Distinct and Atypical Intrinsic and Extrinsic Cell Death Pathways between Photoreceptor Cell Types upon Specific Ablation of <i>Ranbp2</i> in Cone Photoreceptors

<div><p>Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of <i>Ran-binding protein-2</i> (<i>Ranbp2</i>), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific <i>Ranbp2</i> ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of <i>Crx</i> and <i>CoREST</i>, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1⁺-apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of <i>Ranbp2</i> in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of <i>Ranbp2</i>. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial signatures as well as with other etiologically distinct neurodegenerative disorders.</p></div

Cell death in Nr2E3⁺-cell bodies of rod photoreceptors in <i>HRGP-cre:Ranbp2^−/−</i>.

<p>(<b>A</b>) Representative images of TUNEL⁺ (a′–d′), Cre⁺ (a″–d″) and Nr2E3⁺ (a′″–d′″) cell bodies and their colocalization (a″″–d″″) in rod photoreceptors of <i>HRGP-cre:Ranbp2^+/−</i> (+/−; a–a″″) and <i>HRGP-cre:Ranbp2^−/−</i> mice (−/−; b–d″″). d–d′″ are high magnifications of inset boxes of c–c″″. Sections were counterstained with DAPI (a–d). White arrowheads in a′–a″″ indicate Cre⁺Nr2E3⁻ cells, white and green arrows in b′–b″″ point to TUNEL⁺Nr2E3⁺ and TUNEL⁺Nr2E3⁻, respectively. (<b>B</b>) Quantification analysis of <b>A</b>. Among the TUNEL⁺-cell bodies tallied, slightly over 50% were Nr2E3⁺ in −/− mice, whereas they were negligible in +/− mice. No TUNEL⁺Cre⁺-cell bodies were identified in −/− mice. % values are based on % of total DAPI⁺ cells (100%). Data shown represent the mean ± SD, <i>n</i> = 3; *; <i>p</i><0.001. Scale bars = 20 µm (a–c″″), 5 µm (d–d″″).</p

Temporal and morphological profile of degeneration of cone photoreceptors in <i>Ranbp2^−/−</i> mice.

<p>(<b>A–H</b>′″) Immunohistochemistry of retinal sections of <i>HRGP-cre:Ranbp2^+/−</i> (<i>Ranbp2^+/−</i>) and <i>HRGP-cre:Ranbp2^−/−</i> (<i>Ranbp2^−/−</i>) mice at P9 (<b>A–B</b>′″), P13 (<b>C–D</b>′″) P20 (<b>E–F</b>′″) and P27 (<b>G–H</b>′″) of age with the antibodies against Cre recombinase and cone arrestin (Arr4) showing rapid loss of cone photoreceptors in <i>RanBP2^−/−</i>. Cre co-localized with Arr4 only in DAPI-stained cell bodies of cone cells throughout all ages regardless of genotype. Arrowheads in indicate Cre⁺ nuclei retracted to the proximal ONL (outer nuclear layer) in <i>Ranbp2^−/−</i> at P13, P20 and P27 of age, respectively. Arrows point to prominent swellings of synaptic pedicles of cone photoreceptors in <i>Ranbp2^−/−</i> at P20 of age. Scale bars = 25 µm.</p

Temporal profiles of changes in gene expression by ablation of <i>Ranbp2</i> in cone photoreceptors.

<p>(<b>A–G</b>) qRT-PCR of cone-specific (<b>A, B</b>), pan-photoreceptor (<b>C</b>), cone survival (<b>D</b>) and cone transcription factors (<b>E</b>), <i>col6α3</i> and <i>col1α1</i> (<b>F</b>), and <i>Gfap</i> and <i>Hif1α</i> transcripts (<b>G</b>). There is a decrease of cone-specific transcripts beginning at P13 (<b>A, B</b>), whereas pan-photoreceptor (<b>C</b>) and cone survival genes (<b>D</b>) begin transient up-regulations at the same age. The expression of cone survival genes peaks at P20 (<b>D</b>). (<b>E</b>) The transcription factors, <i>Crx</i> and <i>CoREST</i> (<i>Rcor1</i>), are the first nuclear factors whose changes of their transcriptional levels coincides with the genetic ablation of <i>Ranbp2</i> at P7. These events are followed by the up-regulations at P20 of the transcription factors, <i>Nrp1</i>, <i>Trß2</i> and <i>Otx2</i> (<b>E</b>). The up-regulations of transcripts encoding the substrate of MMP11, <i>col6α3</i>, begins at P9 and peaks at P20 (<b>F</b>), whereas those for the hypoxia marker, <i>Hif1α</i>, and inflammatory marker, <i>Gfap</i>, begin at P9 and P20, respectively (<b>G</b>). Legends: Data shown represent the mean ± SD, <i>n = </i>3–4; *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; refer to table S1 for gene designations/symbols.</p

Cre-mediated ablation of <i>Ranbp2</i> selectively in cone photoreceptors.

