the level of expression of neuropeptides under normal physiological conditions?

Give 3 examples of neuropeptides that have different patterns of expression.

Answer 8

• The level of neuropeptide expression under physiological conditions depends on the function of the neuropeptide:

1 - Expression is low but constant for neuropeptides that must be stored for functional availability at any time (e.g. substance P in primary sensory neurones).

2 - Expression is variable for neuropeptides that need to be upregulated following a specific stimulus (e.g. VIP in the GIT).

3 - Expression is transient in development for neuropeptides that are downregulated in adulthood (e.g. somatostatin).

Question 9

List 3 examples of neuropeptides working in conjunction with classical neurotransmitters.

Answer 9

Examples of neuropeptides working in conjunction with classical neurotransmitters include:

1 - CCK with dopamine in the mesolimbic pathway.

2 - Substance P and CGRP with glutamate in primary afferent neurones conveying pain information.

3 - Substance P, enkephalin and dynorphin with GABA in striatal efferents.

Question 10

Describe the mechanism by which substance P, CGRP and NAAG influence pain transmission.

Answer 10

• Substance P and CGRP bind to NK-1 receptors in pain fibres.
• Binding to NK-1 receptors causes PKC-mediated NMDA receptor phosphorylation.
• This increases the affinity of glycine for the glycine site on the NMDA receptor, potentiating NMDA-mediated transmission of pain signals in primary afferent neurones.
• This only occurs when sufficient glutamate is coreleased with the neuropeptide, because sufficient AMPA receptors must first be activated in order to overcome the voltage-dependent Mg2+ block of the NMDA receptors for the neuropeptides to have any effect on transmission.
• NAAG is coreleased with these neuropeptides, which has the ability to bind to presynaptic mGlu3 receptors as a means of negative feedback (NAAG has an analgesic effect).

Question 11

What influences the degree of neuropeptide release from a neurone?

Give examples of situations that would cause different levels of neuropeptide release.

Answer 11

Firing rate of input signals influences neuropeptide release from a neurone:

• Low frequency input results in exocytosis of classical neurotransmitter, but not neuropeptide (the neuropeptide stays in the presynaptic terminal in its large dense core vesicle).
• This might happen during growth / development.
• Medium frequency input results in corelease of moderate volumes of classical neurotransmitters and neuropeptides.
• This might happen in response to stress, anxiety and pain.
• High frequency firing / burst firing results in corelease of large volumes of classical neurotransmitters and neuropeptides.
• This might happen in response to neuronal damage and in pathology.

Question 12

Why do neuropeptide therapies usually have better side effect profiles than other neuropharmacological therapies?

Answer 12

Neuropeptide therapies usually have good side effect profiles because they don’t interfere too much with classic neurotransmission.

*This is despite the fact that there are few selective neuropeptide drugs.

Question 13

List 3 challenges associated with developing peptide drugs.

Answer 13

Challenges associated with developing peptide drugs include:

1 - Poor bioavailability.

2 - Poor CNS penetration.

3 - Expensive to synthesise.

Question 14

List 3 emerging neuropeptide receptor-targeting drugs.

What do they do?

Answer 14

Emerging neuropeptide receptor-targeting drugs include:

1 - CCK antagonists as antidepressants / anxiolytics.

2 - NK1 antagonists as antidepressants / anxiolytics and antiemetics.

3 - NK3 agonists as antidepressants / anxiolytics.

*Surprisingly, NK1 receptor antagonists failed as analgesics!

Question 15

List 2 emerging peptidase enzyme drugs.

How do they work?

What are they used for?

Answer 15

Emerging peptidase enzyme drugs include:

1 - Thiorphan.

• Thiorphan is a membrane metalloendopeptidase (enkephalinase) inhibitor which prolongs the effects of endogenous enkephalins.
• Thiorphan can therefore be used to potentiate morphine-induced analgesia.

2 - NAAG peptidase inhibitors such as ZJ43 and 2-PMPA.

• NAAG peptidase inhibitors limit excessive glutamate release from primary afferent neurons conveying pain information by maintaining high concentrations of intrasynaptic NAAG.
• NAAG peptidase inhibitors can therefore be used as analgesics, but might also work as neuroprotective and antischizophrenic drugs.

Question 16

A*:

How prevalent is NAAG?

Briefly describe the synthesis and breakdown of NAAG.

