Final Report Summary - RBC MIRNA (The role of microRNAs in the Retinal Bipolar Cell)
RBC miRNA
PEOPLE
MARIE CURIE ACTIONS
FP7-PEOPLE-2011-IIF
FINAL REPORT
Introduction
The bipolar cells, positioned post-synaptic to photoreceptors and presynaptic to ganglion cells have a functional role that is fundamental to visual processing in all vertebrates. We wished to investigate the role of miRNAs in maintaining highly specialized neuronal function in the ON-bipolar cell where the stream of visual information for light increments is formed. MicroRNAs function in post-transcriptional regulation of gene expression and as such have been shown to be essential to most biological functions.
Results
The ON-bipolar-cell-specific TrpM1 gene, which is crucial for the depolarizing ON response in bipolar cells, is host to a microRNA, miR-211. This microRNA is found to be highly enriched in the retina’s bipolar cell layer and we find it to be differentially expressed in response to light changes (figure 1). This suggests an important relationship between miRNA regulation of bipolar cell gene expression and the visual responsiveness of the adult retina. To further investigate this effect we developed a mouse model that has an essential factor in the biogenesis of mature microRNAs knocked out only in bipolar cells, the mglur6-dgcr8KO (figure 2) where the promoter mglur6 drives ON-bipolar-cell-specific expression. As dgcr8 is a protein necessary for the targeting of the RNAseIII drosha to the primary microRNA transcript the loss of this protein would mean that the primary transcript should never get processed to produce the mature microRNAs. Prof. Markus Stoffel of the University of Zurich provided the dgcr8 knockout mouse to us.
We carried out the triple cross of mglur6-cre X floxed-dgcr8KO X floxed-tdTomato; this cross took 8 months to complete as some viability issues were presented in establishing the line. The retinas of the mice were FACS-sorted to isolate the dgcr8 deficient ON-bipolar cells. Despite this loss, the visual function of the mglur6-dgcr8KO mouse retina was unchanged compared to controls as assessed by multi-electrode array (MEA) (figure 3). In addition the morphology of the retina appeared as normal with the localization and levels of essential retinal proteins remaining essentially unchanged (figure 4). The RNA from the sorted ON-bipolar population of cells was analysed by qRT-PCR for a number of microRNAs using taqman microRNA assays. We were surprised to find that the mature microRNA population remained unchanged in these mice as they aged despite the loss of dgcr8 (figure 5a). To test whether this was due to a problem with our mouse model we used an AAV expressing both GFP and shRNA to knockdown dgcr8 (AAV-shRNA(dgcr8)-GFP) into mice that had RFP-labelled bipolar cells. Again we failed to see any change in mature miRNA levels of bipolar cells that were transduced with the AAV (figure 5b). The reason for this is unclear but suggests that a dgcr8-independent path may result in the production of microRNAs in the retinal bipolar cells. By contrast the use of an AAV carrying shRNA to silence Drosha, a down-stream member of the microRNA-processing pathway, did result in moderate of the selected microRNAs (figure 5b).
We chose to develop an alternative mouse model, the mglur6-dicer(KO)-tdTomato (from the cross of mglur6-cre X floxed-dicerKO X floxed-tdTomato). Dicer is an RNAseIII enzyme that functions further downstream of dgcr8 and is essential for all microRNA production. The sorted cells from this model did produce the expected reduction in microRNA by P30 (figure 6a). However we did not see a change in the visual function of the mglur6-dicerKO mouse retina at P60 as assessed by MEA (figure 6b). In addition no gross morphology changes were detected at P90 (figure 7). At later ages any remaining microRNAs in this mouse are expected to be entirely eliminated as the turnover of microRNAs becomes complete without replenishment and it is therefore possible that a visual phenotype may emerge in older mice. We will continue to analyze these further time-points.
In the meantime, deep sequencing of these mouse models has revealed a reduction in the synaptic proteins SNAP-25 and Syt1-1 (figure 8, verified by qPCR in 9a). SNAP25 is essential for the assembly of the SNARE complex in ribbon synapses of the retinal bipolar cells, a complex necessary to regulate the exocytosis of synaptic vesicles in ribbon synapses. The Ca(2+)-triggered exocytotic process is regulated by synaptotagmin (Syt), a vesicular Ca(2+)-binding C2 domain protein. We find the levels of the snap25 protein to be reduced in the dgcr8 mouse model at P90 (figure 9b, 9c). A possible dysregulation of the synaptic turnover at the ribbon synapses of the bipolar cells may arise with the disruption of miRNA biogenesis. Physiologically this effect may become apparent with specific MEA protocols such as a paired-pulse paradigm and we plan on pursuing this line of inquiry to complete the project.
Conclusions
It is possible that the dgcr8 KO did not show a reduction in selected miRNAs because by P10 many miRNAs are stable and have reduced or halted turnover. Thus, we may have failed to select the right miRNAs to identify the drop. However miR211, which we have shown to be dynamically expressed in the bipolar cells, is also unaffected in our dgcr8 mouse model. Alternatively the miRNAs are not changed as the dgcr8 may not be expressed in these cells. While, this latter is unexpected, qPCR and western blot data have failed to show the presence of dgcr8 in the ON-bipolar cells. This may otherwise be due to the very low levels of this protein typically found in neurons and to the low sensitivity of the available antibodies and probes. However, it is known that dgcr8 expresses at very low levels in bipolar cells and optimizing the sensitivity of these detection assays may be necessary to identify the protein.
