Hormonal Regulation of Synaptic Plasticity Flashcards
Can LTP be modulated?
Yes; the degree and duration can be influenced
Numerous neuromodulators and nT can affect; the threshold for LTP induction or the magnitude +/- magnitude and duration of LTP
What 2 endocrine hormones markedly influence hippocampal synaptic plasticity?
Leptin
Inslulin
What is Leptin?
“Fat controller”
16 kDa protein encoded by ob gene
Primarily made and secreted by white adipose tissue
Circulates in amounts proportional to body weight
Regulates energy homeostasis via its actions at the hypothalamus
Describe the leptin receptor
Product of the diabetes (db) gene Member of class 1 cytokine superfamily
Multiple splice variants (LepRa - LpRf)
All transmembrane receptors except LepRe (carrier for leptin in plasma)
LepRb (contains tyrosine residues). Long C terminal domain which contains tyrosine residues enabling full complement of leptin receptor signalling
Describe the signalling pathway of leptin receptor signal transduction
Jak2 is phosphorylated
Ras/raf/MAPK kinase
PI-3 kinase
STAT3
Gene transcription and ion channel influence
Where are leptin receptors expressed within the brain?
Specific hypothalamis nuclei (ARC, VHM, DMN = energy balance)
mRNA/protein in cerebellum, hippocampus, brain stem and amygdala
Role of lepin receptors at these extra-hypothalamic areas is unclear
How does leptin enter the brain?
Main route: saturable transport system via triglycerides
Short LepR isoforms; expressed on brain microvessels, these isoforms bind and internalize leptin
Leptin also reaches the brain via CSF
Lepin can be made and released locally in the CNS; as lepin mRNA and protein are widely expressed within the brain
How can the detection of ltpin production be detected in hippocampal neurons?
Immunocytochemical methods
Fixed and permabalised
Exposed to primary Ab against leptin receptor
Secondary antibody with a fluorescent tag
Visualise with laser scanning confocal microscope to visualise the distribution of these rceptors
Describe the cellular distribution of leptin receptors on hippocampal neurons
Expressed at hippocampal synapses
Marker for synapsin 1 (marker for presynaptic synapse)
Is there an association with lepin receptors and NMDA?
Yes; they co-localise
Specifically NR2A
What responses are observed from the Ca1 region?
Dual component EPSC; fast AMPAR EPSC and a slower NMDAR EPSC
GABAergic transmission inhibited by picrotoxin and Cs+ in patch pipette
What is the impact of leptin on hippocampal synaptic plasticity?
Theta burst stimulation protocol
Normal EPSC = short term potentiation; peak then decay
Leptin; small depression in EPSC but then with a theta burst there is a sharp peak which is sustained (LTP like process)
Is there a role for leptin in NMDAR mediated synaptic transmission?
If NMDAR component is isolated via blockade of AMPARs
Gradual then sustained increase in NMDAR conduction
Leptin enhances NMDAR mediated LTP
What is fura 2am?
Race metric calcium dye; measures intracellular calcium levels
Does leptin influence calcium influx?
Yes; it enhances NMDA induced calcium influx
Monitored via fura-2am
Leptin facilitates this calcium rise via NMDAR activation
Shanley et al; 2001
What are the pathways that allow leptin to modulate NMDAR function?
JAK2
Ras/Raf
PI-3 kinase
What is the molecular composition of NMDARs?
At least one GluN1 with one or more GluN2
Can exist with or without a GluN3 subunit
GluN2 subunits determine the biophysics and pharmacology of NMDARS
In hippocampus; NR2A and NR2B
NR2A = synaptic
NR2B = extrasynaptic
Can leptin influence NMDAR evoked currents via NMDAR subunits?
2 electrode voltage clamp receptors in xenopus oocytes
Expression of NR1a and NR2A alone (no leptin receptors); leptin has no impact on NMDAR current
Expression of NR1a/NR2A + Ob-R; leptin can facilitate NMDAR mediated responses due to activation of these leptin receptors
Furthermore, leptin is able to facilitate a maximal NR1A/ NR2A mediated currents.
