TARGETING UNFOLDED PROTEIN RESPONSE IN NEURODEGENERATION

Shahan ullahMphil pharmacology

Shahanpharma@yahoo.com

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.

04/13/2023Shahan 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

Finally…

Thanks!