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. 2020 Sep 22;9:e59422. doi: 10.7554/eLife.59422

Figure 3. Transcriptomic analysis shows neural network adaptation to rod death in early stage retinitis pigmentosa.

RNA-sequencing was performed from whole retinal extracts at 1 month of age in P23H mice (n = 7) and WT (n = 7) littermates. (A) A total of 5404 transcripts showed differential expression (downregulation, green; upregulation, red) between P23H and WT retinas. (B) Gene ontology (GO) term analysis shows expected downregulation in photoreceptor-dominant gene clusters. (C) KEGG analysis shows significant enrichments in cell/neuron adhesion and growth pathways, glutamatergic synapse formation and several pathways associated with both cell stress and synaptic plasticity. (D) GO term analysis illustrates the most significant upregulation in synaptic and neural development and growth pathways in P23H retinas.

Figure 3.

Figure 3—figure supplement 1. Activation of mitogen-activated protein kinase (MAPK) pathway in P23H retinas.

Figure 3—figure supplement 1.

Samples were collected at 1 month of age. Phospho-ERK was first detected using 680 nm fluorescence acquisition. The same membrane was incubated a second time using anti-ERK and anti α-tubulin (loading control) antibodies and detected using 800 nm fluorescence acquisition. MAPK activation is indicated by the increased ERK phosphorylation level. T-test: **p<0.01, ***p<0.001.
Figure 3—figure supplement 1—source data 1. Immunoblot band intensities for a-tubulin, ERK1/2 and phospho-ERK1/2.
Figure 3—figure supplement 2. Response to cell stress overwhelms retinal transcriptomic pathway regulation in intermediate stage retinitis pigmentosa.

Figure 3—figure supplement 2.

Whole retinal extracts from 3-month-old P23H (n = 4) and WT (n = 3) littermate mice were used. (A) Number of differentially expressed genes with an adjusted p<0.05 as a threshold criterion. (B) GO pathway analysis shows progressed downregulation in photoreceptor function-dominant gene clusters. (C) KEGG analysis shows most significant enrichments in extracellular matrix receptor interactions and cell adhesion pathways, and glutamatergic synapse- and axon guidance-associated pathways continue to be significantly upregulated. (D) GO pathway analysis reveals strongest pathway regulation associated with cell stress and growth. Note that this is notably different compared to results obtained at early disease state (refer to main Figure 1D).
Figure 3—figure supplement 3. Top 20 list of GO molecular function terms is similar between 1- and 3-month-old retinas.

Figure 3—figure supplement 3.

Cell growth-related pathways show the strongest upregulation followed by ion channel activity-associated pathways both at 1-month (A) and 3-month-old (B) retinas.
Figure 3—figure supplement 4. P23H retinas show opposing and persistent mRNA expression regulation in genes required for synaptic transmission in pre- versus postsynaptic sites.

Figure 3—figure supplement 4.

EData was obtained from 1-month-old (A) and 3-month-old (B) mice. Expression levels were obtained from FPKM reads. Data are normalized to WT values at 1.0. Note that the decreased mRNA expression of presynaptic genes is likely a consequence of photoreceptor loss in whole retina extracts. Cacna1F encodes Cav1.4 calcium channel, Cabp4 encodes calcium-binding protein that interacts with Cav1.4, and Slc17a7 encodes vesicular glutamate transporter vGlut1. Grm6 encodes metabotropic glutamate receptor 6 (mGluR6), Trpm1 encodes transient receptor potential cation channel subfamily M member 1, and Gpr179 encodes an orphan G protein-coupled receptor GPR179. mGluR6, TRPM1 and GPR179 are required for normal depolarization at ON bipolar cells. Multiple comparisons corrected t-tests: *p<0.05, **p<0.01, ***p<0.001.
Figure 3—figure supplement 5. A subset of important gene regulations was confirmed in 1-month-old retinas by qPCR.

Figure 3—figure supplement 5.

Expression levels were obtained using the ΔΔCt method. Data is normalized to WT values at 1.0. Multiple comparisons corrected t-tests: *p<0.05, ***p<0.001.
Figure 3—figure supplement 5—source data 1. Primary qPCR parameters for each technical and biological replicate.