Single cell RNA sequencing confirms retinal microglia activation associated with early onset retinal degeneration
Asha Kumari1, Raul Ayala-Ramirez2,3, Juan Carlos Zenteno2,3
1Shiley Eye Institute, University of California, San Diego, 9415 Campus Point Drive, La Jolla, CA, 92093, USA.
Abstract:
Mutations in the Membrane-type frizzled related protein (Mfrp) gene results in an early-onset retinal degeneration associated with retinitis pigmentosa, microphthalmia, optic disc drusen and foveal schisis. In the current study, a previously characterized mouse model of human retinal degeneration carrying homozygous c.498_499insC mutations in Mfrp (MfrpKI/KI) was used. Patients carrying this mutation have retinal degeneration at an early age. The model demonstrates subretinal deposits and develops early-onset photoreceptor degeneration. We observed large subretinal deposits in MfrpKI/KI mice which were strongly CD68 positive and co-localized with autofluorescent spots. Single cell RNA sequencing of MfrpKI/KI mice retinal microglia showed a significantly higher number of pan-macrophage marker Iba-1 and F4/80 positive cells with increased expression of activation marker (CD68) and lowered microglial homeostatic markers (TMEM119, P2ry13, P2ry13, Siglech) compared with wild type mice confirming microglial activation as observed in retinal immunostaining showing microglia activation in subretinal region. Trajectory analysis identified a small cluster of microglial cells with activation transcriptomic signatures that could represent a subretinal microglia population in MfrpKI/KI mice expressing higher levels of APOE. We validated these findings using immunofluorescence staining of retinal cryosections and found a significantly higher number of subretinal Iba-1/ApoE positive microglia in MfrpKI/KI mice with some subretinal microglia also expressing lowered levels of microglial homeostatic marker TMEM119, confirming microglial origin. In summary, we confirm that MfrpKI/KI mice carrying the c.498_499insC mutation had a significantly higher population of activated microglia in their retina with distinct subsets of subretinal microglia. Further, studies are required to confirm whether the association of increased subretinal microglia in MfrpKI/KI mice are causal in degeneration.
Insights
Mutations in the Membrane-type frizzled related protein (Mfrp) gene cause early-onset retinal degeneration. Activated microglia, particularly in the subretinal space, are significantly increased in a mouse model, suggesting a role in disease progression.
Area of Science:
- Ophthalmology
- Genetics
- Immunology
Background:
- Mutations in the Membrane-type frizzled related protein (Mfrp) gene are linked to early-onset retinal degeneration.
- This degeneration is associated with conditions like retinitis pigmentosa and microphthalmia.
- A specific homozygous c.498_499insC mutation in Mfrp causes retinal issues in patients and a corresponding mouse model (MfrpKI/KI).
Purpose of the Study:
- To investigate the role of microglial activation in the MfrpKI/KI mouse model of retinal degeneration.
- To characterize the subretinal deposits and cellular changes in the retina of MfrpKI/KI mice.
- To determine if Mfrp mutations lead to a distinct subretinal microglia population.
Main Methods:
- Utilized a previously characterized MfrpKI/KI mouse model.
- Performed single-cell RNA sequencing on retinal microglia.
- Conducted retinal immunostaining and immunofluorescence staining on cryosections.
Main Results:
- MfrpKI/KI mice exhibited large, autofluorescent subretinal deposits.
- Single-cell RNA sequencing revealed a significant increase in activated microglia (Iba-1, F4/80, CD68 positive) with decreased homeostatic markers (TMEM119, P2ry13, Siglech).
- A distinct subretinal microglia population expressing higher APOE and lower TMEM119 was identified.
Conclusions:
- The MfrpKI/KI mouse model displays a significantly higher population of activated microglia in the retina.
- Distinct subsets of subretinal microglia are present in this model.
- Further research is needed to confirm if increased subretinal microglia are causal in Mfrp-associated retinal degeneration.
