Lecture neurodegenerative disorders 6: treatment for neurodegenerative disorders (therapy 3)

Question 1

The Unfolded Protein Response (UPR) is the cell’s stress response. What three protein mediate the UPR?

Answer 1

IRE1α, PERK and ATF6

Question 2

Name examples of UPR target genes.

Answer 2

See picture.

Question 3

IRE1α, PERK and ATF6 all contribute to the transcription of UPR target genes. Shortly describe the contribution of IRE1α.

Answer 3

• When IRE1α senses ER stress, it dimerizes as a homodimer.
• The cytoplasmic tails of IRE1α get autophosphorylated..
• Phosphorylation causes activation of a ribonucleuase domain, which splices XBP1 mRNA.
• The exonic XBP1 mRNA acts as an transcriptional activator and can induce transcription of UPR target genes.

Question 4

IRE1α, PERK and ATF6 all contribute to the transcription of UPR target genes. Shortly describe the contribution of PERK.

Answer 4

• When PERK senses ER stress, it dimerizes as a homodimer.
• The cytoplasmic tails of PERK get autophosphorylated.
• This leads to phosphorylation of the α subunit of eIF2α, therefore activating eIF2α.
• eIF2α activates ATF4.
• ATF4 acts as a transcription factor and can induce transcription of UPR target genes.

Note: eIF2α also inhibits translation, to limit further ER stress. The action of eIF2α can be deactivated through ATF4, which activates the transcription factor CHOP, which in its trun activates PP1C and GADD34. These two proteins can inhibit eIF2α action.

Question 5

IRE1α, PERK and ATF6 all contribute to the transcription of UPR target genes. Shortly describe the contribution of ATF6.

Answer 5

• ATF6 is a membrane-bound protein. When ATF6 senses ER stress, it translocates to the ER lumen.
• In the Golgi apparatus, site-1 and -2 protease (S1P an S2P) cleave ATF6 between the extracellular and cytoplasmic domain.
• The cytoplasmic domain is cleaved and is now called ATF6f.
• ATF6f can induce transcription of UPR target genes.

Question 6

Some processes in the Unfolded Protein Response (UPR) can be measured. Name these.

Answer 6

• There are three phosphorylation processes (IRE1α, PERK and eIF2α phosphorylation), measured by phosphor-specific antibodies.
• Splicing of XPB1 mRNA
• All three pathways lead to upregulation of transcription. The mRNA can be measured here.
• In all three pathways, there’s translocation of proteins, like transcription factors that translocate to the nucleus. This can me monitored by immunocytochemistry.

Question 7

For measuring phosphorylation and translocation, in situ tests can be useful. What are advantages of in situ tests?

Answer 7

• No dilution if sparse
• Co-localization with pathology

Question 8

In what disease is UPR activation an early event?

Answer 8

In Alzheimer’s disease.

Question 9

With what is the UPR specifically associated in Alzheimer’s disease?

Answer 9

With early tau pathology in the subregions of the hippocampus (CA1-4)

Question 10

What can be concluded based on these pictures?

Answer 10

Depicted in the pictures is AT8 (Tau) in red and pPERK (UPR) in green. If you combine the two in one picture (pPERK/AT8), you hardly see any green (left picture). This is if tau aggregates are already dense (in later Braak stages). However, in earlier stages, where tau aggregates are no so dense yet, you do see some green left (right picture). Indicating that in earlier stages of AD and tau aggregation, there is UPR activation.

Question 11

Besides the UPR target genes that are regulated by IRE1α, PERK and ATF6, a part of the UPR is also responsible for the inhibition of further protein synthesis. Therefore preventing further accumulation of misfolded protein and ER stress. By what protein is this regulated?

Answer 11

This is regulated by PERK. When its cytoplasmic domains are autophosphorylated and eIF2α is also phosphorylated and activated, eIF2 will reduce translation.

Question 12

What happens if prion proteins are injected into the brain?

Answer 12

There’s activation of PERK (→ pPERK) and also p-eIF2α.

When injection prion proteins in the brain, we see pPERK activation. And also activation of P-elF2-alpha. Does this also lead to a reduction in protein synthesis?

Question 13

What does injection of prion proteins, followed by activation of PERK and p-eIF2α, lead to?

Answer 13

It leads to the inhibition of protein synthesis.

Question 14

Why does UPR activation also lead to a reduced level of synaptic proteins?

Answer 14

Because when the protein synthesis gets reduced, it doesn’t get back to normal synthesis. This also affects other essential proteins, like synaptic proteins SNAP25 (presynaptic) and PSD95 (postsynaptic). This will cause neurodegeneration.

Question 15

There are several ways to intervene in the PERK pathway. What two things are commonly used?

Answer 15

Lentiviral activation or use of short hairpin RNA (shRNA) for knockdown

Question 16

A mouse model was made with several mice with different transcription patterns:

• Control
• Prion only
• Prion + Salubrinal (inhibition of dephosphorylation of eIF2α)
• Prion + Lentiviral control
• Prion + Lentiviral-GADD34 (increases dephosphorylation of eIF2α)

Describe shortly what patterns of synaptic functions the mice annotated in bold letters displayed.

