TARGETING UNFOLDED PROTEIN RESPONSE IN NEURODEGENERATION
Shahan ullahMphil pharmacology
Institute of Basic Medical SciencesKhyber Medical University Peshawar
INTRODUCTION Neurodegenerative diseases are one of the greatest
challenges facing society at present Ageing population
Increased prevalence Increased morbidity 2nd most important morbidity factor in 2040
Alzheimer’s, Parkinson, Prion and huntingtin diseases are neuro diseases with a shared similarity of Protein aggregation in brain Fatal neuronal loss
This group of diseases are termed as “Protein misfolding disorders”
DISEASESDisorder Misfolded protein Cause
Alzheimer’s disease Amyloid-β Toxic to synapse,reduced synaptic transmission,reduced no of synaptic dendrites
Parkinson disease α synuclein Mitochondrial damage,Cellular death in substantia nigra
Huntingtin’s disease Huntingtin Inclusion bodies formation-Interfere normal cellular process-Induce protein misfolding
Prion disease Prion proteins Neuronal blockage mainly and neuronal loss
NEURODEGENERATION Starts with; Synaptic disfunction Loss of dendritic spines Loss of post synaptic density
ultimate neuronal network failure Cell death
Cellular process: Mitochondrial dysfunction Protein recycling
Recently “Unfolded protein response” has been emerged as a central player in prion disease and shared a common feature in neurodegenerative disorders
UNFOLDED PROTEIN RESPONSE In eukaryotic cells endoplasmic reticulum is
responsible for Secretory and membrane protein synthesis Folding(b/c only folded proteins are forwarded to golgi
apparatus and un/misfolded ones are degraded by ERAD ) Assembly Modification Quality control
Sensitivity: Calcium depletion Oxidative stress Hypoxia Energy depletion Increased protein synthesis Increased misfloded proteins
UNFOLDED PROTEIN RESPONSE Misfolded proteins accumulation trigger the ER
signaling events and multiple strategies are followed;
1. Increased ER folding capacity2. Decreased global protein synthesis3. Enhanced Endoplasmic reticulum associated
degradation(ERAD) of misfolded proteins4. Production of chaperone proteins
i. Ensure correct foldingii. prevent protein aggregation
THREE ARMS OF UPR
1.PROTEIN KINASE RNA LIKE ER KINASE(PERK)Eukaryotic translation
initiation factor 2 alpha((eIF2a)
1. Repress translation2. Halts global protein
synthesis3. Alleviate overload of
misfolded proteins in ER
Activating transcription fator 4(ATF4)
targets;1. NRF2: regulates
function of variety of antioxidant genes
2. Regulate genes associated with1. Protein
folding2. Amino acid
metabolismCHOP: A key in activation of apoptotic pathways and cell death
2. INOSITOL REQUIRING ENZYME 1(IRE-1)
IRE-1 are two of the kind: IRE-1 alpha: kinase and endonuclease Upon activation, catalyze
Splicing of “mRNA encoding transcription factor XBOX binding protein-1”
Results in potent transcription factor that regulates Subset of UPR target genes involved in
ER protein synthesis Protein folding ERAD Redox metabolism
IRE-1 beta: controls site specific cleavage of 28S rRNA Which contributes in translational repression
3. ACTIVATING TRANSCRIPTION FACTOR 6(ATF6)
Chaperones:
GRP78/BiP, GRP 94
TARGETING UPR IN PRION DISEASE Prion proteins(PrP) production and
accumulation; Disease progress;
Reduction in number of synapses, synaptic proteins level
Loss of object recognition memory Reduction in burrowing activity Reduction in hippocampal synaptic transmission
Severe case after several weeks Increased levels of misfolded PrP Clinically ill PrP levels inversely proportional to incubation period
and onset of death
CONT… Reduction in synaptic protein levels either
result from Increased degradation Reduced synthesis
Ubiquitin proteasome pathway is known to be inhibited in prion disease causing Reduction in synthesis NOT increased degradation.
IS THAT SO?
CONT… Hypothesis: Translational repression by UPR was the cause of decrease
in proteins As levels of misfolded Prp increased during disease as its
synthesized over there Found out: Progressively increased
phosphorylated PERK eIF-2alpha
Level of eIF2alpha increases but GADD34 levels were not altered
Which shows that there is insufficient level of GADD34 to dephosphorylate eIF2 alpha
This shows that in Prion disease PERK/eIF2alpha arm of UPR was activated
Leading to inhibition of protein translation Reduction of synaptic protein levels
CONT… Confirmation: Total protein synthesis rates were measured in
hippocampus via uptake of radioactive Methionine(S35) Reduced ribosome 9wpi Reduced mRNA translation Reduction in active translation of SNAP-25 and beta-actin
BUT: ATF4 mRNA active translation was increased
b/c it escapes inhibition by eIF2alpha due to 5’-Untranslated regions(5’-UTR)
PrP didn’t show reduce translation because of similar translational control elements (5’-UTR of PrP gene)
As total mRNA levels remain unchanged; So reduction in protein synthesis in Prion disease is
controlled at Translational level Not transcription level
CONT… eIF2alpha phosphorylation is beneficial to
cells in ER stress But its persistent increased level is
detrimental To test that eIF2a-P is REALLY involved in
Prion neurodegenrative disorder invivo? It was tested that reducing the eIF2a-P
levels will be then neuroprotective SO…
CONT… GADD34 is over expressed using lentivirus vector To reduce eIF2α-P level
Targetted RNA interference(RNAi) of PrP was used to remove
UPR activation Prevent eIF2α-P formation
Also it’s checked that whether increased level of eIF2α-P is neurotoxic?
“Salubrinal” eIF2α-P dephosphorylation inhibitor was used
CONT…1. At 9 wpi mice injected with lentivirus
vector over expressing GADD34 had same level of PERK-P as those in prion infected mice with no treatment at all
Showing UPR was activated But eIF2α-P level was reduced2. RNAi against PrP prevented Prion induced
PERK-P & eIF2α-P as those seen in untreated mice• Confirming prevention of UPR activation
So GADD34 over expression and PrP knockdown restored global translational rates at 9wpi.
05/02/2023Shahan ullah Mphil pharmacology
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CONT…3. As a result
Less burrowing effect Neuronal protection synaptic protein levels Synaptic transmission No of synapses
were protected and restored as compared to uninfected control mice
It was measured that the over expression of GADD34 eIF2α-P level reduction
significant survival effect
CONT… The mice administered with Salubrinal
have an high eIF2α-P levels at 9 wpi than prion only controls
Causing Repression of global translation Accelerated disease Early neuronal loss
DISCUSSION All these data demonstrate that UPR manipulation
represents a novel target for treatment in prion disease
Genetic manipulation was helpful in mice models Bur carry risk in humans regarding
Immune reaction Insertional mutagenesis
An attractive target is PERK inhibition for drug discovery as eIF2α phosphorylation is inhibited Downstream pathological translational repression
Allowing Chaperone proteins expression via
IRE-1 ATF6 arms of UPR
CONT… Importantly some PERK inhibitors are
used now a days as anti tumor agents it’s possible that these or related compounds optimized for penetration of blood brain barrier would be potential therapeutic agents to allow for the development of new compounds for treatment of over activation of PERK branch of UPR
Thanks!