Biology subjects

Dalvi, S.

Publications and source records attributed to Dalvi, S..

7 recordsLinked to original sources

bioRxiv2025

A 3D iPSC retina model reveals non-cell-autonomous and non-neuronal mechanism of photoreceptor degeneration in a lysosomal storage disorder.

Disruption of photoreceptor-retinal pigment epithelium (RPE) interface with loss of photoreceptor outer segments (POSs) in the retina is a pathological hallmark of several neurodegenerative and retinal diseases including lysosomal storage disorders like CLN3 disease. However, the retina is a functional composite in vivo; and in vitro stem cell models of retina that enable investigation of the photoreceptor-RPE interface in healthy and diseased retina are lacking. Here, we developed a 3D human pluripotent stem cell (hPSC)-derived retina model to investigate the photoreceptor-RPE interface in healthy and disease tissue. Using this 3D hPSC retina model, we demonstrated that the most common disease causing CLN3 mutation (CLN3{Delta}ex7-8) leads to reduced levels of acid ceramidase (AC) and consequently altered sphingolipid metabolism and signaling and POS loss in CLN3 disease. Consistent with the 3D hPSC retina model, altered sphingolipid metabolism and signaling coincided with POS loss in a large animal model of CLN3 disease, CLN3 miniswine. Therapeutically, recombinant human acid ceramidase (rhAC) targeted both altered sphingolipid metabolism and retina degeneration in the CLN3 hPSC retina model and the CLN3 miniswine eye. These findings demonstrate a proof-of-concept that rhAC can rescue disease phenotype in a large animal model of CLN3 disease and suggest that rhAC could be a therapeutic approach for CLN3 disease. One Sentence SummaryAcid ceramidase deficiency and consequently altered sphingolipid signaling promotes disease phenotype(s) in a lysosomal storage disorder, CLN3 disease.

neuroscience↗

bioRxiv2024

Genetic and cellular basis of impaired phagocytosis and photoreceptor degeneration in CLN3 disease.

PurposeCLN3 Batten disease (also known as Juvenile Neuronal Ceroid Lipofuscinosis; JNCL) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease iPSC-RPE cells show defective phagocytosis of photoreceptor outer segments (POSs). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect. MethodsIsogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9{-}mediated biallelic deletion of exons 7 and 8. A transgenic CLN3{Delta}7-8/{Delta}7-8 (CLN3) Yucatan miniswine was also used to study the impact of CLN3{Delta}7-8/{Delta}7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis of CLN3{Delta}7/8 and wild-type miniswine eyes were carried out at 6-, 36-, or 48-month age. ResultsCLN3{Delta}7-8/{Delta}7-8 RPE (CLN3 RPE) displayed reduced POS binding and consequently decreased uptake of POS compared to isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosed CLN3{Delta}7-8/{Delta}7-8 POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36 months-of-age and was followed by almost complete loss of photoreceptors at 48 months of age. ConclusionsCLN3{Delta}7-8/{Delta}7-8 mutation (that affects up to 85% patients) affects both RPE and POSs and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.

cell biology↗

bioRxiv2024

A large attachment organelle mediates interaction between a novel Nanobdellota archaeon YN1 and its host

DPANN archaea are an enigmatic superphylum that are difficult to isolate and culture in the laboratory due to their specific culture conditions and apparent ectosymbiotic lifestyle. Here we successfully isolated and cultivated a co-culture system of a novel Nanobdellota archaeon YN1 and its host Sulfurisphaera ohwakuensis YN1HA. We characterised the co-culture system by complementary methods, including metagenomics and metabolic pathway analysis, fluorescence microscopy, and high-resolution electron cryo-tomography (CryoET). We show that YN1 is deficient in essential metabolic processes and requires host resources to proliferate. CryoET imaging revealed an enormous attachment organelle present in the YN1 envelope that forms a direct interaction with the host cytoplasm, bridging the two cells. Together our results unravelled the molecular and structural basis of ectosymbiotic relationship between YN1 and YNHA. This research broadens our understanding of DPANN biology and the versatile nature of their ectosymbiotic relationships.

microbiology↗

bioRxiv2024

Tissue Inhibitor of Metalloproteinase 3 (TIMP3) mutations increase glycolytic activity and dysregulate glutamine metabolism in RPE cells