Leptin can influence expression of NMDARs at membrane of oocytes
Leptin increases NMDAR receptor surface expression (within 5 mins)
Harvey et al (2005)
Summarise the effect of leptin at NMDAR receptors?
Leptin enhances NMDAR function and facilitates synaptic plasticity (Shanley et al; 2001)
Leptin modulates NMDAR function which involves PI1 kinase and MAPK
Enhanced trafficking of NMDARs contributes to the effects of leptin (Harvey et al; 2005)
Is there a difference in leptin activity at excitatory synapses in younger or older mice?
Juvenile studies; leptin to hippocampal slices; depression in synaptic strength which resolves with washout
Adult mice; facilitates an increase in EPSC which is sustained with washout
Describe the molecular composition of AMPARs
Tetrameric complex of 4 subunits (GluA1-4)
Cytoplasmic C-terminal tails are key for regulation of AMPAR function
GluA1 +4 = long tails
GluA2 +3 = short tails
Specific C-terminal regions determine the trafficking properties of AMPAR complexes
GluA1: calcium permeable, displays inward rectification due to polyamine block
GluA2; calcium impermeable, linear current-voltage relationship
Does leptin have an influence on GluA1 expression?
Yes; leptin increases GluA1 surface expression
Moult et al 2010
What are the mechanisms that underlie leptin induced LTP
Insertion of GluA2-lacking AMPARs into synapse
Moult et al (2010)
You can determine this via recording of the rectification index
Increase in GluA1 surface expression in adult hippocampal slices
Leptin promoters GluA1 trafficking to the synapses (immunocytochemistry)
Leptin induced increase in surface GluA1 is accompanied by an increase in PIP3
Inhibition of PTEN mimics the effects of leptin
Leptin occludes the effects of BpV on synaptic strength (selectively inhibits PTEN)
Inhibition of PETN mimics and occludes the effects of leptin
Describe the role of PI 3K?
Involved with trafficking of AMPARs to synapse
PIP3- signalling molecule to activate downstream signalling molecules and kinases
How does leptin increase PIP3 levels?
Leptin increases PIP3 levels via increased PI3-kinase activity
Leptin increases PIP3 levels via inhibition of PTEN
Summarise the effects of leptin on AMPARs
Increasing excitatory synaptic strength via increasing GluA1 surface expression
Increase in surface GluA1 is assoc with an increase in PIP3 levels
Inhibition of PTEN mimics and occludes the effects of leptin
Leptin regulation of surface GluA1 involves PTEN inhibition
Overall, summarise the effects of leptin at the synaptic level
Facilitates hippocampal LTP via enhancement of NMDAR function
Leptin driven changes in dendritic morphology may contribute to increases in synaptic strength
Trafficking of GluA1 subunits is likely to underlie leptin facilitation of synaptic strength
Leptin inhibition of PTEN underlies trafficking of GluA1 to CA1 synapses
Is there a role for leptin in hippocampal dependent learning and memory?
Leptin insensitive rodents (db/db mice; fa/fa rats) display impaired hippocampal LTP and LTD
Direct administration of leptin into the hippocampus improves memory processing (wayer et al; 2004)
Diabetics often display cognitive impairments (increased risk of AD); obesity (leptin resistance) is a common risk factor for T2DM (gispen et al 2000)
What can be seen in leptin insensitive rodents hippocampal LTP?
Zucker lean (normal leptin function): HFS, induced robust LTP Zucker fa/fa (leptin insensitivity); HFS, marked reduction in hippocampal LTP
Are there behavioral impact associated with this reduction in hippocampal LTP within zucker fa/fa animals?
Yes; spatial memory deficits
Longer swimming distance in fa/fa animals in MWM
Fewer crossings of original platform locations in fa/fa rats than controls
No difference in swimming speed between fa/fa rats and cntl rats
Is there a therapeutic potential of leptin?
Leptin modulates plasticity of hippocampal excitatory circuits and is a potential cognitive enhancer
AD?
