Sreebhavan, S. | bioRxiv Science

bioRxiv2021

Cystinosin deficient rats recapitulate the phenotype of nephropathic cystinosis

BackgroundThe lysosomal storage disease cystinosis is caused by mutations in CTNS, encoding a cystine transporter, and in its severest form leads to proximal tubule dysfunction followed by kidney failure. Patients receive the drug-based therapy cysteamine from diagnosis. However, despite long-term treatment, cysteamine only slows the progression of end-stage renal disease and a kidney transplant is inevitable. Pre-clinical testing in cystinotic rodents is required to evaluate new therapies; however, the current models are sub-optimal. To solve this problem we generated a new cystinotic rat model. MethodsWe utilized CRISPR/Cas9-mediated gene editing to disrupt exon 3 of Ctns and measured various parameters over a 12-month time-course including blood and tissue cystine levels, urine and serum electrolytes, and analysed the histopathology and immunohistochemistry of the kidney. ResultsCtns-/- rats display hallmarks of cystinosis by 3-6 months of age as seen by a failure to thrive, excessive thirst and urination, cystine accumulation in tissues, corneal cystine crystals, a loss of Lrp2 in proximal tubules and immune cell infiltration. High levels of glucose, calcium, albumin and protein are excreted at 6-months of age, consistent with the onset of Fanconi syndrome, with a progressive diminution of urine urea and creatinine from 9-months of age, indicative of chronic kidney disease. The kidney histology and immunohistochemistry showed proximal tubule atrophy and glomerular damage as well as classic swan neck lesions. Overall, Ctns-/- rats show a disease progression that more faithfully recapitulates nephropathic cystinosis than existing rodent models. ConclusionsThe Ctns-/- rat provides an excellent new rodent model of nephropathic cystinosis that is ideally suited for conducting pre-clinical drug testing and a powerful tool to advance cystinosis research. Translational StatementGood animal models of disease are essential to perform pre-clinical testing of potential new drugs and therapies before they can progress to human clinical trials. Unfortunately, the cystinosis field has been hampered by a lack of suitable animal models that fully recapitulate the disease. We have overcome this by generating a new rat model of cystinosis by CRISPR-Cas9 gene editing. These rodents closely model the human condition in a time-frame that makes them an excellent model ideally suited for pre-clinical drug testing as well as being a powerful tool to advance cystinosis research.

physiology↗

bioRxiv2018

A revised biosynthetic pathway for the cofactor F420 in bacteria

Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420 has not been fully eluci-dated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the {gamma}-glutamyl ligase (FbiB) in bacteria are known. Here we show that the guanylyltransferases FbiD and CofC accept phosphoenolpyruvate, rather than 2-phospho-L-lactate, as their substrate, leading to the formation of the previously uncharacterized intermediate, dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420 species when combined with the {gamma}-glutamyl ligase activity of the N-terminal domain. This new insight has allowed the heterologous expression F420 from a recombinant F420 biosynthetic pathway in Escherichia coli.

molecular biology↗