Neuropeptides PCTH 502

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NeuropeptidesPCTH 502

• Dr. Stephanie L. Borgland
• borgland_at_interchange.ubc.ca

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Outline

• What are Neuropeptides?
• Neuropeptide-mediated neurotransmission
• Synthesis
• Storage
• Release
• degredation
• Differences between classical neurotransmitters
  and neuropeptides
• Overview of Neuropeptides in
• Feeding
• Stress
• Orexin/hypocretin system
• Peptides in drug development

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Neuropeptides

• At least 3 of the seven deadly sins can be
  mediated by neuropeptides
• Charles F. Stevens
• Peptides consist of 2 or more amino acids (linked
  by peptide bonds)
• Neuropeptides Can act as neurotransmitters,
  neuromodulators or neurohormones

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Examples of Neuropeptides
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Why so many neurotransmitters?

• Classical transmitters (Glu, GABA), monamines
  (DA, 5HT), gases (NO), cannabinoids,
  neuropeptides
• If transmitters simply serve as a chemical bridge
  that conveys information between two spatially
  distinct cells, why have so many transmitters?

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Why so many neurotransmitters?

• Many nerve terminals synapse onto a single neuron
• How can a neuron distinguish between multiple
  inputs that carry different information?
• Segregate the place on the neuron at which an
  input arrives, such as the soma, axon or dendrite
  
• however so many afferents terminate in close
  proximity ---thus require another means of
  distinguishing inputs and their information is
  necessary
• Chemical coding of the inputs by
  neurotransmitters
• Most neurons contain more than one transmitter
• Different transmitters by a neuron may be used to
  signal different functional states to its target
  cell

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Neuropeptide Synthesis and Transport
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Synthesis of Peptide Neurotransmitters

• Peptides are synthesized in the soma and
  transported to axon terminal in vesicles
• Genes encoding peptide transmitters give rise to
  a prohormone which is incorporated into a
  secretory granule.
• prohormone is cleaved by peptidases to form the
  peptide transmitter
• A small number of peptide transmitters are
  synthesized enzymatically
• Slow, energy intensive synthesis (compared to
  classical transmitters)

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POMC encodes a number of neuropeptides

• POMC is a prepropeptide and encodes for ACTH,
  melanocyte-stimulating hormone, ß-endorphin
• The prepropeptide is translocated to the ER where
  it is translated into propeptides
• The propeptides are processed by prohormone
  convertases 1 and 2 (PC1 and PC2) step-wise
  reaction, cleaving certain dibasic residues
• (e.g., Lys-Arg Lys-Lys, Arg-Arg
  Arg-Lys)?smaller peptides with Lys and Arg at
  their N- and C-termini
• carboxypeptidase E and aminopeptidase remove
  these basic residues

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Processing of POMC

• N-terminal acetylation via N-acetyltransferases
  can occur which regulates the activity of the
  neuropeptide
• a-MSH activity is increased by N-terminal
  acetylation
• ?-endorphin activity is decreased by N-terminal
  acetylation
• NOTE the specific neuropeptides synthesized from
  a prepropeptide gene depend upon the tissue
  because different tissues contain different
  prohormone convertases

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Storage of peptide neurotransmitters
Dense core vesicles 100 nm
Synaptic vesicles 50 nm
Fischer-Colbrie et al., 1982 Obendorf et al.,
1988
Neuropeptides are stored in dense core vesicles
assembled in the golgi network
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Release of neuropeptides peptides

• Dense core vesicles are transported down the axon
  to terminals
• DSVs are docked outside the active zone
• Released upon large, sustained calcium entry into
  the cell
• High frequency action potentials
• Burst firing

2001 Sinauer Associates, Inc.
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Neuropeptides are co-localized with other
transmitters

• Many neuropeptides are co-localized with small
  neurotransmitter molecules
• Under low stimulation conditions, the small
  neurotransmitter vesicles will be mobilized and
  fuse with the membrane
• Under higher stimulation conditions, the dense
  core vesicles will mobilize and fuse with the
  membrane

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Neuropeptide Pharmacology

• in the vast majority of cases, the receptors for
  neuropeptides are G-protein-coupled
• disconnect between receptor and neuropeptide
  localization in brain ? far reaching effects
• neuropeptides bind with very high affinity (nM
  range, vs. small neurotransmitters which bind in
  µM range)
• neuropeptides are flexible molecules which may
  account for their ability to interact with their
  receptors