List 2 receptors that interact with NAAG.

Answer 16

• NAAG is the 3rd most common neurotransmitter in the body, behind GABA and glutamate.
• NAAG synthetases convert NAA and glutamate into NAAG.
• NAAG is broken down by NAAG peptidases such as GCPII into NAA and glutamate.

1 - NAAG is a partial agonist at NMDA receptors.

2 - NAAG is an mGluR3 agonist.

Question 17

A*:

Describe the role of NAAG in development.

Answer 17

The role of NAAG in development involves neurone-glial and glial-glial signalling:

• Neurones release NAA and NAAG in the extracellular fluid.
• NAA signals to oligodendrocytes to promote myelination.
• NAAG signals to astrocytes to promote neuronal metabolic support.
• Oligodendrocytes also synthesise and release NAAG to signal to astrocytes, and conversely astrocytes hydrolyse NAAG into NAA, which is released to signal to oligodendrocytes.
• These signalling pathways are thought to be involved in:

1 - Controlling release of trophic factors.

2 - Transmitting information regarding the direction and distance between neurones and glia. A process involved in metaplasticity - see A card 37 in novel neuronal signalling mechanisms.

• Deficiency of this pathway is thought to result in poor formation of the brain in developmental abnormalities. This reflects the neuroprotective function of NAAG (see card below for examples).

Question 18

A*:

List 3 potential therapeutic roles of NAAG-targeting drugs.

Answer 18

1 - NAAG peptidase inhibitor ZJ43 reduces PCP-induced motor symptoms of schizophrenia.

• NAAG is a partial agonist at NMDA receptors, meaning in low concentrations, NAAG has an antagonistic effect, and in high concentrations has an agonistic effect. In schizophrenia, it has been hypothesised that decreased expression of NAAG underlies the hypoglutamatergic state. This has been evidenced by functional imaging studies comparing NAA and NAAG expression in schizophrenia patients and healthy individuals. Furthermore, a negative relationship exists between NAAG expression and positive and negative symptoms scale (PANSS) (Jessen et al., 2013). Hence, ZJ43 is thought to improve symptoms of schizophrenia by reducing NAAG metabolism, which in turn alleviates NAAG-mediated inhibition of glutamatergic transmission at NMDA receptors. Also see below for general neuroprotective functions of NAAG.

2 - NAAG peptidase inhibitor 2-PMPA protects against neuronal apoptosis and neurite degeneration in diabetic neuropathy.

• The mechanisms underlying the therapeutic effect of NAAG in diabetic neuropathy are unclear, however cell culture models of diabetic neuropathy have shown that mGluR activation leads to an increase in synthesis of NAAG, which in turn inhibits proapoptotic caspase enzymes. This could underlie the neuroprotective effect (and also see point 3 below).

3 - NAAG peptidase inhibitor 2-PMPA and GCPII inhibitors protect against motoneurone death in amyotrophic lateral sclerosis, a neurodegenerative disease characterised by motor deficits arising from degeneration of upper and lower motoneurones.

• 2-PMPA: Owing to NAAG’s neuroprotective effect, NAAG peptidase inhibitors have been proposed to have therapeutic potential for the treatment of neurodegenerative diseases such as ALS. NAAG is a potent agonist at mGluR3s. Activation of mGluR3s is known to reduce glutamate release through action at inhibitory mGluR3s, preventing excitotoxicity and stimulating release of growth factors such as TGF-beta that prevent neuronal apoptosis (and also see point 2 above).
• GCPII inhibitors: This has a dual mechanism. Firstly, inhibition of GCPII leads to increased concentrations of NAAG, which as described above, exerts a neuroprotective effect. Secondly, NAAG is metabolised into NAA and glutamate. By inhibiting NAAG metabolism, glutamate concentrations decrease, protecting against excitotoxicity.

Question 19

A*:

Describe the role of galanin in modulating pain pathways.

Answer 19

• Many neuropeptidergic neurones exhibit strong neuroplasticity.
• In the dorsal root ganglion (DRG), the phenotype of neurones is influenced by peripheral nerve lesions.
• For example, galanin and is upregulated in the DRG in response to nerve injury.
• Galanin has analgesic effects at Gal1 receptors and pronociceptive effects at Gal2 receptors.

Question 20

A*:

Describe the role of nociceptin and nocistatin in the brain.

