Genetic Reactivation of Cone Photoreceptors...

Question 1

Common pathology of retinitis pigmentosa

Answer 1

rod photoreceptors die early while light-insensitive, morphologically altered cone photoreceptors persist longer

Question 2

Paper’s main finding is that

Answer 2

Expression of archaeobacterial halorhodopsin in light-insensitive cones can substitute for native phototransduction cascade and restore light sensitivity.

Question 3

Question addressed

Answer 3

can light-insensitive cells be reactivated and can information from them still flow to downstream visual circuits

Question 4

how was light-evoked activity restored in cone photoreceptors?

Answer 4

targeted light-activated chloride pump (halorhodopsin) to photoreceptors using AAVs (adeno-associated viruses)

Question 5

why was halorhodopsin a good choice?

Answer 5

both eNpHR-expressing cells and normal photoreceptor cells hyperpolarize in response to light intensity increase

Question 6

how was the effectiveness of the promoters used with halorhodopsin tested?

Answer 6

eNpHR was fused with fluorescent protein

Question 7

why is it important to restrict the expression of eNpHR to photoreceptors only?

Answer 7

If they were in downstream retinal circuit elements they might inhibit the flow of info

Question 8

suggests translational down-regulation of opsins with retinal degeneration (RD)

Answer 8

AAV-transduced cells had opsin mRNA but not the protein

Question 9

in retinitis pigmentosa, the first to go are the

Answer 9

rod photoreceptors

Question 10

1- Describe the main characteristics of Retinitis pigmentosa. What is the question the authors want to answer in this study?

Answer 10

1. caused by mutations in rod photoreceptors
2. the rods degenerate, causing night vision loss, and then cones lose their outer segments

The question is whether the cones can be reactivated

Question 11

2- Which experiments prove that the reactivation of cone photoreceptors is feasible in mice and humans?

Answer 11

1. tested whether eNpHR dirves cone light responses in RD retinas after many or all rods are dead by measuring photocurrents
2. recorded excitatory currents from ganglion cells to test for potential signal flow in morphologically reorganized photoreceptor-to-bipolar synapses

Question 12

3- What is halrhodopsin (eNpHR) and how is it activated? Why did the authors use this channel?

Answer 12

light-activated chloride channel. both halorhodopsin and normal photoreceptors hyperpolarize in response to light intensity increase

Question 13

4- Describe the differences between the s-RD and f-RD mice.

Answer 13

s-RD are slow forms of retinal degeneration

f-RD are fast

Question 14

5- Why is important to restrict the expression of eNpHR to photoreceptors only? Explain the figure that shows this result.

Answer 14

expression in downstream retinal circuit elements coudl inhibit info flow across retina.

Question 15

6- hRO and mCAR promoters are used in the s-RD mice experiments. Explain the reasoning behind this statement.

Answer 15

have ability to selectively drive expression of eNpHR-EYFP in high percentage of cone photoreceptors

Question 16

7- The ability of eNpHR-reactivated RD photoreceptors to convey information to downstream retinal circuits depends on the presence of functional photoreceptor-to-bipolar cell synapses. Are these synapses functional in RD mice transduced with eNpHR-EYFP?

Answer 16

they are morphologically reorganized, but still functional

Question 17

8- Mention differences described in the study between normal and transduced cones.

Answer 17

light sensitivity of eNpHR is less and have a fixed range of sensitivity

Question 18

9- Which are the evidences that demonstrated that resensitized photoreceptors are able to drive visually guided behavior in f-RD?

Answer 18

• dark-light box tests performance was better with eNpHR and correlated with illumination level
• only f-RD with eNpHR performed better in optomotor reflex test

Question 19

10- What are some potential limitations of this approach if used to treat human patients?

Answer 19

the amount of time cone cell bodies last