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Ex. Galanin receptor distribution is different
than peptide distribution
Developmental expression of Galanin and Galanin
receptors in rat hypothalamus And other brain
regions
Gundlach et al., 2001
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Inactivation of peptide transmitters

• Peptides are released from all parts of the
  terminal and after release are enzymatically
  degraded
• Can also diffuse from the terminal
• typically no transporter-mediated reuptake
• Peptide neurotransmitter inactivating enzymes are
  specific for certain dipeptide sequence and are
  not specific to any single peptide.
• Ex. Enzyme that degrades enkephalin is referred
  to as enkephalinase, but also cleaves other
  peptide transmitters
• Receptor internalization is another method of
  inactivation

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Inactivation can lead to biologically active
metabolites

• Some peptide fragments from degradation can also
  be biologically active
• Eg. Angiotensin
• Angiotensin I is metabolized to yield angiotensin
  II and III
• Each is successively more active than the parent
  angiotensin I
• Can be difficult to distinguish between
  transmitter synthesis and transmitter
  inactivation
• the peptide stored in vesicles is considered the
  transmitter, but peptidases may lead to other
  biologically active fragments

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Classical neurotransmission vs peptide
transmitters

• Classical transmitters
• released and lower firing rates
• Mediate fast neurotransmission
• glutamate release, ionotropic glutamate receptor
  activation fast
• Reuptake
• Rapidly replaced synthesis occurs in nerve
  terminals
• Stored in small synaptic vesicles (50 nm)

• Neuropeptides
• released at higher firing rates and particularly
  under burst-firing patterns
• Mediate slower neurotransmission
• Metabotropic receptor activation slower
• Degradation after release
• Must be synthesized in cell body and transported
  to the terminal
• Stored in large dense core vesicles (100 nm)

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Neuropeptides neurohormones or neurotransmitters?

• Neurohormones when neurons secrete their
  peptides into the vascular system to be
  transported to a relatively distant target
• Neurotransmitter Many axon terminals of
  neurosecretory cells secrete their products at
  the synapse to directly affect a post synaptic
  cell
• Neuropeptides can do both depends on nerve
  terminal

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Neuropeptides as neuromodulators

• What is a neuromodulator?
• Secreted in response to Ca2 influx
• Capable of modulating an excitatory or inhibitory
  post synaptic potential (EPSP/IPSP)
• But not sufficient to evoke an EPSP or IPSP
• Facilitate or Inhibit EPSPs or IPSPs
• Neuropeptides may act as neurotransmitters at
  some synapses and neuromodulators at others

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Volume Transmission

• Characterized by a 3-dimensional conduction of
  the signal within extracellular fluid
• Autocrine, paracrine transmission for short
  distances
• Neuroendocrine transmission for longer distance
• One reason why neuropeptide receptors are very
  high affinity

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Feeding-related peptides
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Hypothalamic feeding peptides
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Hypothalamic Pituitary Adrenal (HPA) Axis
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Inhibition activation
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Orexin projections

• Orexin-producing neurons are exclusively
  localized to the lateral hypothalamus (LHA) and
  adjacent regions and project widely. (Sakura et
  al., 1998 de Lecea et al., 1998).

VTA
Sakurai,2003 Curr Opin Clin Nutr Metab Care 6
353.
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Orexin Pharmacology

• Orexin A binds with high affinity to both
  inhibitory GPCRs receptors, OXR1 and OXR2
• Orexin B binds to only OX2R.

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Physiological Roles of Orexin

• Feeding
• ICV injections stimulate food consumption (Sakura
  et al., 1998)
• orexin-stimulated food consumption is mediated in
  part by activation of NPY neurons (Yamananaka et
  al., 2000).
• Regulation of arousal
• Disruption of prepro-orexin gene (Chemelli et
  al., 1999) or OX2R knockouts (Lin et al., 2000)
  results in a syndrome similar to human
  narcolepsy.
• ICV injection promotes wakefulness, supresses
  non-REM and REM sleep (Hagan et al., 1999).
• ICV injection increases locomotor activity
  (Nakamura et al., 2000)
• Modulates energy metabolism
• Prepro-orexin knockouts are hypometabolic (Hara
  et al. 2001).
• ICV injection of orexin A increases oxygen
  consumption and respiratory quotient through an
  increase in carbohydrate metabolism (Lubkin et
  al., 1998).