These results are of particular relevance for future studies of mouse models with disrupted miRNA biogenesis.
PEOPLE
MARIE CURIE ACTIONS
FP7-PEOPLE-2011-IIF
FINAL REPORT
Introduction
The bipolar cells, positioned post-synaptic to photoreceptors and presynaptic to ganglion cells have a functional role that is fundamental to visual processing in all vertebrates. We wished to investigate the role of miRNAs in maintaining highly specialized neuronal function in the ON-bipolar cell where the stream of visual information for light increments is formed. MicroRNAs function in post-transcriptional regulation of gene expression and as such have been shown to be essential to most biological functions.
Results
The ON-bipolar-cell-specific TrpM1 gene, which is crucial for the depolarizing ON response in bipolar cells, is host to a microRNA, miR-211. This microRNA is found to be highly enriched in the retina’s bipolar cell layer and we find it to be differentially expressed in response to light changes (figure 1). This suggests an important relationship between miRNA regulation of bipolar cell gene expression and the visual responsiveness of the adult retina. To further investigate this effect we developed a mouse model that has an essential factor in the biogenesis of mature microRNAs knocked out only in bipolar cells, the mglur6-dgcr8KO (figure 2) where the promoter mglur6 drives ON-bipolar-cell-specific expression. As dgcr8 is a protein necessary for the targeting of the RNAseIII drosha to the primary microRNA transcript the loss of this protein would mean that the primary transcript should never get processed to produce the mature microRNAs. Prof. Markus Stoffel of the University of Zurich provided the dgcr8 knockout mouse to us.
We carried out the triple cross of mglur6-cre X floxed-dgcr8KO X floxed-tdTomato; this cross took 8 months to complete as some viability issues were presented in establishing the line. The retinas of the mice were FACS-sorted to isolate the dgcr8 deficient ON-bipolar cells. Despite this loss, the visual function of the mglur6-dgcr8KO mouse retina was unchanged compared to controls as assessed by multi-electrode array (MEA) (figure 3). In addition the morphology of the retina appeared as normal with the localization and levels of essential retinal proteins remaining essentially unchanged (figure 4). The RNA from the sorted ON-bipolar population of cells was analysed by qRT-PCR for a number of microRNAs using taqman microRNA assays. We were surprised to find that the mature microRNA population remained unchanged in these mice as they aged despite the loss of dgcr8 (figure 5a). To test whether this was due to a problem with our mouse model we used an AAV expressing both GFP and shRNA to knockdown dgcr8 (AAV-shRNA(dgcr8)-GFP) into mice that had RFP-labelled bipolar cells. Again we failed to see any change in mature miRNA levels of bipolar cells that were transduced with the AAV (figure 5b). The reason for this is unclear but suggests that a dgcr8-independent path may result in the production of microRNAs in the retinal bipolar cells. By contrast the use of an AAV carrying shRNA to silence Drosha, a down-stream member of the microRNA-processing pathway, did result in moderate of the selected microRNAs (figure 5b).
We chose to develop an alternative mouse model, the mglur6-dicer(KO)-tdTomato (from the cross of mglur6-cre X floxed-dicerKO X floxed-tdTomato). Dicer is an RNAseIII enzyme that functions further downstream of dgcr8 and is essential for all microRNA production. The sorted cells from this model did produce the expected reduction in microRNA by P30 (figure 6a). However we did not see a change in the visual function of the mglur6-dicerKO mouse retina at P60 as assessed by MEA (figure 6b). In addition no gross morphology changes were detected at P90 (figure 7). At later ages any remaining microRNAs in this mouse are expected to be entirely eliminated as the turnover of microRNAs becomes complete without replenishment and it is therefore possible that a visual phenotype may emerge in older mice. We will continue to analyze these further time-points.
In the meantime, deep sequencing of these mouse models has revealed a reduction in the synaptic proteins SNAP-25 and Syt1-1 (figure 8, verified by qPCR in 9a). SNAP25 is essential for the assembly of the SNARE complex in ribbon synapses of the retinal bipolar cells, a complex necessary to regulate the exocytosis of synaptic vesicles in ribbon synapses. The Ca(2+)-triggered exocytotic process is regulated by synaptotagmin (Syt), a vesicular Ca(2+)-binding C2 domain protein. We find the levels of the snap25 protein to be reduced in the dgcr8 mouse model at P90 (figure 9b, 9c). A possible dysregulation of the synaptic turnover at the ribbon synapses of the bipolar cells may arise with the disruption of miRNA biogenesis. Physiologically this effect may become apparent with specific MEA protocols such as a paired-pulse paradigm and we plan on pursuing this line of inquiry to complete the project.
Conclusions
It is possible that the dgcr8 KO did not show a reduction in selected miRNAs because by P10 many miRNAs are stable and have reduced or halted turnover. Thus, we may have failed to select the right miRNAs to identify the drop. However miR211, which we have shown to be dynamically expressed in the bipolar cells, is also unaffected in our dgcr8 mouse model. Alternatively the miRNAs are not changed as the dgcr8 may not be expressed in these cells. While, this latter is unexpected, qPCR and western blot data have failed to show the presence of dgcr8 in the ON-bipolar cells. This may otherwise be due to the very low levels of this protein typically found in neurons and to the low sensitivity of the available antibodies and probes. However, it is known that dgcr8 expresses at very low levels in bipolar cells and optimizing the sensitivity of these detection assays may be necessary to identify the protein.
These results are of particular relevance for future studies of mouse models with disrupted miRNA biogenesis.
