多能干细胞分化来源视网膜色素上皮细胞移植治疗视网膜变性研究进展

视网膜色素上皮（RPE）对视觉功能的维持起着至关重要的作用。视网膜变性是全球不可治愈性致盲疾病的重要原因,它由视网膜色素上皮功能失常所引起。因此,视网膜色素上皮移植是视网膜变性患者恢复视力的一种最有前景的手段之一。随着干细胞技术的快速发展,从多能干细胞（PSC）到有功能的视网膜色素上皮细胞的体外分化诱导技术已经成熟,其中包括胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）等。此外,从患者特异性iPSCs分化而来的RPE更能用于阐明发病机理并有针对性地个体治疗。更值得一提的是,经诱导得到RPE的移植不论在动物模型中,还是在临床试验里都已经得到了可喜的治疗效果。本文回顾PSC来源RPE干预治疗视网膜变性的最新研究进展。     

Facilitation between extensor carpi radialis and pronator teres in humans: A study using a post-stimulus time histogram method

Group I muscle afferents modulate the excitability of motor neurons through excitatory and inhibitory spinal reflexes. Spinal reflex relationships between various muscle pairs are well described in experimental animals but not in the human upper limb, which exhibits a fine control of movement. In the present study, spinal reflexes between the extensor carpi radialis (ECR) and pronator teres (PT) muscles were examined in healthy human subjects using a post-stimulus time histogram method. Electrical stimulation of low-threshold afferents of ECR nerves increased the motor neuron excitability in 31 of 76 PT motor units (MUs) in all eight subjects tested, while stimulation of low-threshold afferents of PT nerves increased the motor neuron excitability in 36 of 102 ECR MUs in all 10 subjects. The estimated central synaptic delay was almost equivalent to that of homonymous facilitation. Mechanical stimulation (MS) of ECR facilitated 16 of 30 PT MUs in all five subjects tested, while MS of PT facilitated 17 of 30 ECR MUs in all six subjects. These results suggest excitatory reflex (facilitation) between PT and ECR. Group I afferents should mediate the facilitation through a monosynaptic path.     

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28''s SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.     

Differential Light-induced Responses in Sectorial Inherited Retinal Degeneration

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking. M39R showed a faster rate for transducin activation than WT rhodopsin with a faster metarhodopsinII decay, whereas N55K presented a reduced activation rate and an altered photobleaching pattern. N55K also showed an altered retinal release from the opsin binding pocket upon light exposure, affecting its optimal functional response. Our data suggest that these sector RP mutations cause different protein phenotypes that may be related to their different clinical progression. Overall, these findings illuminate the molecular mechanisms of sector RP associated with rhodopsin mutations.     

Human Retinal Progenitor Cell Transplantation Preserves Vision

Cell transplantation is a potential therapeutic strategy for retinal degenerative diseases involving the loss of photoreceptors. However, it faces challenges to clinical translation due to safety concerns and a limited supply of cells. Human retinal progenitor cells (hRPCs) from fetal neural retina are expandable in vitro and maintain an undifferentiated state. This study aimed to investigate the therapeutic potential of hRPCs transplanted into a Royal College of Surgeons (RCS) rat model of retinal degeneration. At 12 weeks, optokinetic response showed that hRPC-grafted eyes had significantly superior visual acuity compared with vehicle-treated eyes. Histological evaluation of outer nuclear layer (ONL) characteristics such as ONL thickness, spread distance, and cell count demonstrated a significantly greater preservation of the ONL in hRPC-treated eyes compared with both vehicle-treated and control eyes. The transplanted hRPCs arrested visual decline over time in the RCS rat and rescued retinal morphology, demonstrating their potential as a therapy for retinal diseases. We suggest that the preservation of visual acuity was likely achieved through host photoreceptor rescue. We found that hRPC transplantation into the subretinal space of RCS rats was well tolerated, with no adverse effects such as tumor formation noted at 12 weeks after treatment.     

Bond torsion affects the product distribution in the photoreaction of retinal model chromophores

Ab initio molecular dynamics (MD) calculations have been performed to study the photoisomerization of a 3-double-bond retinal model chromophore, the all-trans-4, 6-dimethylpenta-3, 5-dieniminium cation, and the possible influence of non-planar distortions on the product distribution. In total, 171 trajectories have been generated for four different conformations of the structure, a planar one and three in which the C4–C5 and the C5=C6 bonds were increasingly twisted out of plane. Starting geometries randomly distributed about the equilibrium geometry were generated by zero-point energy sampling; trajectories were calculated using CASSCF-BOMD methodology and were followed until the photoproduct and its configuration could be assigned. For the latter, two different approaches were applied, one involving the CASSCF configuration vectors, the other an analysis of the MD at the first possible hopping event. Isomerization was found to occur almost exclusively about the central C3=C4 double bond in the case of the planar model compound. Twisting the conjugated π-system shifts the isomerization site from the central double bond to the terminal C5=C6 double bond. With both the C4–C5 and the C5=C6 bonds twisted by 20°, about 35% of the trajectories lead to the configurationally inverted 5-cis product. The results are discussed with reference to the highly selective and efficient photo-induced isomerization of the retinal chromophore in rhodopsin. Figure Product distribution in the MD simulations of models 1–4. The percentage of trajectories that lead to either C3=C4 or C5=C6 rotation is given beside the bar graphs. The green and the red portions of the bars represent the productive and the unproductive events, respectively, with respect to that particular rotation estimated from the torsion angles at the first close approach of the energy surfaces Dedicated to Professor Dr. Paul von Ragué Schleyer on the occasion of his 75th birthday.     

