RanBP2 Modulates Cox11 and Hexokinase I Activities and Haploinsufficiency of RanBP2 Causes Deficits in Glucose Metabolism

The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2(−/−) are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2(+/−) mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies

3D motion capture system for assessing patient motion during Fugl‐Meyer stroke rehabilitation testing

The authors introduce a novel marker‐less multi‐camera setup that allows easy synchronisation between 3D cameras as well as a novel pose estimation method that is calculated on the fly based on the human body being tracked, and thus requires no calibration session nor special calibration equipment. They show high accuracy in both calibration and data merging and is on par with equipment‐based calibration. They deduce several insights and practical guidelines for the camera setup and for the preferred data merging methods. Finally, they present a test case that computerises the Fugl‐Meyer stroke rehabilitation protocol using the authors’ multi‐sensor capture system. They conducted a Helsinki‐approved research in a hospital in which they collected data on stroke patients and healthy subjects using their multi‐camera system. Spatio‐temporal features were extracted from the acquired data and machine learning‐based evaluations were applied. Results showed that patients and healthy subjects can be correctly classified at a rate of above 90%. Furthermore, they show that the most significant features in the classification are strongly correlated with the Fugl‐Meyer guidelines. This demonstrates the feasibility of a low‐cost, flexible and non‐invasive motion capture system that can potentially be operated in a home setting

Photopic ERG negative response from amacrine cell signaling in RCS rat retinal degeneration. Invest Ophthalmol Vis Sci.

PURPOSE. The authors investigated photopic electroretinographic changes during degeneration in the Royal College of Surgeons (RCS) and transgenic P23H rhodopsin rat models, including the cellular origins of a large corneal-negative component that persists in the RCS rat. METHODS. Photopic and scotopic electroretinograms (ERGs) were recorded from dystrophic RCS (RCS-p ϩ /Lav) rats (4 -18 weeks old) and transgenic rhodopsin Pro23His line 1 (P23H) rats (4 -30 weeks old). Age-matched congenic (RCS-rdy ϩ p ϩ / Lav) and Sprague-Dawley rats were used as controls. N-methyl-DL-aspartic acid (NMA), dopamine, and ␥-aminobutyric acid (GABA) were injected intravitreally, and optic nerve sectioning (ONS) was performed to suppress or remove inner retinal neuron activity. Retinal morphology for cone cell counts and immunohistochemistry for quantification of Kir4.1 channels were performed at various stages of degeneration. RESULTS. As degeneration progressed, the photopic ERG of RCS dystrophic rats was distinctly different from that of P23H rats, primarily because of the growth of a corneal-negative response (RCS-NPR) after the b-wave in RCS rats. This response had a peak time similar to the photopic negative response (PhNR) in controls but with a more gradual recovery phase, and it was not affected by ONS. The PhNR in P23H rats declined linearly with the b-wave. NMA and GABA eliminated the RCS-NPR and uncovered a larger b-wave in RCS rats at late stages of degeneration, but NMA had little effect on the ERG in P23H rats. The NMA-sensitive negative response in RCS rats declined with age more slowly than did the NMA-isolated b-wave. The density of Kir4.1 channels at the endfeet of Müller cells and in the proximal retina increased significantly between 6 to 10 weeks and 14 weeks of age in the RCS rat retina but not in the P23H rat retina. CONCLUSIONS. The photopic ERG of the dystrophic RCS rat retina becomes increasingly electronegative because of an aberrant negative response, originating from amacrine cell activity, which declines more slowly than the b-wave with degeneration. The absence of this response in the P23H rat indicates that the inner retinal cone pathway pathology is different in the two models. A relative increase in Kir4.1 channels on Müller cells of RCS retina may contribute to the enhanced negative ERG response in the RCS rat. (Invest Ophthalmol Vis Sci. 2008;49:442-452

The direct effect of platelets on EPCs functional properties and its comparison to the platelets’ indirect effect.

<p><b>A.</b> The average number of colonies per field ± SE in EPCs cultured with vs. without platelets, p<0.05, n = 13 (Wilcoxon matched-pairs signed-rank test). The capacity to form colonies was higher in EPCs cultured with platelets compared to EPCs cultured alone. <b>B.</b> Culture viability expressed as OD (560 nm) ± SE in EPCs cultured with vs. without platelets, p<0.05, n = 11 (Wilcoxon matched-pairs signed-rank test). EPCs cultured with platelets have enhanced culture viability. <b>C.</b> FACS analysis of the average number of Tie-2 expressing cells ± SE in EPCs cultured with vs. without platelets, p<0.05, n = 7 (Wilcoxon matched-pairs signed-rank test. A higher percent of Tie-2 expressing cells appear in EPCs cultured with platelets compared to EPCs cultured alone. D–F Direct vs indirect effect of platelets on EPCs ability to form colonies (D), culture viability (E) and the expression of Tie-2(F), for n = 13 or 11 or 7 respectively, p = NS for all. There was no significant difference in any of the tested parameters in EPCs incubated with platelets directly vs. indirectly.</p

bFGF inhibition and its effect on EPCs differentiation.