<p>(<b>A</b>) Schematic diagram for targeted <i>Ranbp2</i> allele upon HGRP-driven Cre excision of exon 2 and production of the recombinant mRNA of <i>Ranbp2.</i> Pr1 and Pr2 are specific to fused exons 1 and 3, and exon 5, respectively, and were used to monitor the functional excision of <i>Ranbp2^Flox</i> allele. (<b>B</b>) Schematic diagram of a constitutively targeted <i>Ranbp2</i> allele upon insertion of a promoterless bicistronic (β-geo-PLAP) cassette with a splicing acceptor site (SA) between exons 1 and 2. (<b>C</b>) RT-PCR of recombinant <i>Ranbp2</i> mRNA without exon 2 using Pr1 and Pr2 primers. Excision of exon 2 (<b><i>Δ</i></b>E2) was detectable in <i>HRGP-cre:Ranbp2^−/−</i> (−/−) but not wild-type (+/+) mice at P7 of age, a day after expression of <i>Cre</i> recombinase. (<b>D</b>) Co-expression (a″″–c″″) of Arr4 (a′), M-opsin (b′), S-opsin (c′), Cre (a″–c″) and PNA (a′″–c′″) in cone photoreceptor neurons of the superior (dorsal, a–b″″) or inferior (ventral, c–c″″) regions of the retina of <i>HRGP-cre:Ranbp2^+/−</i> mice at P15. Sections were counterstained with DAPI (a–c). Legends: HGRP, L/M opsin promoter; Arr4, cone arrestin 4; PNA, Peanut Agglutinin. Scale bars = 25 µm.</p

Ultrastructural changes of cone and rod photoreceptors upon ablation of <i>Ranbp2</i> in cones at P20.

<p>(<b>A</b>) Ultrastructural image of rod and cone photoreceptors outer and inner segments of <i>HRGP-cre:Ranbp2^+/−</i> (+/−). All structures look unremarkable. (<b>B–E</b>) Representative ultrastructural images of multiple features rod and cone photoreceptors outer and inner segments of <i>HRGP-cre:Ranbp2^−/−</i> (−/−). (<b>B</b>) shows the collapse of the cone outer segment and formation of a large electron lucent area, (<b>C</b>) shows extended discs and partial erosion of cone outer segment, (<b>D</b>) shows the accumulation of amorphous electron dense material at the connecting cilium of a cone photoreceptor, (<b>E</b>) shows the formation of electron lucent areas in the inner segment of a cone photoreceptor. White stars, euchromatic nuclei of cone photoreceptors. (<b>A′–E′</b>) are magnifications of boxed areas in <b>A–E</b> showing the aforementioned pathological features. (<b>F</b>) Synaptic pedicles of cones with normal morphology of <i>HRGP-cre:Ranbp2^+/−</i> (+/−). (<b>G</b>) Multi-lamellar body with electrodense material in a cone synaptic pedicle of <i>HRGP-cre:Ranbp2^−/−</i> (−/−). (<b>H</b>) Electron lucent area surrounding subcellular debris in a cone synaptic pedicle of <i>HRGP-cre:Ranbp2^−/−</i> (−/−). <b>F′–H′</b> are magnified images of boxed areas in <b>F–H</b>. (<b>I</b>) Synaptic spherule of a rod photoreceptor containing unremarkable mitochondria (black arrows) in <i>HRGP-cre:Ranbp2^+/−</i> (+/−). (<b>J</b>) Synaptic spherule of a rod photoreceptor with abnormal mitochondria (black arrows) containing disorganized cristae and electron lucent areas in the matrix in <i>HRGP-cre:Ranbp2^−/−</i> (−/−). Scale bars =  5 µm (<b>A–E</b>), 1 µm (<b>A′–E′</b>), 500 nm (<b>F–H</b>) and 200 nm (<b>F′–H′</b>).</p

Activation of matrix metalloproteinase 11 (MMP11) upon ablation of <i>Ranbp2</i> in cone photoreceptors.

