Localization of RanBP2 and Its LD Molecular Partners

<p>(A–F) are thin cryosections of an area of the hipocampus (CA1 neurons) and cerebral cortex, respectively, immunostained against HKI (A and D), RanBP2 (B and E), and merged images thereof (C and F). Note that while RanBP2 and HKI are widely expressed among and colocalize to hippocampal neurons (C), HKI expression and localization with RanBP2 is restricted to a subset of cortical neurons (likely interneurons) (F). Images of the distal region of bovine retinal cryosections comprising part of the nuclear layer of photoreceptor neurons and their inner (myoid and ellipsoid) segment compartment (G–O) are immunostained against mHsp70 (G) and RanBP2 (H), mHsp70 (J) and Cox11 (K), HKI (M) and Cox11 (N), and merged images thereof (I–O). Note the prominent localization of RanBP2, mHsp70, and Cox11 at the mitochondria-rich ellipsoid compartment of photoreceptors and the colocalization of RanBP2 and Cox11 with mHsp70 (I and L), while HKI colocalization with Cox11 was limited to restricted foci (R, arrowheads). High-resolution images of dissociated primary cerebral neurons and glial cells confirmed that the colocalization of HKI and Cox11 was highly restricted (P–R), while RanBP2 extensively colocalized with HKI (S–U) and mHsp70 (V–Z). Scale bars in A–O and P–Z are 40 and 10 μm, respectively. ONL, outer nuclear layer.</p

Haploinsufficiency of <i>RanBP2</i> Causes a Decrease in HKI Protein and ATP Levels

<div><p>(A) Quantitative analysis of NPCs in dissociated hippocampal neurons of wild-type (+/+) and heterozygote (+/−) mice upon immunostaining with mAb414. No difference in the density of NPCs (3–4 NPC/μm²) at the nuclear envelope was found between <i>RanBP2^+/+</i> and <i>RanBP2^+/−</i> mice.</p><p>(B) Immunoblots with anti-RanBP2/Nup153/Nup62 (mAb414), −HKI, −mHsp70, and −Cox11 antibodies of retinal (top panel) and hippocampal homogenates of +/+ and +/− mice. In comparison to <i>RanBP2^+/+, RanBP2^+/−</i> mice exhibit a reduction in the expression levels of RanBP2 and HKI but not of other proteins.</p><p>(C) Quantitative analysis of relative protein expression levels of RanBP2, Cox11, HKI, and mHsp70 in the hippocampus of <i>RanBP2^+/+</i> and <i>RanBP2^+/−</i>mice. There is ~2- and 4-fold reduction of RanBP2 and HKI in heterozygote mice.</p><p>(D) The level of HKI is reduced in the brain but not in other non-neuronal tissues tested (muscle, spleen, and liver).</p><p>(E) The total ATP level is reduced in the CNS tissues (brain and retina) but not in non-neuronal tissues tested (e.g., spleen).</p></div

Insertion Mutagenesis of the Murine <i>RanBP2</i> Gene

<div><p>(A) Diagram of the genomic region of <i>RanBP2</i> disrupted by insertion trap mutagenesis with a bicistronic reporter vector between exon 1 and 2. The bicistronic transcript produces two proteins under regulation of RanBP2. Upon splicing of <i>RanBP2,</i> a fusion between exon 1 and β-geo (a fusion between the <i>β-gal</i> and <i>neo</i> genes) is generated, while human placental alkaline phophatase (PLAP) is independently translated using the internal ribosome entry site. Consistent with previous studies, the expression of the former is directed to cell bodies, while expression of the latter is targeted to the axonal processes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020177#pgen-0020177-b067" target="_blank">67</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020177#pgen-0020177-b068" target="_blank">68</a>]. Transcriptional 5′ RACE analysis detects a fusion between exon 1 and β-geo.</p><p>(B) Southern analysis of the <i>RanBP2</i> locus of wild-type and heterozygous genomic DNA of tails of F1 mice digested with <i>Ppu</i>MI (left panel) and <i>Hind</i>III (right panel) with probes at the 3′ (left panel) and 5′ (right panel) flanking regions of the insertion breakpoint. Q1 is a cosmid containing the <i>RanBP2</i> gene up to exon 20 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020177#pgen-0020177-b004" target="_blank">4</a>].</p><p>(C) Lateroventral view of a whole-mount stain of a ~12.5 dpc heterozygous embryo for PLAP and β-gal (inset picture) activities. Although PLAP was broadly expressed (e.g., somites, limbs, and CNS), the PLAP and β-Gal (inset picture) expression was particularly high in the optic vesicle (arrow). X-gal single (D) and combined staining with PLAP (E) of a retinal section of a 3-mo-old RanBP2^+/− mouse. Consistent with previous immunocytochemistry studies, β-Gal activity is detected in the neuroretinal bodies and inner segment compartment of photoreceptors with conspicuously strong expression in ganglion cells. PLAP expression is found throughout the plexiform/synaptic layers and outer segment of photoreceptors (E). GC, ganglion cell; PLAP, human placental alkaline phophatase; ROS, rod outer segment; RIS, rod inner segment; ONL, outer nuclear layer; OPL, outer plexiform (synaptic) layer; INL, inner nuclear layer; IPL, inner plexiform (synaptic) layer; GC, ganglion cell layer.</p></div