- Circulant levels of leptin are lower in AD patients and tg mice with fAD
- Leptin decrease beta amyloid levels in tg mice with elevated b-amyloid load
- Leptin improves memory in a mouse model of b-amyloid toxicity
Can leptin influence LTP in AD/ amyloid beta slices?
Doherty et al (2013); leptin can prevent the detrimental effects of AB on hippocampal LTP
Can amyloid interfere with AMPAR trafficking?
Yes; AB promotes AMPAR internalization (hu et al; 2009)
Leptin can prevent this AB induced internalization (Doherty et al; 2013)
Conclude the ability of leptin as a therapeutic
Reduced detrimental effects of Ab on synaptic plasticity
Leptin prevents AB induced internalisation of AMPARs
Leptin system is a potential novel target for AD
Specific fragments of leptin peptide mimic the anti-obesity actions of leptin
Small-leptin fragments that mimic the cognitive enhancing and neuroprotective actions of leptin may have therapeutic effects in AD
Can leptin fragments mimic leptin action?
Yes; Malekizadeh et al (2017)
Amino acids 22-56 and 116-130
116-130 is involved with assay of increasing surface expression of GluA1
Leptin (116-130); protects against the aberrant effects of AB
Conclude the evidence for leptin fragments and their therapeutic benefits
Leptin (116-130) displays cognitive enhancing properties
Leptin (116-130) mirrors the neuroprotective effect of leptin against AB
Establishes further the role of the leptin system as a therapeutic target for AD
Leptin (116-130) is a possible template for small molecules that mimic leptin action
What are the peripheral actions of insulin?
Main hormone that controls intermediate metabolism having actions in liver, muscle and fat
Conserves fuel by enhancing uptake, utilisation and storage of glucose
Growing evidence that insulin has widespread actions in the CNS
Describe the insulin receptor
Large transmembrane glycoprotein complex
Consists of two alpha and two beta subunits linked by a disulphide bride
Alpha subunit: extracellular and have an insulin binding domain
Beta subunit; transmembrane protein with intrinsic tyrosine kinase activity
How can the insulin receptor expression be investigated within the CNS?
Immunocytochemistry; antibodies against IR subunits to determine cellular localisation of IR
In situ hybridisation: [S]dATP labelled antisense oligonucleotide probes for IR subunit. This detects IR mRNA in specific brain regions (NB. mRNA expression does not indicate functional protein)
Western blots and PT-PCR; used to determine specific localisation of IR mRNA and protein within different brain regions
Are insulin receptors present within the hippocampus?
Schwartz et al; 1992: Insulin receptor immunolabelling and mRNA expressed in the hippocampus
Havrankova et al; 1978: expression of insulin mRNA in the hippocampal neurons
Folli and Baskin; 1994: key components of the insulin receptor driven signalling pathways (IRS protein and PI 3-kinase) have been found in the hippocampus
Describe the work of Zhao et al (1999) who used in situ hybridization to localise IR mRNA
IR mRNA present in the hippocampal CA1 cells
What is MAP-2?
Somatodendritic marker
Where on the neuron does the IR localie?
Good expression on neurons
Synapsin 1 and IR co-localise, and therefore they localise at the synapse
How does the peripherally driven insulin enter the brain?
Active transport across the BBB
Saturable insulin transporter exists
Insulin transporter is a regulated system i.e. shut off during hibernation and alerted with fasting and diabetes
Insulin can also be released locally within the CNS:
Insulin mRNA expressed in neurons
Insulin released from neurons by depolarisation
What is the functional role of insulin within the CNS?
In periphery: regulated glucose metabolism
CNS: insulin mediates cerebral glucose uptake but only in glial cells, not neurons
Insulin has effects on hippocampal synaptic function
Is there a role for insulin in learning and memory?
Epidemiology studies indicate that long term hyper-insulinemia is a risk factor for dementia
High insulin levels results in a resistance to insulin - and therefore a reduced level of insulin to the brain
Craft et al; 1999: insulin administration to healthy individuals (glucose levels kept constant) improves memory formation
How does insulin influence learning and memory?
