Early synaptic defects in tulp1-/- mice

Abstract

Purpose: Mutations in the photoreceptor-specific tubby-like protein 1 (TULP1) underlie a form of autosomal recessive retinitis pigmentosa. To investigate the role of Tulp1 in the photoreceptor synapse, the authors examined the presynaptic and postsynaptic architecture and retinal function in tulp1(-/-) mice

Methods: The authors used immunohistochemistry to examine tulp1(-/-) mice before retinal degeneration and made comparisons with wild-type (wt) littermates and retinal degeneration 10 (rd10) mice, another model of photoreceptor degeneration that has a comparable rate of degeneration. Retinal function was characterized with the use of electroretinography.

Results: In wt mice, Tulp1 is localized to the photoreceptor synapse. In the tulp1(-/-) synapse, the spatial relationship between the ribbon-associated proteins Bassoon and Piccolo are disrupted, and few intact ribbons are present. Furthermore, bipolar cell dendrites are stunted. Comparable abnormalities are not seen in rd10 mice. The leading edge of the a-wave had normal kinetics in tulp1(-/-) mice but reduced gain in rd10 mice. The b-wave intensity-response functions of tulp1(-/-) mice are shifted to higher intensities than in wt mice, but those of rd10 mice are not.

Conclusions: Photoreceptor synapses and bipolar cell dendrites in tulp1(-/-) mice display abnormal structure and function. A malformation of the photoreceptor synaptic ribbon is likely the cause of the dystrophy in bipolar cell dendrites. The association of early-onset, severe photoreceptor degeneration preceded by synaptic abnormalities appears to represent a phenotype not previously described. Not only is Tulp1 critical for photoreceptor function and survival, it is essential for the proper development of the photoreceptor synapse.

FIGURE 1

Tulp1 is localized to the photoreceptor synapse. (A) Immunofluorescent localization of Tulp1 (green) and nuclei (DAPI; blue) in wt mouse retinal sections. (B) Higher power image of the OPL of the wt retina. Tulp1 (green), synaptic protein Bassoon (red), and nuclei (blue). The final panel is a digital magnification; arrows indicate that Tulp1 and Bassoon colocalize to the photoreceptor synaptic terminals. bv, blood vessel.

FIGURE 2

Photoreceptor synaptic ribbon-associated proteins are abnormal in the tulp1^−/− retina. Deconvolution-generated images of immunofluorescent localization of Bassoon (green) and Piccolo (red) in the OPL of mouse retinal sections. In the wt OPL at P16 (A), rd10 at P16 (C), and P21 (D), the horseshoe-like appearances of the photoreceptor synaptic ribbons are clearly visible. Arrows highlight the tight coupling between Bassoon and Piccolo, composing individual ribbons. In the tulp1^−/− OPL at P16 (B), the ribbons appear to exhibit morphologic abnormalities, possibly indicating a structural ribbon defect. To best show the distinction between the proximity and coupling of Bassoon and Piccolo at P16, three-dimensional surface plots were generated from a portion of the 2-µm Z-stacks of wt (A) and tulp1^−/− (B) retinas. In the wt retina (E), arrows point to the union of Bassoon and Piccolo, forming the compact horseshoe-shaped ribbons. In the tulp1^−/− retina (F), both proteins are present and in immediate proximity; however, they are not spatially fixed, as they are in the wt retina, into the normal horseshoe-shaped ribbon formation (arrow points to the union of Bassoon and Piccolo; arrowheads point to the separate ribbon staining of Bassoon and Piccolo). Early in the development of the wt functional synapse (P13), the coupling of the two proteins is not as robust (G) compared with the more mature P16 wt OPL (A); however, individual synaptic ribbons can be observed (arrows show coupling). In the tulp1^−/− OPL at P13 (H), the ribbons exhibit the same abnormalities as P16, possibly indicating that the deficits of the ribbon are present throughout development. Scale bar, 5 µm; gridlines, 10 µm.

FIGURE 3

The central ribbon protein, ribeye, is abnormal in the tulp1^−/− retina. Deconvolution-generated images of immunofluorescent localization of Ribeye/CtBP2 (green) in the OPL of mouse retinal sections. In the wt OPL at P16 (A), rd10 at P16 (C), and P21 (D), the horseshoe-like appearance of the synaptic ribbons is distinct (arrows). This is also the case in the wt OPL early in development (E). In the tulp1^−/− OPL at P13 (F) and P16 (B), the ribbons appear to exhibit morphologic abnormalities. The ribbons change from the classic horseshoe shape to diffuse immunoreactive areas, further raising the possibility of a synaptic malformation in the tulp1^−/− retina. Scale bar, 5 µm.