Beta-catenin is essential for lamination but not neurogenesis in mouse retinal development

During vertebrate retinal development, the seven retinal cell types differentiate sequentially from a single population of retinal progenitor cells (RPCs) and organize themselves into a distinct laminar structure. The purpose of this study was to determine whether beta-catenin, which functions both as a nuclear effector for the canonical Wnt signaling pathway and as a regulator of cell adhesion, is required for retinal neurogenesis or lamination. We used the Cre-loxP system to either eliminate beta-catenin or to express a constitutively active form during retinal neurogenesis. Eliminating beta-catenin did not affect cell differentiation, but did result in the loss of the radial arrangement of RPCs and caused abnormal migration of differentiated neurons. As a result, the laminar structure was massively disrupted in beta-catenin-null retinas, although all retinal cell types still formed. In contrast to other neural tissues, eliminating beta-catenin did not significantly reduce the proliferation rate of RPCs; likewise, activating beta-catenin ectopically in RPCs did not result in overproliferation, but loss of neural retinal identity. These results indicate that beta-catenin is essential during retinal neurogenesis as a regulator of cell adhesion but not as a nuclear effector of the canonical Wnt signaling pathway. The results further imply that retinal lamination and retinal cell differentiation are genetically separable processes.     

血塞通对糖尿病视网膜病变患者视网膜微循环的影响

目的:探究血塞通对糖尿病视网膜病变(DR)患者视网膜微循环的影响。方法:收集2013年2月至2016年2月在我院接受治疗的95例双眼糖尿病视网膜病变患者,并随机分为对照组(48例)和观察组(47例)。对照组患者给予常规降糖治疗,观察组在对照组的基础上给予血塞通治疗。观察比较两组治疗前后的视网膜微循环指标,包括视网膜中央动脉(CRA)的舒张末期血流速度(EDV)、收缩期峰值流速(PSV)及阻力指数(RI)以及视网膜中央静脉(CRV)的最低血流速度(Vmin)、最高血流速度(Vmax)及平均血流速度(MV),血液流变学指标包括全血低切黏度(NBL)、全血高切黏度(NBH)、红细胞变形指数(DE)、红细胞压积(Hct)、红细胞聚集指数(AE)及血沉(ESR)以及临床疗效。结果:治疗后,观察组的临床总有效率为87.8%,明显高于对照组的61.4%(P0.05),对照组的EDV、Vmax、Vmin均较治疗前明显改善(P0.05),NBL和NBH较治疗前明显降低,且观察组的PSV、EDV明显高于对照组,而MV、RI、Vmax、Vmin均低于对照组(P0.05),NBL、NBH、DE、Hct、AE及ESR均较治疗前明显改善,且相较于对照组改善幅度更显著(P0.05)。结论:血塞通联合常规降糖治疗糖尿病视网膜病变患者的疗效显著,可有效改善患者的视网膜微循环和血液流变学。     

Apoptosis of Central and Peripheral Neurons Can Be Prevented with Cyclin-Dependent Kinase/Mitogen-Activated Protein Kinase Inhibitors

Abstract: Previous studies have indicated that certain members of the cyclin-dependent kinase/mitogen-activated protein kinase superfamily are involved in apoptosis of neuronal cells. Here, we have examined programmed cell death induced by withdrawal of neurotrophic support from CNS (rat retinal) and PNS (chick sympathetic, sensory, and ciliary) neurons. All four neuron types were equally rescued by the purine analogues olomoucine and roscovitine. Olomoucine inhibits multiple cyclin-dependent and mitogen-activated protein kinases with similar potency. Roscovitine is a more selective cyclin-dependent kinase inhibitor; but, so is butyrolactone I, which did not prevent retinal ganglion cell death. The specific p38^MAPK inhibitor SB-203580 did not prevent apoptosis in retinal ganglion cells. Death of these cells in the absence of neurotrophic factors was accompanied by morphological changes indicative of apoptosis, including nuclear condensation and fragmentation. Treatment with olomoucine or roscovitine not only prevented these apoptotic changes in retinal ganglion cells but also blocked neurite outgrowth. The survival-promoting activity of olomoucine correlated with its in vitro IC₅₀ for c-Jun N-terminal kinase-1 and its potency to repress c- jun induction in live PC12 cells. Roscovitine was more potent in rescuing neurons than in inhibiting Jun kinase. Thus, the antiapoptotic action of roscovitine might be due to inhibition of additional kinases.