IRs are concentrated in brain regions involved in learning and memory (hippocampus and amygdala)
Man et al; 2000: insulin induces changes in excitatory synaptic strength
Injection of streptozotocin, a diabetic inducing agent into the brain results in significant memory impairment
Defective insulin action has been reported in AD (metabolic imbalance in AD)
T2DM; impairment of cognitive function is well documented
Does insulin modulate NMDAR function?
Yes;
Facilitates native NMDAR responses (hippocampal)
Recombinant NMDAR mediated currents
Insulin enhances:
- NMDAR single channel currents
- NMDAR mediated synaptic transmission
Insulin selectively enhances NR1/NR2A and NR1/NR2B recombinant NMDAR isoforms (Lui et al; 1995)
Does insulin potentiate NMDAR currents?
Skeberdis et al; 20001:
Xenopus oocytes expressing NR1-4a/ NR2A subunits, under -60mV voltage clamp
- Insulin enhances the inward currents evoked by NMDAR
- The general tyrosine kinase inhibitor, genistein did not prevent insulin action
- Tyrphostin A47, a selective inhibitory or IR tyrosine kinase blocked insulin action
Describe IR signal transduction
Insulin binds to alpha subunit causing autophosphorylation of tyrosine residues on beta subunits
This stimulates intrinsic tyrosine kinase activity of IR
Phosphorylated IR triggers activation of different signalling cascades including: IRS proteins and various Src homology domain proteins (PI3-kinase)
Shc (adaptor protein); mediates association of IR with Grb-2SOS complex (activates Ras-Raf-MAPK cascade)
Do the signalling pathways that mediate insulin facilitate NMADR responses?
Skeberdis et al 2001: tyrphostin blocks NMDAR responses
Liao et al; 1999; PKC inhibitors block insulin action at NMDARs
Liao and Leonard; 1999: Src TK inhibitors block insulin action at NMDARs
Christie et al 1999: insulin causes tyrosine phosphorylation of NR2A/ NR2B subunits
Does insulin enhance the surface expression of NMDARS?
Skeberdis et al; 2001:
Recordings from oocytes expressing NR1-4a/ NR2A. Insulin increases maximal NMDAR currents
Insulin increased number of functional channels/ cells. Currents recorded in MK-801 (use dependent NMDAR antagonist). NMDAR currents peaked and they decayed as the channels opened and were blocked by MK-801. Cumulative charge transfer, Q (area under curve) is larger after insulin indicating an increase in no. of NMDAR channels
Normalised currents to analyse decay time constants. Insulin did not change decay time i.e. no change in NMDAR channel kinetics
How does insulin act to increased NMDAR surface expression?
Skeberdis et al; 2001
Increases exocytosis of NMDARs:
- Injection of botox into oocytes reduced insulin enhancement of NMSAR currents
- Botox cleaves SNAP-25 and prevents SNAP-25 dependent exocytosis
- PKC can directly phosphorylate SNAP-25. Insulin simulation of atypical PKC downstream of PI 3-kinase may underlie this effect
Using calcium imaging studies, can insulin modulate Ca2+ influx via NMDAR channels?
Neurons loaded with Ca2+ sensitive dye, Fura 2-AM
CCD-based imaging system used to measure changes in Ca2+
What signalling pathways are involved with the insulin induced NMDAR responses?
PI 3-kinase
MAPK
Summarise the effects of insulin potentiation of NMDARs
Insulin does not affect basal Ca2+ levels
Insulin potentiates NMDAR induced Ca2+ influx in a reproducible and reversible manner
Inhibitors of PI 3-kinase and MAPK (ERK) attenuate this action
Increased NMDAR exocytosis and trafficking to cell membrane
Describe the impact of insulin on excitatory synaptic transmission
Insulin facilitates NMDAR mediated synaptic transmission = promotion of LTP induction
Man et al; 2000
- Insulin depresses AMPAR mediated EPSCs in the hippocampal neurons (reducing surface AMPAR expression) = modulates LTD
Does insulin induce LTD?
