Disease Flashcards
(40 cards)
Stroke 1
- Reduction in mRNA encoding ADAR2 -down-regulation of ADAR2 expression = more CP-AMPARs (less Q/R editing of GluA2-AMPARs)
- Expressing ADAR2 ischemia rescues change of rectification seen + rescues neurones from cell death (less FJ+, more NeuN)
Stroke 2
Activation of REST (Repressor Element Silencing Transcription Factor)
Physiological conditions:
- P by CKI kinase
- Ubiquitanated by beta-TrCp
- Lysosomal degradation
Stroke
- Down-regulation of CKI kinase + beta-TrCp
- REST not degraded = repress GluA2 transcription (amongst other genes)
Propidium iodide
-Knock-down REST = less fluorescence cells (neuronal cell death) compared to control
Markers of cell death
FJ+ = dying cells (non-specific)
Activated caspase-3 = marker of apoptotic cells
Propidium iodide - fluorescent; bind to DNA in dying cells (enters via rupturing membrane)
Stroke - AMPAR trafficking
Increase in PICK1-dependent GluA2-internalisation
Hippocampal brain slices, 30 min OGD (buffer deprived of oxygen + glucose) - rectification seen immediately after 30 min exposure
Peptide - block PICK-GluA2 = blocks changes in rectification
Increase in GluA2-containing AMPARs internalisation = synapse is more Ca-permeable!
44,000 compounds - identified FSC-231 - inhibit PDZ domain on PICK1, been used in vivo!
Increase in trafficking to the lysosome = increased lysosomal degradation!
-no immediate change
-15-min post-OGD = small increase in co-localisation
- <30-min post-OGD = increase in co-localisation
-1-hour post-OGD = no co-localisation = had been degraded
**GluA2 lysosomal degradation relatively late event during stroke = novel target for therapeutics????
Acute application of a peptide to block GluA2 degradation and redirect to the synapse
Hippocampus vs Cortex
Hippocampus = PICK1-dependent internalisation of GluA2-containing AMPARs = synapse is more Ca-permeable
Cortex = no internalisation of GluA2-containing AMPARs = less neuronal cell death!!!!
Due to less excitotoxicity
Cortical neurones shrink more
Hippocampus = more activated Rac (increased inactivation of cofillin) - due to increased expression of Tiam1 (Rac GEF)
Knock-down Tiam1 (shRNA) = more spine shrinkage, hippocampus acted like cortical neurones, decreased neuronal cell death
Why don’t hippocampal neurones enlarge in addition to remaining stable at the synapse (Rac is activated)? There must be other factors inhibiting the enlargement of spines (ie. inhibiting Arp2/3) - just preventing de-P of cofillin is enough to prevent shrinkage!
Spine size reduction = neuroprotective - less CP-AMPARs at the synapse
Increased rac (hippocampus) = more NOX
- lipid perioxidation
- DNA oxidation (alter gene expression; up-reg GluA3; replace GluA2 = GluA1-GluA3 CP-AMPARs)
- histone phosphorylation (involved in cell necrosis)
Hippocampus: CA3 vs CA1
CA1 = more vulnerable - more cell death!