<p><b>A.</b> The average number of colonies per field ± SE in EPCs cultured with platelets compared to EPCs cultured with platelets and FGF inhibitor or alone, P<0.05, n = 23 (Friedman test followed by Wilcoxon matched-pairs signed-rank test) The ability to form colonies was higher in EPCs cultured with platelets compared to EPCs cultured alone or with platelets and FGF inhibitor. <b>B–C.</b> FACS analysis of the average number of VE-cadherin (A) and Tie-2 (B) expressing cells ± SE, p<0.05, n = 11 for A, p<0.05 n = 10 for B. EPCs cultured with platelets have a higher proportion of Tie-2 and VE-cadherin expressing cells compared to EPCs cultured alone or with platelets and bFGF inhibitor.</p

The indirect effect of platelets on EPCs’ functional properties.

<p><b>A.</b> The average number of colonies per field ± SE in EPCs cultured with vs. without platelets, p<0.05, n = 13 (Wilcoxon matched-pairs signed-rank test). The capacity to form colonies was higher in EPCs cultured with platelets compared to EPCs cultured alone<b>. B.</b> Culture viability expressed as OD (560 nm) ± SE in EPCs cultured with vs. without platelets, p<0.05, n = 11 (Wilcoxon matched-pairs signed-rank test). EPCs cultured with platelets have enhanced culture viability. <b>C.</b> FACS analysis of the average number of Tie-2 expressing cells ± SE in EPCs cultured with vs. without platelets, p<0.05, n = 7 (Wilcoxon matched-pairs signed-rank test. A higher percent of Tie-2 expressing cells appear in EPCs cultured with platelets compared to EPCs cultured alone. <b>D.</b> Representative EPC colonies with platelets (right) compared to EPCs cultured alone (left). <b>E.</b> FACS analysis representative figure of Tie-2 expressing cells.</p

PDGF and FGF protein levels on EPC-PLT supernatant and relative PDGF B/C mRNA levels in EPCs following co incubation with platelets.

<p><b>A–B.</b> PDGF(A) and FGF(B) levels expressed as pg/ml ± SE in supernatants of EPCs cultured with vs. without platelets, n = 11, p<0.05 for A, n = 5 p = NS for B (Wilcoxon matched-pairs signed-rank test). PDGF levels were significantly higher in EPCs cultured with platelets compared to EPCs cultured alone. There were no significant differences in FGF levels between the two groups. <b>C–D.</b> PDGFC(C) and PDGFB(D) relative expression appears as AU ± SE in EPCs cultured with vs. without platelets, n = 6 p<0.05 for A, n = 8, p = NS for B (Wilcoxon matched-pairs signed-rank test). PDGFC mRNA levels were significantly higher in EPCs cultured with platelets compared to EPCs cultured alone (C). There were no significant differences in PDGFB levels between the two groups (D).</p

PDGF inhibition and its effect on EPCs functional properties.

<p><b>A.</b> The average number of colonies per field ± SE in EPCs cultured with platelets compared to EPCs cultured with platelets and PDGF inhibitor or alone, n = 23, p<0.05, p<0.001. (Friedman test followed by Wilcoxon matched-pairs signed-rank test) The ability to form colonies was higher in EPCs cultured with platelets compared to EPCs cultured alone or with platelets and PDGF inhibitor. <b>B.</b> Culture viability expressed as OD (560 nm) ± SE in EPCs cultured with platelets compared to EPCs cultured with platelets and PDGF inhibitor or alone p<0.05, n = 11(Friedman test followed by Wilcoxon matched-pairs signed-rank test). EPCs cultured with platelets had greater culture viability compared to EPCs cultured with platelets and PDGF inhibitor or alone<b>. C–D</b> FACS analysis expressed as the average number of VE-cadherin (C) and Tie-2(D) expressing cells ± SE in EPCs cultured with platelets compared to EPCs cultured with platelets and PDGF inhibition or alone, p<0.05, n = 8 for C, p<0.05, n = 11 for D (Friedman test followed by Wilcoxon matched-pairs signed-rank test)<b>.</b> EPCs cultured with platelets have a higher percent of VE-cadherin (C) and Tie-2(D) expressing cells compared to EPCs cultured alone or with platelets and PDGF inhibitor. <b>E.</b> Representative EPC colonies with platelets (center) compared to EPCs cultured alone (left) or with platelets and PDGF inhibitor (right). <b>F.</b> FACs analysis representative figure of VE-cadherin expressing cells in EPCs cultured with platelets with or without PDGF inhibitor.</p