Metabolic Phenotypes of <i>RanBP2^+/−</i> Inbred Mice on High-Fat Diet

<div><p>(A) 3-mo-old inbred <i>RanBP2^+/−</i> mice (<i>n</i> = 5) have normal glucose clearance rates upon glucose challenge and overnight fasting.</p><p>(B) In contrast, 6-mo-old inbred <i>RanBP2^+/−</i> mice (<i>n</i> = 5) have significantly decreased glucose clearance rates upon glucose challenge and overnight fasting.</p><p>(C) Fasted 6- to 8-mo-old <i>RanBP2^+/+</i> and <i>RanBP2^+/−</i> mice have no difference in insulin-mediated glucose uptake as assayed by insulin tolerance test (<i>n</i> = 5).</p><p>(D) Pyruvate tolerance test shows normal rise in glucose but decreased glucose clearance between inbred <i>RanBP2^+/+</i> and <i>RanBP2^+/−</i> mice (<i>n</i> = 5).</p></div

Effect of Cox11 and RanBP2 on HKI Activity

<div><p>(A) Saturation kinetics, rate versus glucose of HKI (0.24 μg) in the absence (solid circles) and presence of Cox11 (open circles, 0.25 nM; solid triangles, 7.5 nM). The activity of HKI decreases with increasing concentrations of Cox11. No measurable HKI activity was recorded in the presence of 15 nM of Cox11 (unpublished data).</p><p>(B) Hanes-Wolf plot of (A) (1/rate versus glucose) in the absence and presence of fixed concentrations of Cox11. Linearity of reciprocal plots also supported the hyperbolic behavior of the reactions (unpublished data). Cox11 behaves as a noncompetitive inhibitor of HKI by reducing the <i>V</i>_max of HKI but not its <i>K</i>_m toward glucose.</p><p>(C) HKI rate is plotted as a function of LD concentration at saturating glucose and fixed Cox11 (7.5 nM) concentrations. Note that increasing concentrations of the LD of RanBP2 reverse the inhibition of HKI activity by Cox11. A half-maximal effect of the LD of RanBP2 on HKI activity in the presence of 7.5 nM of Cox11 was observed at a concentration of ~0.05 nM of LD.</p><p>(D) Rate versus glucose plot in the absence and presence of the LD of RanBP2. At a saturating concentration of the LD of RanBP2 (3.75 nM), the HKI activity was reduced by about 20%. <i>v,</i> rate; S, glucose.</p></div

Electroretinograms from 6-Mo-Old <i>RanBP2^+/−</i>and <i>RanBP2^+/+</i> Inbred Mice Showing Photoreceptor and Postreceptor Neuron Electrophysiological Response Phenotypes

<div><p>(A) Scotopic (dark-adapted) responses from <i>RanBP2^+/−</i> mice to light stimuli of increasing intensity, beginning at threshold, have reduced amplitudes compared to those observed in <i>RanBP2^+/+</i> mice. The three lower intensities represent responses generated in the rod photoreceptor neuronal pathway. The upper intensities are comprised of responses generated in both the rod and cone pathways.</p><p>(B) Photopic (light-adapted, cone photoreceptor pathway) responses of <i>RanBP2^+/−</i> mice to increasing light stimulus intensities also exhibited reduced amplitudes compared to those observed in <i>RanBP2^+/+</i> mice.</p><p>(C) Average ± SE (<i>n</i> = 9) scotopic <i>b</i>-wave amplitudes from <i>RanBP2^+/−</i> (open circles) and <i>RanBP2^+/+</i> (filled squares) mice representing postreceptoral neuron function. (Note: log amplitude scale.)</p><p>(D) Average ± SE (<i>n</i> = 5) scotopic <i>a</i>-wave amplitudes, representing photoreceptor function, for <i>RanBP2^+/−</i> and <i>RanBP2^+/+</i> mice in response to bright flashes. Amplitudes of responses from <i>RanBP2^+/−</i> mice were lower over the entire range of stimulus intensities for both <i>b</i>- and <i>a</i>-waves. Asterisks represent significant differences between the groups (Student's <i>t</i> test, <i>p</i> < 0.05). Statistical significance was found across all intensities for <i>b</i>-wave amplitudes (2-way ANOVA, <i>p</i> < 0.0001), but not for the <i>a</i>-wave.</p></div