CA1 = AMPAR EPSCs recover more quickly CA3 = PICK1-mediated internalisation of all GluA2 BUT then kept away from the synapse
CA1 neuroprotective = lose all AMPARs, no CP, reduce excitoxicity
Block effect in CA3:
-peptide which blocks PICK1-GluA2
-incomplete recovery of AMPARs to the surface
-INCOMPLETE = suggests another mechanism was acting to keep the AMPARs internalised
or that peptides did not completely block PICK1-GluA2 interaction
Novel stroke therapies (molecular)
FSC231 = inhibiting PDZ domain on PICK1 - less PICK1-mediated internalisation
Inhibiting PDZ domain on PSD-95 - cannot bind TARPs - cannot slot GluA1-AMPARs at the synapse
Hypothermia = decrease in hippocampal AMPARs/NMDARs
Decreased apoptosis of penumbra
Cerebellar head trauma
Traumatic brain injury = TBI
PICK1-dependent GluA2 internalisation = FSC231
TBI = increases P @ Ser880 on GluA2 -modelled = stretch + NMDA = increase phosphospecific ABs via Western blotting
Stretch + NMDA
- decreased spine density
- high levels of NO = increase P @ Ser818 = increased surface GluA1 expression (enhances GluA1-4.1N interaction)
Mechanical trauma = reduces the Mg block in NMDARs - therefore larger Ca influx in stretched neurones > uninjured
Mechanical trauma = elevates IC superoxide levels in cortical neurones
- superoxides regulate PKC
- oxidative modification of PKC = preferential binding of PKC to PSD-95 = anchor PKC to P-GluA2 @ Ser880 = GluA2-internalisation
What do high levels of NO do?
Cause P of Ser818 on GluA1 = internalisation
Altered ADAR2: Diseases
Stroke - down-regulation due to decreased mRNA
Sporadic MND
SZ + Bipolar = proposed as a molecular mechanism underlying the disorder
Thiamine deficiency
Leads to regional defective neuronal loss
Shown to cause alterations to GluA2 pre-mRNA
Inhibits the Q/R editing of GluA2 = more unedited, CP-GluA2
Stroke + K channels
Endogenous mechanism to stroke
Kv beta-1 subunit = N-type inactivation of K channels; chain + ball
DR –> fast, transient A current
Ball = ball of cysteine residues
Stroke = cysteine residues get oxidised, cannot work to occlude the pore - therefore A current –> slow DR current
= decrease excitability due to increased K+ efflux
Amyolateral Sclorosis/Motoneurone Disease
ALS/MND
What is it?
Selective degeneration of motoneurones = muscle weakening + atrophy
General body movement is affected - speaking, walking, breathing, swallowing
Vast majority, 95% = sporadic (no inherited genetic defects)
Some familial cases (SOD, ALS)
None of the genes associated with familial ALS are associated with sporadic ALS
Familial ALS
SOD (superoxide _____) = ‘mops up’ superoxide radicals - O2. –> H2O2
Increase free radicals = DNA oxidation damage
Up-regulation of GluA3 translation
Displace GluA2 –> GluA1-GluA3 = CP-AMPARs
ALS2 gene = LOF mutation
Normally - interacts with + localises GRIP
K/O = GRIP mislocalisation = less GRIP at synapses, more in the spinal motoneurones soma - therefore less synaptic GluA2
K/O mice = subtle motor deficits + neuropathological abnormalities
BUT - fail to develop MND/ALS
Sporadic ALS
-Reduced expression of ADAR2 = more unedited, Ca-P GluA2-AMPARs
-Reduction in the expression of Ca-buffering proteins
Increased IC [Ca]
-Reduced expression of astrocytic glutamate transporters (EAAT1/2) - over-stimulation of AMPARs
PAIN: Activation of macrophages w/ P2X7 = recruitment of pannexin hemichannels, release interleukin-beta, cause direct down-regulation of EAAT1/2
Hyperphosphorylated tau results in…
Mislocalised tau results in…
Decreasing synaptic density
Stimulates endocytosis of AMPARs/NMDARs; prevents exocytosis - maybe due to being unable to deliver IC machinery via microtubules (ie. recycling endosomes)
Inhibits LTP + increases LTD
Tau is normally localised in axons - some in dendrites to regulate LTD
HyperP tau = mislocalisation to dendrites
-Spine loss = increasing GluA2-PICK1 interaction
-Microtubule breakdown (less normal tau to regulate/associate with microtubular network)
-Increase in cytosolic [Ca] (less surface GluA2-AMPARs)
-Abberant kinase activity
Tau + LTD
LTD = phosphorylation of Ser396
Mutant tau, cannot be P @ Ser396 = no LTD occurs!!!