<p>(<b>A</b>) Light microscopy images of methylene blue stained semi-thin retinal sections showing the presence of prominent interstitial spaces (black arrows) originating from cone cell bodies (white arrows) and between cell bodies of rod photoreceptors in <i>HRGP-cre:Ranbp2^−/−</i> (−/−) compared to <i>HRGP-cre:Ranbp2^+/−</i> (+/−) mice. * denote euchromatic nuclei of cones. (<b>B</b>) Electron micrographs depicting ultrastructural changes in the lower fiber of a −/− cone photoreceptor. Note the swelling of the lower fiber (arrows) traced to the cell body of a cone nucleus characterized by its prominent euchromatin (*). *, cone nuclei. (<b>C</b>) Temporal profile of MMP11 activity of retinal extracts of <i>HRGP-cre:Ranbp2^−/−</i> mice (−/−) mice relative to age-matched +/− controls shows significant MMP11 activity at P13 and P20. The MMP11 activity of +/− and +/+:Cre controls shown are at P20. (<b>D</b>) Immunoblots of MMP11 from retinal homogenates of <i>HRGP-cre:Ranbp2^−/−</i> (−/−) compared to +/− mice at P20 showing an increase of the active form of MMP11 in −/−. Hsc70 is cytosolic heat shock protein 70 used as loading control. (<b>E</b>) Quantitation analysis of the levels of active MMP11 in (<b>D</b>) of <i>HRGP-cre:Ranbp2^−/−</i> (−/−) relative to age-matched +/− mice at P20. (<b>F</b>) Retinal sections immunostained with antibodies against cone arrestin (Arr4) and MMP11. (a–d) central retinal region of <i>HRGP-cre:Ranbp2^+/−</i> (+/−); (e–f) central retinal region of <i>HRGP-cre:Ranbp2^−/−</i> (−/−); (e′–f′) magnification of boxed regions shown in e–f; (i<b>–</b>l) peripheral retinal region of <i>HRGP-cre:Ranbp2^+/−</i> (+/−); (m<b>–</b>p) peripheral retinal region of <i>HRGP-cre:Ranbp2^−/−</i> (−/−); (m′–p′) magnification of lower fibers of cones of boxed regions shown in m–p. MMP11 predominantly localizes to cone photoreceptors, such as interstitial space around cell bodies, lower fibers and inner segments. Legend: Data shown represent the mean ± SD, <i>n</i> = 4; *, <i>p</i><0.05; **, <i>p</i><0.01; ^o, <i>p</i><0.0001; scale bars = 25 µm (A, F), 5 µm (B).</p

Topographic degeneration of M-cone photoreceptors and their outer segments in <i>HRGP-cre:Ranbp2^−/−</i>.

<p>(<b>A</b>) Retinal flat mount images of <i>HRGP-cre:Ranbp2^−/−</i> (−/−) and <i>HRGP-cre:Ranbp2^+/−</i> (+/−) immunostained with an antibody against M-opsin showing progressive and severe M-cone cell loss in the retina of −/− mice. (a′–f′) Magnifications of the superior/central inset regions of a–f. (a″–f″) 3D-reconstruction images from a′–f′. (<b>B–C</b>) Quantitative and temporal analyses of the number (<b>B</b>) and length of outer segments (<b>C</b>) of M-cone photoreceptors in the superior (S)/central regions of retina of −/− and +/− mice. (<b>D</b>) Comparison of topographic distribution of Cre⁺ cells in the retina of −/− and +/− mice at P13 showing the proximal localization of a few displaced cone cells in −/− mice. White dashed line represents a virtual midline boundary between proximal and distal areas of the outer nuclear layer (ONL) used for tallying the topography of Cre⁺-cell bodies in (<b>E</b>). (<b>E</b>) Quantitative and morphometric analyses of the localization of Cre⁺ cells in the proximal <i>versus</i> distal ONL and central <i>versus</i> peripheral retina between −/− and +/− mice. About 20% of Cre⁺ cells are present in the proximal ONL region of −/− at P13 and there is greater number of Cre⁺-cell loss in the central retina of −/− mice at P20. Legend: Boxes in <b>A</b> (a–f) are ROIs used for quantitation of data shown in <b>B</b> and <b>C</b>. Data shown represent the mean ± SD, <i>n</i> = 3–5; **, <i>p</i><0.01; ***, <i>p</i><0.001. Scale bars = 150 µm (a–f), 20 µm (a′–f′), 50 µm (<b>D</b>).</p

Spatiotemporal modalities of cell death between Cre⁺, Arr4⁺ and Arr4⁻-photoreceptor cell bodies in <i>HRGP-cre:Ranbp2^−/−</i>.

