Biology subjects

Ramamurthy, V.

Publications and source records attributed to Ramamurthy, V..

7 recordsLinked to original sources

bioRxiv2025

Loss of PIKfyve in Rod Photoreceptors and RPE Cells Leads to Endolysosomal Dysfunction and Retinal Degeneration

Photoreceptor outer segment (OS) degradation is primarily mediated by retinal pigment epithelial (RPE) cells through daily phagocytosis of shed distal OS tips. In contrast, much less is understood about the cell-autonomous mechanisms photoreceptors use to clear mislocalized molecules caused by protein misfolding or trafficking defects. Mislocalized or excess rhodopsin that fails to reach the OS is retained in the inner segment or cell body, where it is presumably degraded via the endolysosomal system. We identify PIKfyve, a phosphoinositide kinase that generates PI(3,5)P2, as a key regulator of this pathway. Using Translating Ribosome Affinity Purification (TRAP), we find that PIKfyve is highly expressed in rod photoreceptors. Rod-specific PIKfyve deletion causes progressive retinal degeneration, marked by inner segment vacuolation, elevated LAMP1/2, thinning of the outer nuclear layer, and eventual loss of rod and cone function. Loss of one copy of PIKfyve in rod photoreceptors accelerates degeneration in P23H rhodopsin mutant mice. In RPE cells, PIKfyve loss disrupts phagocytosis and autophagy, leading to accumulation of rhodopsin, LAMP1, LC3A/B, and lipid droplets, along with metabolic disturbances. These findings demonstrate that PIKfyve is essential for photoreceptor and RPE health by regulating lysosomal function, phagocytosis, autophagy and metabolism, and suggest that enhancing PIKfyve activity could be a therapeutic strategy for retinal degenerative diseases.

neuroscience↗

bioRxiv2025

HSP90α is specifically required for rod photoreceptor function and cannot be replaced by HSP90β.

Heat Shock Protein 90 (HSP90) is a critical molecular chaperone that exists as two cytosolic paralogs, HSP90 and HSP90{beta}, which share high sequence identity but may perform non-redundant functions in vivo. Loss of HSP90 in mice results in progressive rod photoreceptor degeneration despite normal retinal development and expression of HSP90{beta}. To investigate whether HSP90{beta} can substitute for HSP90 in photoreceptors, we generated adeno-associated virus (AAV) vectors expressing HA-tagged HSP90 or HSP90{beta} under the control of a short rhodopsin promoter. In Hsp90 -/- mice, subretinal delivery of AAV-Hsp90aa1 (HSP90) restored rod function and prevented photoreceptor degeneration, as measured by electroretinography (ERG). In contrast, AAV-mediated expression of HSP90{beta} failed to rescue rod function despite comparable expression levels. Overexpression of either paralog in wild-type mice had no adverse effects on retinal function. These findings reveal a paralog-specific and intrinsic requirement for HSP90 in rod photoreceptors, demonstrating that HSP90{beta} cannot compensate for its loss despite structural similarity.

biochemistry↗

bioRxiv2022

The Musashi proteins are post-transcriptional activators of protein expression and alternative exon splicing in vertebrate photoreceptors

The Musashi proteins, MSI1 and MSI2, are conserved RNA binding proteins with a role in the maintenance and renewal of stem cells. Contrasting with this role, retina and terminally differentiated photoreceptor cells express high levels of MSI1 and MSI2, indicating that the two proteins have a role unrelated to maintaining undifferentiated cell state. Here we show that the Musashi proteins are essential in mature photoreceptors. Combined knockout of Msi1 and Msi2 lead to loss of the retina response to light and progressive photoreceptor cell death. The two proteins are fully redundant as individual deletion of Msi1 or Msi2 did not produce a phenotype. To define the molecular functions underlying the requirement for Musashi in photoreceptors, we delineated their RNA targets and analyzed the effect of the combined Msi1/Msi2 knockout on transcript levels, pre-mRNA splicing, and protein expressions. We show distinct nuclear and cytoplasmic functions for the Musashi proteins in photoreceptor cells. In the nucleus, Musashi binding to the downstream proximal intron promotes splicing of alternative exons. Surprisingly, four conserved photoreceptor-specific alternative exons in genes critical for vision proved to be dispensable, leaving open questions about the selective pressures that lead to the conservation of these exons and the contribution of alternative splicing to the phenotype of the Musashi knockout. In the photoreceptor cell cytoplasm MSI1 and MSI2 act as activators of protein expression. The combined knockout of Msi1 and Msi2 reduced the levels of multiple proteins including proteins required for vision and photoreceptor survival.

neuroscience↗

bioRxiv2022

The 13A4 monoclonal antibody to the mouse PROM1 protein recognizes a structural epitope

