Quantification of dolichol in the human lens with different types of cataracts

PURPOSE: To quantify and characterize dolichol species in cataractous and clear human lenses. METHODS: Whole lenses were collected from cadaver eyeballs from the C.H. Nagri Eye Bank and Red Cross Society Eye Bank (Ahmedabad, India). Cataractous nuclei were collected after extracapsular cataract extraction (ECCE). Wet weight for all the lenses was taken and were stored at -50 degrees C until used. Dolichol was extracted using a standard protocol and then analyzed using High Performance Liquid Chromatography (HPLC) on a 4.6 mmx60 mm Hypersil-Octadecylsilane (ODS; 3 microm) reversed phase column using a Waters dual pump apparatus, a Waters gradient programmer, and an ultraviolet (UV) detector set at 210 nm. Dolichol 13 was used as an internal standard, and dolichol mixture from the liver was used as an external qualitative standard. RESULTS: The highest dolichol concentration was found in nuclear cataract (2.54+/-0.6 microg) followed by posterior subcapsular cataract (1.4+/-0.35 microg), and the lowest levels were observed in cortical cataract (0.37+/-0.06 microg). The level of dolichol concentration in cataractous lenses was statistically significantly higher than the levels in clear lenses (1.0+/-04.3 microg; p<0.01). CONCLUSIONS: The dolichol concentration was significantly higher in lenses with nuclear cataract. A significant difference in dolichol concentration was observed between the different types of cataract. It suggests that dolichol and other isoprenoids may be associated with cataractogenesis

Analysis of single nucleotide polymorphisms of <i> CRYGA</i> and <i> CRYGB</i> genes in control population of western Indian origin

<b>Aim:</b> Polymorphisms in <b>&#947;-</b>crystallins (<i> CRYG</i> ) can serve as markers for lens differentiation and eye disorders leading to cataract. Several investigators have reported the presence of sequence variations within crystallin genes, with or without apparent effects on the function of the proteins both in mice and humans. Delineation of these polymorphic sites may explain the differences observed in the susceptibility to cataract observed among various ethnic groups. An easier Restriction Fragment Length Polymorphism (RFLP)-based method has been used to detect the frequency of four single nucleotide polymorphisms (SNPs) in <i> CRYGA</i> /<i> CRYGB </i> genes in control subjects of western Indian origin. <b> Materials and Methods:</b> A total of 137 healthy volunteers from western India were studied. Examination was performed to exclude volunteers with any ocular defects. Polymerase chain reaction (PCR)-RFLP based method was developed for genotyping of G198A (Intron A), T196C (Exon 3) of <i> CRYGA</i> and T47C (Promoter), G449T (Exon 2) of <i> CRYGB</i> genes. <b> Results: </b> The exonic SNPs in <i> CRYGA</i> and <i> CRYGB </i> were found to have an allele frequency 0.03 and 1.00 for ancestral allele respectively, while frequency of non-coding SNP in <i> CRYGA</i> was 0.72. Allele frequency of T90C of <i> CRYGB</i> varied significantly (<i> P</i> = 0.02) among different age groups. An <i> in-silico</i> analysis reveals that this sequence variation in <i> CRYGB</i> promoter impacts the binding of two transcription factors, ACE2 (Member of CLB2 cluster) and Progesterone Receptor (PR) which may impact the expression of <i> CRYGB</i> gene. <b> Conclusions:</b> This study establishes baseline frequency data for four SNPs in <i> CRYGA</i> and <i> CRYGB</i> genes for future case control studies on the role of these SNPs in the genetic basis of cataract

Retinal ganglion cell repopulation for vision restoration in optic neuropathy: a roadmap from the RReSTORe Consortium

Abstract Retinal ganglion cell (RGC) death in glaucoma and other optic neuropathies results in irreversible vision loss due to the mammalian central nervous system’s limited regenerative capacity. RGC repopulation is a promising therapeutic approach to reverse vision loss from optic neuropathies if the newly introduced neurons can reestablish functional retinal and thalamic circuits. In theory, RGCs might be repopulated through the transplantation of stem cell-derived neurons or via the induction of endogenous transdifferentiation. The RGC Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) Consortium was established to address the challenges associated with the therapeutic repair of the visual pathway in optic neuropathy. In 2022, the RReSTORe Consortium initiated ongoing international collaborative discussions to advance the RGC repopulation field and has identified five critical areas of focus: (1) RGC development and differentiation, (2) Transplantation methods and models, (3) RGC survival, maturation, and host interactions, (4) Inner retinal wiring, and (5) Eye-to-brain connectivity. Here, we discuss the most pertinent questions and challenges that exist on the path to clinical translation and suggest experimental directions to propel this work going forward. Using these five subtopic discussion groups (SDGs) as a framework, we suggest multidisciplinary approaches to restore the diseased visual pathway by leveraging groundbreaking insights from developmental neuroscience, stem cell biology, molecular biology, optical imaging, animal models of optic neuropathy, immunology & immunotolerance, neuropathology & neuroprotection, materials science & biomedical engineering, and regenerative neuroscience. While significant hurdles remain, the RReSTORe Consortium’s efforts provide a comprehensive roadmap for advancing the RGC repopulation field and hold potential for transformative progress in restoring vision in patients suffering from optic neuropathies