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Juvenile Neuronal Ceroid Lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 years and is most commonly caused by mutations in the highly conservedCLN3gene. Here, we generatedcln3morphants and stable mutant lines in zebrafish. Although neither morphant nor mutantcln3larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed higher basal activity, hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and a global decrease of bis(monoacylglycero)phosphate (BMP) species, two classes of molecules previously proposed as potential biomarkers forCLN3disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant forCLN3. Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomaticCLN3disease.

T cells undergo a characteristic metabolic rewiring that fulfills the dramatically increased bioenergetic, biosynthetic, and redox demands following antigen stimulation. A robust adaptive immune system requires effector T cells to respond and adapt to fluctuations in environmental nutrient levels imposed by infectious and inflammatory sites in different tissues. Inevitably, such responsiveness and adaptation reflect metabolic plasticity, allowing T cells to elicit immune functions by using a wide range of nutrient substrates. Here, we show that effector T cells utilize inosine, as an alternative substrate, to support cell growth and function in the absence of glucose. T cells metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase (PNP). Using Stable Isotope-Resolved Metabolomics (SIRM), we demonstrated that ribose moiety of inosine can enter into central metabolic pathways to provide ATP and biosynthetic precursors. Accordingly, the dependence of T cells on extracellular glucose for growth and effector functions can be relieved by inosine. On the other hand, cancer cells display diverse capacity to utilize inosine as a carbon resource. Moreover, the supplement of inosine enhances the anti-tumor efficacy of immune-checkpoint blockade or adoptive T cell transfer, reflecting the capability of inosine in relieving tumor-imposed metabolic restrictions on T cellsin vivo.

CHC22 clathrin plays a key role in intracellular membrane trafficking of the insulin-responsive GLUT4 glucose transporter, and so in post-prandial clearance of glucose from human blood. We performed population genetic and phylogenetic analyses of the CLTCL1 gene, encoding CHC22, to understand its variable presence in vertebrates and gain insight into its functional evolution. Analysis of ~50 complete vertebrate genomes showed independent loss of CLTCL1 in nine lineages after it arose from a gene duplication during the emergence of jawed vertebrates. All vertebrates considered here retained the parent CLTC gene encoding CHC17 clathrin, which mediates endocytosis and other housekeeping membrane traffic pathways, as performed by the single clathrin gene product in nonvertebrate eukaryotes. Statistical analysis provides evidence of strong purifying selection over phylogenetic timescales for CLTCL1, as well as for CLTC, supporting preserved functionality of CHC22 in those species that have retained CLTCL1. In population genetic analyses of humans and chimpanzees, extensive allelic diversity was observed for CLTCL1 compared to CLTC. In all human populations, two variants of CLTCL1 segregate at high frequency, resulting in CHC22 protein with either methionine or valine at position 1316. The V1316 variant occurs only in humans, but the same site is polymorphic in non-human primates as well. Analysis of archaic and ancient humans assigned the appearance of the derived V1316 allele to 500-50 KYA. Balancing selection on the two high-frequency CHC22 variants is inferred, with V1316 being more frequent in farming, as compared to hunter-gatherer populations. Together these analyses suggest that CHC22 clathrin is undergoing selection in humans related to its role in nutrient metabolism. Consistent with this conclusion, we observed functional differences between the two CHC22 variants in their ability to control GLUT4 membrane traffic, as predicted by structural modeling and differences in cellular dynamics of the two variants.