PurposeWe endeavored to map the epitope of the rat monoclonal antibody mAB 13A4 to the mouse PROM1 (CD133, AC133) protein. mAB 13A4 is the main reagent used to detect the mouse PROM1 protein. PROM1 is required for the maintenance of primary cilia and mutations in the Prom1 gene are associated with retinal degeneration. MethodsEpitope tagged clones of splice variants and tiled deletion mutants were used to map the mAB 13A4 epitope and test the predicted tertiary structure of PROM1. The proteins were expressed in Neuro 2a cells and analyzed by Western blot with antibodies to PROM1 and the epitope tag. ResultsDeletions in the second and third extracellular domains of the PROM1 protein disrupted the mAB 13A4 epitope. Furthermore, the affinity of mAB 13A4 to the major PROM1 isoform in photoreceptor cells is significantly reduced due to the inclusion of a photoreceptor-specific alternative exon in the third extracellular domain. Interestingly, a deletion in the photoreceptor specific isoform of six amino acids adjacent to the alternative exon restored the affinity of mAB 13A4 to PROM1. ConclusionmAB 13A4 recognizes a structural epitope that is stabilized by two of the extracellular domains of PROM1. The results of our mutagenesis are consistent with the computationally predicted helical bundle structure of PROM1 and point to the utility of mAB 13A4 for evaluating the effect of mutations on the PROM1 structure. We show that the PROM1 isoform composition needs to be considered when interpreting tissue and developmental expression data produced by mAB 13A4.

immunology↗

bioRxiv2020

A critical role for MSI1 and MSI2 in photoreceptor morphogenesis

Musashi family of RNA-binding proteins are known for their role in stem-cell renewal and are negative regulators of cell differentiation. Interestingly, in the retina, Musashi proteins, MSI1 and MSI2 are differentially expressed throughout the cycle of retinal development including robust expression in the adult retinal tissue. To study the role of Musashi proteins in the retina, we generated a pan-retinal and rod photoreceptor neuron specific conditional knockout mouse lacking MSI1 and MSI2. Independent of sex, photoreceptor neurons with simultaneous deletion of Msi1 and Msi2 were unable to respond to light and displayed severely disrupted OS morphology and ciliary defects. The retina lacking Musashi exhibited neuronal degeneration with complete loss of photoreceptors by six months. In concordance with our earlier studies that proposed a role for Musashi in regulating alternative splicing, the loss of Musashi prevented the use of photoreceptor-specific exons in transcripts critical for OS morphogenesis, ciliogenesis and synaptic transmission. Overall, we demonstrate a critical role for Musashi in the morphogenesis of terminally differentiated photoreceptor neurons. This role is in stark contrast with the canonical function of these two proteins in maintenance and renewal of stem cells.

developmental biology↗

bioRxiv2019

Proteoglycan IMPG2 shapes the interphotoreceptor matrix and modulates vision

The extracellular matrix surrounding the photoreceptor neurons, interphotoreceptor matrix (IPM) is comprised of two unique proteoglycans: IPM proteoglycan 1 and 2 (IMPG1 and IMPG2). Although the functions of the IPM are not understood, patients with mutations in IMPG1/2 develop visual deficits with subretinal material accumulation. Here, we generated mouse models lacking IMPG1/2 to decipher the role of these proteoglycans and the pathological mechanisms that lead to vision loss. IMPG1 and IMPG2 occupy specific locations in the outer retina, and both proteoglycans are fundamental for the constitution of the IPM system. Mice lacking IMPG2 show abnormal accumulation of IMPG1, and in later stages, develop subretinal lesions and reduced visual function. Interestingly, removal of IMPG1-2 showed normal retinal morphology and function, suggesting that the aberrant localization of IMPG1 causes the alterations observed in IMPG2 KO mice. In conclusion, our results demonstrate the role of IMPG2 in shaping the IPM, shed light on the potential mechanisms leading to subretinal lesions, and show that the secreted proteoglycans depend on the extracellular matrix environment to properly integrate into the matrix.

cell biology↗

bioRxiv2018

The Novel Mnk1/2 Degrader VNLG-152 Potently Inhibits TNBC Tumor Growth and Metastasis

Currently, there are no effective therapies for patients with triple-negative breast cancer (TNBC), an aggressive and highly metastatic disease. Activation of eukaryotic initiation factor 4E (eIF4E) by mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (Mnk1/2) play a critical role in the development, progression and metastasis of TNBC. Herein, we undertook a comprehensive study to evaluate the activity of a first-in-class Mnk1/2 protein degraders, in clinically relevant models of TNBC. These studies enabled us to identify racemic VNLG-152R as the most efficacious Mnk1/2 degrader. By targeting Mnk1/2 protein degradation (activity), VNLG-152R potently inhibited both Mnk-eIF4E and mTORC1 signaling pathways and strongly regulated downstream factors involved in cell cycle regulation, apoptosis, pro-inflammatory cytokines/chemokines secretion, epithelial-mesenchymal transition (EMT) and metastasis. Most importantly, orally bioavailable VNLG-152R exhibited remarkable antitumor and antimetastatic activities against cell line and patient-derived TNBC xenograft models, with no apparent host toxicity. Collectively, these studies demonstrate that targeting Mnk-eIF4E/mTORC1 signaling with a potent Mnk1/2 degrader, VNLG-152R, is a novel therapeutic strategy that can be developed as monotherapy for effective treatment of patients with primary/metastatic TNBC.

cancer biology↗