Mandy Flashcards
EAATs, VGLUTs, BIII-spectrin
How is EAAC1/EAAT3 an anti-oxidant?
EAAT3 also uptakes L-cystine, which is rate limiting factor for gluthionine (anti-oxidant), and loss of EAAT3 increases oxidation of lipids and proteins, and neurodegeneration
Implicated in PD since those neurons have a lot of EAAT3-> trying to combat susceptibility to oxidative stress
PD= parkinsons disease
How is glutamate transporter dysfunction implicated in aberrant neurotransmission and other neurological disorders?
Loss of EAAT1= episodic ataxia, P209R reduces iCl- and glu cleraence, and impairs glilal morphology
Loss of EAAT2= motor neuron disease and PD, downregulation (bc miRNAs) or decreased recycling (bc LRRK2) in astrocytes means glu-toxciticy-mediated neurodegeneration
Loss of EAAT3= PD, alzheimers, dicarboxcylic amioaciduria- increased oxidative stress and reduced memory/learning (role in synaptic plasticity)
Loss of EAAT4= ataxia, in area of cerebellum with high EAAT4+ causes depolarisation and burst firing from increased mGluR activation. (also associated with loss of betaIII-spectrin
Enhanced VGLUTs= epilepsy, increased Cl- facilitates inc packaging, more packaging leads to greater release and excitability
Loss of GLAST impairs differentiation of fine bergmann glial cell processes which wrap around purkinje cell dendritess and synaptic inputs
EAAT3 decreases mGluR1 activation and increases D1R activation and ensures LT synaptic plasticity - new role for shaping glutamatergic and dopaminergic signalling and controlling repeated movement execution
cerebellar dysfunction associated with ASD/ depression/ alhziemers/schziophrenia
What is the evidence implicating loss of GLT1 (EAAT2) in motor neuron disease (ALS)?
Decreased transport of glutamate in ALS post-mortem tissue
Can see decreased EAAT2 in ALS patients (via antibody staining)
Used THA to block glutamate transport which resulted in MN death (implicates role of EAAT in ALS)
EAAT2 controls the glutamate-glutamine shuffle in the CNS, and disrupting this results in increased Glu conc at the synapse = motor neuron death.
What are the different ways expression of plasma membrane glutamate transporters can be modulated?
- Neuron conditioned media
- Astrocyte conditioned media
- Beta-lactam antibiotics
- miRNAs
anything that affects the above factors would impact Glu clearance and thus action
What is the experimental data implicating a role for EAAC1 (EAAT3) in transporting cystine?
- Radiolabelled glutamate, then observed uptake in presence of L-cysteine; see that L-cysteine is competing with glutamate for EAAT3 uptake
- Fluorescently tagged thial groups in glutathione and saw that there was decreased levels in EAAT3 KO; implicating that there was more L-cysteine (which reduces glutathione synthesis) due to KO of EAAT3
Where are each of the EAATs located?
EAAT1: astrocytes in cerebellum
EAAT2: glilal cells in the CNS
EAAT3: neurons in CNS (hippocampus, cerebellum, basal ganglia)
EAAT4: purkinje cells in the cerebellum (banded expression) (located perisynaptically)
EAAT5: retina
EAAT4 and 5 high Cl- conductance possiblely to prevent local depolarization (since Glu is uptook with Na+), Cl conductance could maintain membrane potential so its optimized for Glu intake
What is the role of betaIII spectrin in cerebellar disease?
Beta III-spectrin mutations= spinocerebellar ataxia type 5
Needed for stabilisasion of EAAT4 in the membrane, in KO there is loss of EAAT4 at the membrane, so extra glutamate in the synapse binds mGluR which increases iCa2+ which activates CaMKII which phosphorylates GluA1 subunit on existing AMPARs which increases excitability
Loss of BIII-spectrin= loss of GLAST= cerebellar ataxia
Loss of BIII changes morphology of purkinje cells and disrupts the planarity which is key for maintaining synaptic input
Where are the VGLUTs located?
VGLUT1= glutamatergic neurons in hippocampus/cortex/cerebellum
VGLUT2= glutamaterigic neurons in brainstem
In cerebellum, VGLUT2 is expressed in climbing fibres and VGLUT1 is expressed in granule cells
Both are essential for life, vglu 2 KO mice are not viable at birth, vlgut1 KO mice survive to 2 weeks post birth
What is the role of VGLUT in disease?
Epilepsy- ketone diet used to reduce seizures since acetoacetate (ketone body) competes with Cl- for transport binding by VGLUTs thus reduces packaging into vesicles and thus reduces glutamate release and recduces seizures
What are the function of EAATs?
- Take up and transport Glutamate
- prevent glutamate toxcitiy
- Modulate excitability
- Determine EPSC duration
- Supply precursor for GABA and glutathione
Glu toxcitiy arises bc it activates intracellular Ca2+ release which has multiple downstream pathways and results in neurodegeneration
Rank the EAATs from fastest to slowest
EAAT4> EAAT2~EAAT3 > EAAT1 >EAAT 5
How do EAATs maximise their glutamate clearence capacity?
Especially since their kinetics are super slow
- Shape (bowl shape allows glutamate sequestering/imobilisation)
- Density (high density of EAATs at the membrane allows them to take up a large amt of glutamate
How does VLGUT isoform affect release probability?
VGLUT1= lower release probability (PPF)
VGLUT2= higher release probability (PPD)
Because VLGUT1 can bind endophilin (preventing it from doing its job) and VGLUT2 cannot, so by preventing endophilin from faciliating release of vesicles VGLUT1 is lowering the release probability
endophilin is needes to bend the membrane, aka vesicle fusion
How does expression level of EAATs alter synaptic plasticity?
- The more EAAT4 expressed, the less glutamate there is to activate mGluR, the less LTD; thus different EAAT4 levels contribute to LTD
- The more EAAT4 expressed, the less glutamate there is to activate AMPAR on glial cells; thus different EAAT4 levels modulate extrasynaptic receptors