Mutations in Tissue Inhibitor of Metalloproteinases 3 (TIMP3) cause Sorsbys Fundus Dystrophy (SFD), a dominantly inherited, rare form of macular degeneration that results in vision loss. TIMP3 is synthesized primarily by retinal pigment epithelial (RPE) cells, which constitute the outer blood-retinal barrier. Quantitative proteomics and RNAseq analysis on the choroid/RPE of mice expressing mutant TIMP3 identified a dysregulation in metabolic processes. We examined the effects of mutant TIMP3 on RPE metabolism using human ARPE-19 cells expressing mutant S179C TIMP3 and patient-derived induced pluripotent stem cell-derived RPE (iRPE) carrying the S204C TIMP3 mutation. Stable isotope tracing experiments demonstrated enhanced glucose utilization and glycolytic activity in mutant RPE concomitantly with altered glutamine utilization. This study provides important information on the dysregulation of the metabolome of RPE cells in SFD and implicates a potential commonality with other retinal degenerative diseases, emphasizing RPE cellular metabolism as a therapeutic target.

biochemistry↗

bioRxiv2021

Structural insights on the effects of mutation of a charged binding pocket residue on phosphopeptide binding to 14-3-3 ζ Protein

Mutation of an invariant aspartate residue in the binding pocket of 14-3-3{zeta} isoform to alanine dramatically reduced phosphopeptide binding and induced opening of the binding pocket. Here we use extensive molecular dynamics simulations to understand the role of D124 residue in ligand binding. The simulations show that in the absence of phosphopeptide, the D124A mutation leads to binding pocket reorganization including widening up of the binding pocket at the major groove and repositioning of N173, a key residue that interacts with the main chain of phosphopeptide. These structural changes would interfere with the efficient binding of the peptide, corroborating the experimental observations. Both gain and loss of electrostatic interactions in the form of salt bridges strongly indicate a rearrangement of the network of interactions within the binding pocket. Limited proteolysis coupled mass spectrometry (lip-MS) of the apo and holo forms of WT and mutant protein shows a peptide binding helix otherwise buried in the WT protein was particularly accessible to trypsin in the apo form of the mutant protein and the region was mapped to 158-186 amino acid residues of 14-3-3{zeta}). These results further confirm the dynamic nature of D124A mutant. Unlike other basic residues, the invariant D124 facilitates peptide binding by maintaining the geometry of interacting residues and by enforcing the structural integrity of amphipathic pocket.

biophysics↗

bioRxiv2021

DESIGNER DNA HYDROGELS TO STIMULATE 3D CELL INVASION BY ENHANCED RECEPTOR EXPRESSION AND MEMBRANE ENDOCYTOSIS

DNA has emerged as one of the smartest biopolymers to bridge the gap between chemical science and biology to design scaffolds like hydrogels by physical entanglement or chemical bonding with remarkable properties. We present here a completely new application of DNA based hydrogels in terms of their capacity to stimulate membrane endocytosis, leading to enhanced cell spreading and invasion for cells in ex-vivo 3D spheroids models. Multiscale simulation studies along with DLS data showed that the hydrogel formation was enhanced at lower temperature and it converts to liquid with increase in temperature. DNA hydrogels induced cell spreading as observed by increase in cellular area by almost two-folds followed by increase in receptor expression, endocytosis and 3D invasion potential of migrating cells. Our first results lay the foundation for upcoming diverse applications of hydrogels to probe and program various cellular and physiological processes that can have lasting applications in stem cells programming and regenerative therapeutics.

bioengineering↗

bioRxiv2019

14-3-3γ prevents centrosome duplication by inhibiting NPM1 function.

14-3-3 proteins bind to ligands via phospho-Serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between 14-3-3 proteins and their ligands remain unclear. We identified two conserved acidic residues in the 14-3-3 peptide-binding pocket (D129 and E136) that potentially regulate complex formation and dissociation. Altering these residues to Alanine led to opposing effects on centrosome duplication. D129A inhibited centrosome duplication while E136A stimulated centrosome amplification. These results were due to the differing abilities of these mutant proteins to form a complex with NPM1. Inhibiting complex formation between NPM1 and 14-3-3{gamma} led to an increase in centrosome duplication and overrode the ability of D129A to inhibit centrosome duplication. We identify a novel role of 14-3-3 in regulating centrosome licensing and a novel mechanism underlying the formation and dissociation of 14-3-3 ligand complexes dictated by conserved residues in the 14-3-3 family.

cell biology↗