Answer 16

• Prion + Salubrinal → Inhibition of dephosphorylation of eIF2α decreases synaptic function, because phosphorylation leads to inhibition of protein synthesis.
• Prion + Lentiviral-GADD34 → Increasing dephosphorylation of eIF2α decreases inhibition of protein synthesis, so the levels of protein synthesis are resued. This also rescues to amount of neurons, the synaptic number and the synaptic function and therefore prevents (further) neurodegeneration.

Question 17

What happens if PERK is used as a therapeutic target by inhibiting PERK?

Answer 17

PERK inhibition leads to inhibition of all the downstream steps (so no phosphorylation of eIF2α, no reduction of translation, no loss of critical proteins and no neurodegeneration). It’s seen that inhibition of PERK, rescues protein synthesis and synaptic proteins.

Question 18

Besides that PERK inhibition rescues protein synthesis, can it do something else?

Answer 18

Yes, it doesn’t only rescue protein synthesis, but also interferes with tau-pathology. This is seen by the fact that, compared to control mice with tau pathology, there’s less tau aggregates in the brain and more CA1 neurons (and increased total brain weight). This is also seen from the outside: a mouse with tau pathology has poor grooming, hunched posture, hind-leg clasping and poor mobility, while the mouse with PERK inhibition doesn’t have this.

Thus: PERK inhibition inhibits neuronal loss and accumulation of p-Tau.

Question 19

Are there side effects of PERK inhibition/knockout?

Answer 19

Yes, it causes a severe side effect → diabetes. We could have known about this, since PERK knockout mice also have reduced levels of insulin.

Question 20

What is a solution to the fact that PERK inhibition doesn’t work due to the severe side effect of diabetes?

Answer 20

By inhibiting the effects of the effector protein downstreams of PERK → eIF2α. This is done with compound ISRIB. Since it doesn’t affect PERK, you avoid pancreatic toxicity (i.e. diabetes).

Note: so ISRIB doesn’t affect eIF2α itself, but it affects the effects of eIF2α.

Question 21

What happens to transcription when mice with prions are treated with ISRIB?

Answer 21

First of all, when looking at the transcription intensity of eIF2α(-P) no difference is seen. This makes sense, since ISRIB affects proteins downstreams of eIF2α. So what you do see is a reduced expression of ATF4 (unfortunately this effect is not as strong as it is with PERK inhibition).

Question 22

What happens to neurodegeneration when mice with prions are treated with ISRIB?

Answer 22

Neurodegeneration is reduced and there’s no pancreatic toxicity. Unfortunately, compared to PERK inhibition, the effect is not as strong.

Question 23

Two drugs that are already registered (licensed as antidepressants) can be used for neurodegenerative treatment. These drugs are Salubrinal and Guanabenz. What do these drugs do and what are advantages/disadvantages?

Answer 23

• Salubrinal inhibits all dephosphorylation, including dephosphorylation of eIF2α (not convenient, since this is the problem with persistent UPR activation → it leads to constitutive inhibition of protein synthesis).
• Guanabenz only inhibits stress induced desphosphorylation, which is more convenient since it can regulate eIF2α-P protein synthesis inhibiton in a more specific way. Only guanabenz is normally used for hypotension, so the side effect is that it interacts with α2 adrenergenic receptors and causes hypertension.

Question 24

How can we solve this side effects of Guanabenz?

Answer 24

By looking for a compound that is struturally similar to Guanabenz. This compound has been identified as Sephin1, which doens’t have the same side effects and it is able to prevent neurodegeneration.

Question 25

So we’ve discussed several interventions in the PERK pathway. A huge opposite effect is seen between Sephin1, Salubrinal and Guanabens and ISRIB and PERK inhibitors. What differnence is seen?

Answer 25

• Sephin1, Salubrinal and Guanabenz prevent dephosphorylation of eIF2α. So more eIF2α remains active and protein synthesis remains inactive.
• ISRIB and PERK inhibitors act more upstreams in the cascase and have a broader inhibiton of the UPR.

Question 26

So if one group of drugs is responsible for stimulation of the UPR and the other is responsible for inhibition of the UPR. What is important to consider before you start thinking of using the drugs for treatment?

Answer 26

If you want to cure or prevent the pathology.

Question 27

What happens if you want to prevent development of pathology by using Sephin1?

Answer 27

So the treatment with Sephin1 was started first, before they induced pathology. This means that the cells are already ‘ready’ to react to the problem when they encounter it. Unfortunately, pathology is already induced by the fact that you stimulate the UPR with the use of Sephin1 (because you stimulate inhibition of dephosphorylation of eIF2α and therefore inhibit protein synthesis), before pathology has even been induced.

Question 28

What happens if pathology development is first induced, before you start treatment?

Answer 28

Then drug treatment is not enough