Tau K/O =
- Reduce GluA2-PICK1 interaction during LTD
- Co-immunoprecipitation
Tau is required for increased GluA2-PICK1 binding during LTD (not during basal trafficking)
GluA2-PICK1 requires tau - tau promotes internalisation/AMPAR retention only in LTD (not basal constitutive recycling)
How it regulates this interaction is unclear!!!!
Chicken + egg scenario = what comes first???
Increased P of tau therefore increased LTD
OR increased LTD therefore increased tau P?
AB42 + tau
AB42 oligomers cause misregulation of kinases/phosphatases = hyperP tau
ADDLs (Amyloid Derived Diffusable Ligands) = tau mislocalisation (more in dendrites > axons) + increased P @ various sites (not just Ser396 which is required for LTD)
Mutations in AD + FTD
AD = model amyloidopathy alpha-secretase = ADAM10, ADAM17 beta-secretase = BACE10 gamma-secretase = presenellin 1/2
Frontotemporal dementia (tauopathy) - mutations in MAPT ie. F301L = causes hyperP of tau at various sites
P301L Experiments
P301L = causes behavioural + motor deficits; age-dependent development of NFT
wt = doesn’t enter spines
P301L = enters dendritic spines
Mutant which mimics P = enters dendritic spines
Mutant which blocks P = blocks tau co-localisation in spines
= the phosphorylation of tau is required to enter dendritic spines
P301L = causes reduced basal synaptic transmission (depresses AMPAR responses)
Mutant which mimics P = same effect
Block P = do not get the reduction of EPSPs
= P tau causes a depression in AMPAR responses
Over-express P301L mutant = fewer GluA1 @ synapse
Decreased co-localisation of GluA1 w/ PSD-95 BUT no decrease in PSD-95 puncta
Dendritic spine stability is compromised by loss of AMPARs preceeding spine loss -
- the stability and the existence of dendritic spines can be compromised by prolonged absence of functional synaptic AMPARs, the loss of AMPARs might be a cellular alteration that leads to the degeneration of dendritic spines
- supports hypothesis that GluA2 (via N-cadherin binding) is responsible for dendritic morphology
Too much synaptic depression = cognitive deficits
KIBRA
Gene associated with memory performance
- binds PICK1 to regulate AMPAR trafficking
- works w/ PICK1 to restrict recycling of GluA2
KIBRA has been associated with late-onset AD (GWAS studies)
- via increased acetylated tau
- supports the concept that altered AMPAR trafficking is a critical component of the cognitive deficits observed in AD
Facilitates LTP = regulating GluA1-insertion
Facilitates LTD = works w/ PICK1 to restrict GluA2 away from synapse
KO KIBRA
- impaired LTP expression (no GluA1-insertion) (EPSPs graph)
- impaired LTD = faster recycling of GluA2 to the synapse (look at capacitance graph)
Similar cellular and behavioural phenotype to PICK K/O - suggest they act on the same pathway to regulate GluA2-containing AMPAR trafficking
-severe deficits in contextual fear learning + memory (impaired LTP + LTD)
Acetylated tau
Lysine (K) –> glutamine (Q) = mimics acetylation
= Reduces post-synaptic KIBRA levels
- Less GluA1-inserted = less LTP
- Faster recycling of GluA2-AMPARs
Restore effects:
- Restoring KIBRA expression
- Promoting actin polymerisation
Sources of experimental AB42
ADDLs = Amyloid Derived Diffusable Ligands
Over-express APP w/ familial mutations (ADAM10/17, BACE1, presenellin1/2) in a cell line; produce a lot of AB42 which is secreted into the medium; apply medium onto hippocampal slices
AD Novel Drug
BAN2401 = mouse monoclonal AB which selectively targets large, insoluble AB protofibrils (most toxic)
Very promising in vivo mouse studies
Currently in Phase III
ALSO
Calcineurin inhibitor
-can alleviate AB-induced spine loss
-also could block hyperP tau (hyperactivated calcineurin leads to disrupted interactions between tau + phosphatases, contributing to AB-induced hyperP-tau)