Answer 20

• Nociceptin and nocistatin are opioid-related neuropeptides that are produced from the same prepropeptide.
• Nocistatin is not biologically active on its own but can antagonise the effects of nociceptin.
• Nociceptin (considered an anti-opioid) causes allodynia, and is therefore a potential target for pain therapy.
• Nociceptin has been shown to reduce the reward of abused drugs such as morphine, cocaine and amphetamines, so is a potential target for treating drug addiction.

Question 21

A*:

What is neurotrypsin?

What is its role in the brain?

What is the therapeutic potential of drugs targeting neurotrypsin?

Answer 21

• Neurotrypsin is a peptidase enzymes expressed in the cortex, amygdala and hippocampus.
• It accumulates around the apical membrane of presynaptic terminals and is released into the synaptic cleft by exocytosis in response to neuronal activity.
• In the synapse, it cleaves agrin to produce agrin-22 and agrin-90.
• Agrin-22 is thought to stimulate the growth of dendritic spines, hence it is thought to enhance synaptogenesis.
• Neurotrypsin therefore has a role in memory and cognition.
• Individuals lacking neurotrypsin suffer from mental retardation.
• This system is a potential target for cognition-enhancing drugs.

Question 22

A*:

Give an example of a potential neuropeptide target for fat metabolism.

Answer 22

• Adipose tissue is innervated by noradrenergic neurones.
• These neurones corelease noradrenaline and neuropeptide Y, which have a synergistic effect on fat metabolism in adipocytes.
• The effect of noradrenaline / neuropeptide Y corelease on fat metabolism depends on:

1 - The presence of different noradrenaline / neuropeptide Y receptor subtypes.

2 - The distribution of noradrenaline / neuropeptide Y receptors in the adipocyte.

3 - The relative quantities of noradrenaline / neuropeptide Y release.

• This system is a potential target for anti-obesity drugs, however further research into the signalling mechanism is required before a drug can be developed.

Question 23

A* feeding

Answer 23

Just a reminder to look in neurobiology of feeding behaviour lecture because that has lots of neuropeptides.

Question 24

A*:

List 3 neuropeptides that are released in the cerebrovasculature.

Describe the role of neuropeptides in the cerebrovasculature.

Give an example of a neuropeptidergic treatment for migraines.

Answer 24

• Neuropeptides released in the cerebrovasculature include neuropeptide Y, CGRP and PACAP.
• They are coreleased with primary neurotransmitters such as acetylcholine, but also with other neuromodulators such as noradrenaline and NO.
• Their supposed effect is in modulating vascular tone, although this is subject of much debate.
• There is a possibility for blockade of CGRP in the cerebrovasculature in order to prevent migraine, as CGRP known to potently induce vasodilation, which in turn is implicated in the pathogenesis of migraines.
• CGRP is thought to induce vasodilation both directly and through upregulation of the vasodilator, NO. This is thought to occur by CGRP-induced activation of PKA, which in turn activates eNOS - an enzyme responsible for the production of endothelial NOS. In individuals suffering from migraines, this process is thought to become dysregulated, leading to excessive vasodilation (Favoni et al., 2019).
• The vasodilatory effect of CGRP has been well-studied. For example, intravenous administration of CGRP has been shown to be beneficial for patients suffering from congestive heart failure. However, the current understanding of the role of CGRP in migraines is challenged by various lines of evidence:
• Edvinsson et al. (2014) have suggested that CGRP-induced vasodilation works by a process that is independent of the endothelium, and instead acts directly on the vascular smooth muscle.
• Furthermore, CGRP-induced vasodilation was not shown to exert a significantly different effect in endothelium derived from migraine sufferers compared to healthy individuals (Edvinsson and Edvinsson, 2008). This would indicate that increased sensitivity to CGRP-induced vasodilation does not alone underpin migraine pathophysiology.
• On the other hand, clinical trials using anti-CGRP antibodies (e.g. fremanezumab), anti-CGRP receptor antibodies (e.g. erenumab) and CGRP antagonists (e.g. ubrogepant) have shown efficacy in clinical trials for treating migraines, and have also shown ideal safety profiles.
• Furthermore, higher levels of CGRP have been identified in women compared to men. This aligns with the fact that women are approximately 3 times more likely than men to suffer from migraines.
• Overall, CGRP blockade poses an attractive alternative to classical serotonergic migraine treatments, but the mechanisms underpinning their action require further investigation.