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Physiological Roles of Orexin
Sakurai,2003 Curr Opin Clin Nutr Metab Care 6
353.
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Channelrhodopsin2

• Light activated ion channel isolated from algae
• unspecific cation channels, conducting H, Na,
  K, and Ca2 ions.
• Blue light opens ChR2 (absorbs 480 nm)
• Neuron depolarizes

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Selective expression of ChR2 in hypocretin
neurons
Adamantidis et al., 2007
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Photostimulation at the same frequency inducing
firing causes mice to wake up

• http//www.nature.com/nature/journal/v450/n7168/ex
  tref/nature06310-s2.mov

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Physiological Roles of Orexin

• Orexin may act by coordinating behavioral and
  physiological responses of these complementary
  homeostatic functions.

Drive State Ghrelin Leptin Glucose
DA
Locomotor activation Foraging
Orexin
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Circuitry of Addiction
Reward Prediction/ Salience
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Orexin and Dopamine

• Terminals of LHA orexin neurons are opposed to
  dendrites and somata of dopamine VTA neurons
  (Fadel and Deutch, 2002).

Fadel and Deutch, 2002 Neurosci 111 379.
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In the Rat VTA there are very few orexin-DA or
GABA neuron synapses
Balcita-Pedicino Sesack, 2007
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Orexin and Dopamine

• Orexins increase firing frequency in VTA neurons
  and in some cases cause burst firing (Korotkova
  et al., 2003).

Korotkova et al., 2003, J. Neurosci. 23 7.
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How does orexin mediate the reinforcing effects
of drugs?
NMDA dependent plasticity in the VTA is Important
for - transition from tonic to phasic firing
(increased DA release) - Long-term
potentiation - behavioural (locomotor)
sensitization
Borgland et al., 2006 Neuron
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Orexin receptor-1 antagonist blocks cocaine
induced neural plasticity and cocaine
sensitization
Borgland et al., 2006 Neuron
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Hypothesis

• Because orexin neurons primarily function in
  mediating arousal, and orexin in the VTA plays an
  important role in reward, orexin may function to
  promote arousal for goal directed behavior or
  motivation
• Does orexin signaling mediate motivated
  behaviour?

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Breakpoint
Sanchis-Segura Spanagel, 2007
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Orexin receptor antagonist, SB, reduces
breakpoint for cocaine and high fat food
SB 334867 10 mg/kg ip
Borgland et al., 2009 J. Neurosci
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Self-administration of cocaine and high fat food
increases orexin-mediated plasticity in the VTA
Borgland et al., 2009 J. Neurosci
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How are ox/hcrt effects occurring in the VTA?
Kalivas, 2006
Modified from
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Can one use neuropeptides as drugs?

• Problems
• First pass metabolism
• Crossing the blood brain barrier
• Hypothalamus has leaky blood brain barrier

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Drug Delivery

• Intranasal route?
• Oxytocin, vasopressin, melanococortin, insulin
  delivered intranasally CSF levels increase
  after 30 min (Nat Neurosci 2002 5 5146)
• small molecules and peptides can pass directly
  from the submucous space of the nose to the CSF
  of the olfactory lobe and then circulate within
  the CSF flow tracts of the brain
• However
• transnasal delivery does not deliver a drug deep
  into the brain parenchyma
• The drug is only delivered to the CSF and will be
  cleared back into the peripheral blood before
  there is time for it to be carried to brain
  tissue

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Other options?

• Taking advantage of receptor-mediated
  transcytosis (insulin)
• In animals can use TAT-fusion proteins
• TAT from HIV easily transports peptides across
  BBB
• Cationic Lipid delivery (Liposomal delivery
  systems)
• Electroporation TMS (makes BBB leaky)
• Intracranial infusion pumps (invasive)

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Summary
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Suggested reading

• Neuropeptides opportunities for drug discovery.
  Hokfelt, Bartfai, Bloom (2003) Lancet Neurology