<p>(<b>A</b>) Comparison of the relative 3-D distribution between Cre⁺-cone and TUNEL⁺ cells in the outer nuclear layer of retinas of <i>HRGP-cre:Ranbp2^+/−</i> (+/−, top) and <i>HRGP-cre:Ranbp2^−/−</i> mice (−/−, bottom) at P20 shows that all TUNEL⁺ cells are Cre⁻. (<b>B</b>) Morphometric and topographic distribution of TUNEL⁺ cells in outer nuclear layer (ONL, proximal <i>vs</i> distal) and regions of retina (peripheral <i>vs</i> central) between <i>HRGP-cre:Ranbp2^+/−</i> (+/−) and <i>HRGP-cre:Ranbp2^−/−</i> mice (−/−) at P13, P20 and P27. TUNEL⁺ cells become prominent at P20 with most localizing at the distal ONL of central and peripheral retina. No TUNEL⁺ cells were identified by P27. Data shown represent the mean ± SD, <i>n</i> = 4–5; ***, <i>p</i><0.001; ^o, <i>p</i><0.0001. (<b>C</b>) Identification and localization of TUNEL⁺ (apoptotic) and EthD-III⁺ (necrotic) in Arr4⁻-photoreceptor cell bodies of P20 retinas of <i>HRGP-cre:Ranbp2^+/−</i> mice (+/−; a–a′″) and <i>HRGP-cre:Ranbp2^−/−</i> mice (−/−; b–b′″). Representative images of TUNEL⁺ (a′–b′) or EthDIII⁺ (a″–b″) cell bodies of Arr4⁻-photoreceptors (a′″–b′″) and co-localization of these (a″″–b″″) are shown. No TUNEL⁺ and EthD-III⁺-cell bodies in Arr4⁺-photoreceptor cell bodies were identified. Sections were counterstained with DAPI (a–b). White arrows in b′–b′″ indicate the localization of TUNEL⁺EthD-III⁺ cell bodies. (<b>D</b>) Quantification analysis of <b>C</b>. Among the Arr4⁻-photoreceptor cell bodies tallied, approximately 50, 30 and 20% were TUNEL⁻EthD-III⁺, TUNEL⁺EthD-III⁻ and TUNEL⁺EthD-III⁺, respectively, in −/− mice, whereas they were negligible in +/− mice. Data shown represent the mean ± SD, <i>n</i> = 3; ***, <i>p</i><0.001; **, <i>p</i><0.02; *, <i>p</i><0.005; Scale bars = 50 µm (<b>A</b>), 20 µm (<b>C</b>).</p

Spatiotemporal activation of caspases 3 and 7 and Parp1 in photoreceptors of <i>HRGP-cre:Ranbp2^−/−</i> mice.

<p>(<b>A</b>) Temporal profile of caspase 3/7 activation of retinal extracts of <i>HRGP-cre:Ranbp2^−/−</i> mice (−/−) mice relative to age-matched +/− controls shows significant caspase3/7 activation at P13 and P20. The caspase 3/7 activities of +/− and +/+:Cre controls shown are at P20. (<b>B</b>) Quantitative, temporal and relative distributions between activated (cleaved) caspase 3 (c-casp3⁺), Cre⁺ and TUNEL⁺ in cell bodies of photoreceptors of <i>HRGP-cre:Ranbp2^+/−</i> (+/−) and <i>HRGP-cre:Ranbp2^−/−</i> mice (−/−) at P13 and P20. The majority of photoreceptor cell bodies are c-casp3⁺Cre⁻. The levels of any type of c-casp3⁺ cell bodies are negligible by P20. (<b>C</b>) Relative immunolocalizations of Cre⁺ and c-casp3⁺ (a–a′″, d–d′″), Cre⁺ and c-casp7⁺ (b–b′″, e–e′″), and Cre⁺ and activated Parp1 (c-Parp1⁺) (c–c′″, f–f′″) cell bodies of photoreceptors of <i>HRGP-cre:Ranbp2^−/−</i> mice at P13 (a–c′″) and P20 (d–f′″). c-casp3⁺ develop at P13, c-casp7⁺ and c-Parp1⁺ at P20 and all c-Parp1⁺ cell bodies are Cre⁻, whereas c-casp7⁺ are Cre⁺. Legend: Data shown represent the mean ± SD, <i>n</i> = 4–5; *; <i>p</i><0.05, **; <i>p</i><0.01; scale bars = 25 µm; cleaved caspases 3 and 7, c-casp3 and c-casp7, respectively.</p