Huang et al 2004:
Insulin can induce a novel form of hippocampal LTD
Concentration dependent effects of insulin on synaptic efficacy
Low conc of insulin = transient synaptic depression
High concentrations = LTD
What receptor does insulin LTD involve?
Huang et al; 2004
IR; not IGF-1R
Insulin induced LTD is unaffected by anti-IGF1 Ab
IGF-1 induced LTD is blocked by anti-IGF1 antibody
Where is this form of insulin induced LTD expressed?
Post-synaptic locus of expression
Depresses AMPAR EPSCs but not NMDAR EPSCs
Describe the role for AMPAR internalization in insulin induced LTD
Man et al (2000); insulin reduced GluA2 but not GluA1 surface expression
Insulin accelerates endocytosis of GluA2 subunits
Increased endocytosis results in LTD of excitatory synaptic transmission
Surface Rs labeled with Ab in non-permanent conditions (green). Total Rs were probed under permeant conditions
Insulin has no effect on GluA1 but results in a reduction of GluA2 surface expression
Insulin promotes clathrin mediated endocytosis of AMPARs;
Surface AMPARs are pre-labelled with a time dependent reduction in surface AMPARs
Insulin accelerates the loss of AMPARs compared to control
Hypertonic sucrose blocked insulin induced loss of surface AMPARs
How does AMPAR internalization occur?
Involves a series of events;
Formation of clathrin coated vehicles and an intracellular pool of receptors
Hypertonic sucrose can block this
Summarise the role of insulin on AMPARs
Man et al; 2000:
Insulin inhibits AMPARs but not NMDAR EPSCs
IV curves show that insulin reduces EPSC currents
Insulin induced LTD is dependent on post-synaptic activation of IR
Insulin induced LTD can be blocked by postsynaptic dialysis with IR neutralising Ab (anti-IR)
Summarise the role of insulin on LTD
Induces a novel form of hippocampal LTD which is expressed post-synaptically
This process involved PTK, PI 3-kinase and PCK
Involves internalization of Glu-A2 containing AMPARs
Is there a role for insulin during learning and memory in rodents?
Zhao et al (1999): examined changes in IR expression, tyrosine phosphorylation and IR signalling molecules after training in MWM
Training upregulated IR mRNA and protein levels with CA1 and dentate gyrus
An increased in IR immunostaining
After training, staining was clustered within specific intracellular compartments, compared with an even distribution in control animals
Describe the biochemical changes that occur following MWM training (insulin)
Zhao et al (1999):
- 2 proteins (52 and 66 KDa) showed an increase in tyrosine phosphorylation after training
One protein: Shc-66 co-precipitated with IR suggesting these 2 proteins associate in vivo after training
Significant accumulation of Grb (52 KDa) in hippocampal synaptic membrane function
Increase in active form of MAPK were also detected
Spatial training induces a number of changed within the insulin system
Is there a link between insulin and neurodegenerative disorders?
Insulin has cognitive enhancing properties
T2DM; cognitive impairment and AD like phenotype
De Felice et al 2009; bolstering of brain insulin signalling could have significant potential to slow or deter AD
Describe the link between insulin and ADDL
De Felice et al; 2009:
AB derived diffusible ligands (ADDLS) induce hippocampal synaptic loss
Treatment with insulin prevents this loss
Lee et al 2009:
- HPS induced LTP in control slices, but failed to induce LTD in AB-1-42
Co-incubation of slices with insulin prevents this AB1-42 induced block of LTP
Incubation of insulin alone had no significant effect on the LTP induction
Summarise the role of insulin in AD
Cognitive enhancing properties
Insulin markedly influences hippocampal synaptic plasticity and glutamate receptor trafficking
Insulin (like leptin) reduced detrimental effects of AB on synaptic plasticity (LTP and LTD)
Insulin prevents AB induced hippocampal synapse loss
Insulin system is a potential novel therapeutic target for AD