FIGURE 4

Rhodopsin is mislocalized in tulp1^−/− and rd10 retinas. Immunofluorescent localization of rhodopsin (red) in mouse retinal sections (DAPI; blue). In the wt retina at P16 (A), rhodopsin staining in restricted to the OS. Rhodopsin is also confined to the OS in the rd10 retina at P16 (B), although the OS shows some signs of disorganization. In contrast, during the period of maximal degeneration at P21 (C), rhodopsin is severely mislocalized, with staining appearing in the IS, throughout the ONL, and within the photoreceptor terminals of the rd10 retina. As previously reported, rhodopsin is mislocalized in tulp1^−/− retina at P16 (D). Scale bars, 20 µm.

FIGURE 5

Bipolar cell dendrites are abnormal in the tulp1^−/− retina. PKC immunofluorescent staining of rod bipolar cell bodies in the INL with processes extending into the OPL. Insets: high magnification, illustrating representative lengths and branching of the bipolar dendrites. In wt at P16 (A), the dendritic field is robust, with long processes penetrating the OPL and terminating with a high degree of arborization. This is in contrast to tulp1^−/− at P16 (B), where the dendrites appear as short appendages and branching has been severely attenuated. rd10 retinas at P16 (C) and P21 (D) show some signs of disorganization of the bipolar layers; however, it is only at P21 that retraction of the dendrites results in lengths comparable to those of tulp1^−/− at P16. In wt OPL at the early time point of P13 (E), dendrite lengths are shorter and branching is abbreviated compared with the wt at P16. However, in the tulp1^−/− OPL at P13 (F), dendrites are difficult to differentiate from the bipolar cell bodies and branching is not observed, possibly indicating that the dendrites are poorly developed and never mature properly. Scale bars, 10 µm.

FIGURE 6

Bipolar cell dendritic field is attenuated in the tulp1^−/− retina. (A) A montage of photomicrographs of the inferior mouse retina, immunostained for PKC. White boxes indicate the regions analyzed to quantify the dendritic field in P16 mice. Representative images of wt (B, D) and tulp1^−/− (C, E) retinas analyzed from the central and peripheral regions, respectively. Yellow lines indicate the measurements of individual dendrites. The average dendrite length was statistically significantly shorter in both the central (F; P = 0.006) and the peripheral (G; P = 0.005) regions in tulp1^−/− retinas compared with the wt. Data bars are the average ± SEM for four mice. Scale bars, 10 µm.

FIGURE 7

Relationship between presynaptic and postsynaptic elements show deficits in the tulp1^−/− retina. Deconvolution-generated images of immunofluorescence of PKC (red) and Bassoon (green) in the OPL at P16 showing the relationship of the presynaptic and postsynaptic elements. In the wt OPL (A), long and branching dendrites stretch toward the photoreceptor terminals that are decorated with Bassoon-immunoreactive horseshoe-shaped ribbons. In the tulp1^−/− OPL (B), dendrites show shortened appendages with reduced branching, and Bassoon-immunoreactive ribbons appear punctate. However, the presynaptic and postsynaptic elements are still closely apposed. Scale bars, 10 µm.

FIGURE 8

Electroretinography of P16 mice. (A) Representative dark-adapted ERGs obtained from wt (first column), tulp1^+/− (second column), tulp1^−/− (third column), and rd10 (fourth column) mice. Intensity-response functions for the amplitude of the a-wave (B) and b-wave (C) of dark-adapted ERGs obtained from wt, tulp1^+/−, tulp1^−/− and rd10 mice. The amplitudes of the a- and b-waves are comparable in wt and tulp1^+/− mice, and are reduced in tulp1^−/− and rd10 animals. Note that the rd10 b-wave function is shifted downward, whereas the tulp1^−/− b-wave function appears shifted down and to the right. Data points indicate average ± SD for ≥5 mice. (D) Comparison of the leading edge of the dark-adapted ERG a-wave obtained to a high intensity stimulus for each genotype studied. In each case, the plot represents an average of all mice tested, normalized to the a-wave trough. Note that normalized responses for wt, tulp1^+/− and tulp1^−/− mice overlap, whereas those of rd10 mice are delayed. (E) Comparison of A (equation 2) and K (equation 1) values obtained for each mutant genotype relative to the wt values. There is little difference in these parameters between wt and tulp1^+/− mice. In comparison, rd10 mice have reduced values of A but not K, whereas tulp1^−/− mice have increased values of K but not A.