Simultaneous profiling of native-state proteomes and transcriptomes of neural cell types using proximity labeling Christina C. Ramelow, Eric B. Dammer, Hailian Xiao, Lihong Cheng, Prateek Kumar, Claudia Espinosa-Garcia, Maureen M. Sampson, Dilpreet Kour, Ruth S. Nelson, Sneha Malepati, Rashmi Kumari, Wooyoung Eric Jang, Qi Guo, Pritha Bagchi, Duc M. Duong, Nicholas T. Seyfried, Steven A. Sloan, Srikant Rangaraju Nature Communications, 2026 Phenotyping cells at transcriptomic and proteomic levels is an essential step to understanding cellular contributions to development, aging, injury, and disease. Since proteome and transcriptome level abundances modestly correlate, complementary profiling of both is needed. We report a method called simultaneous protein and RNA -omics (SPARO) to capture the cell type-specific transcriptome and proteome simultaneously in vitro using BV2 microglial and HEK293 cell lines and in vivo using astrocytic and neuronal Cre driver mice crossed with Rosa26-TurboID knock-in mice. SPARO leverages TurboID to biotinylate RNA-interacting cytosolic proteins, enabling enrichment of proteins for proteomics and protein-associated RNA for transcriptomics. We validate SPARO first using well-controlled in vitro systems to verify that the proteomes and transcriptomes obtained reflect the global proteomes and transcriptomes. The effect of neuroinflammatory activation by lipopolysaccharide is also faithfully captured. We apply SPARO to obtain native-state proteomes and transcriptomes from astrocytes and neurons, thereby validating the approach in vivo. We interrogate mRNA-protein concordance and discordance, providing insights into molecular processes that exhibit uniform or cell type-specific patterns. The transcriptome and proteome provide complementary information about the cellular phenotype, state and function. Here, the authors introduce SPARO, a method that enables simultaneous profiling of cell type-specific transcriptomes and proteomes in vitro and in vivo by leveraging TurboID-based biotinylation of RNA-interacting cytosolic proteins to enrich both proteins and associated RNAs.
Isobavachalcone ameliorates Alzheimer disease pathology by autophagy-mediated clearance of amyloid beta and inhibition of NLRP3 inflammasome in primary astrocytes and 5x-FAD mice Dilpreet Kour, Parul Khajuria, Kuhu Sharma, Alpa Sharma, Ankita Sharma, Syed Mudassir Ali, Priya Wazir, P. Ramajayan, Sanghapal D. Sawant, Utpal Nandi, Zabeer Ahmed, Ajay Kumar Frontiers in Pharmacology, 2025 Background and AimAlzheimer’s disease (AD) progresses with Aβ plaque deposition and neuroinflammation. Given the complexity of AD pathology, single-target therapies have frequently failed in clinical trials. We hypothesized that a multitarget approach could yield better therapeutic outcomes. To this end, we identified isobavachalcone (IBC), a natural compound with dual pharmacological activity in reducing Aβ plaques and neuroinflammation.Experimental ProcedurePrimary astrocytes were isolated from 3 to 4 days old C57BL/6J mice pups for in-vitro assays, while in-vivo studies were conducted on 5x-FAD mice. Protein alterations were evaluated using ELISA, western blotting, immunocytochemistry, and immunohistochemistry. Behavioral analyses included the radial arm maze, open field, and rotarod tests. Data from all in vitro and in vivo experiments were analyzed by using one-way ANOVA and post-hoc Bonferroni tests.ResultsIn-vitro analyses in astrocytes demonstrated that IBC at 5 and 10 μM concentrations induce AMPK phosphorylation through CAMKK2, promoting autophagy and inhibiting the NLRP3 inflammasome in primary astrocytes. IBC-treated astrocytes exhibited significant clearance of extracellular amyloid beta. Mechanistic studies highlighted autophagy as a key factor in reducing both NLRP3 inflammasome activity and Aβ levels. Two months of treatment of 5x-FAD mice with IBC at 25 and 50 mg/kg significantly improved cognitive functions, as evidenced by enhanced memory and motor performance in behavioral tests. Subsequent brain tissue analysis revealed that IBC upregulated autophagic proteins to reduce the brain’s amyloid beta levels, resulting in decreased expression of neuroinflammation markers.ConclusionIBC effectively ameliorates AD pathology through autophagy-mediated clearance of Aβ and suppressing neuroinflammation in 5x-FAD mice.
Identification of Novel Kv1.3 Channel-Interacting Proteins Using Proximity Labelling in T-Cells , Dilpreet Kour, Christine A. Bowen, , , Upasna Srivastava, Hai M. Nguyen, Rashmi Kumari, Prateek Kumar, Amanda D. Brandelli, Sara Bitarafan, Brendan R Tobin, Levi B. Wood, Nicholas T. Seyfried, Heike Wulff,, Srikant Rangaraju Cellular Physiology and Biochemistry, 2025 Background/Aims: Potassium channels regulate membrane potential, calcium flux, cellular activation and effector functions of adaptive and innate immune cells. The voltage-activated Kv1.3 channel is an important regulator of T cell-mediated autoimmunity and microglia-mediated neuroinflammation. Kv1.3 channels, via protein-protein interactions, are localized with key immune proteins and pathways, enabling functional coupling between K+ efflux and immune mechanisms. Methods: To gain insights into proteins and pathways that interact with Kv1.3 channels, we applied a proximity-labeling proteomics approach to characterize protein interactors of the Kv1.3 channel in activated T-cells. Biotin ligase TurboID was fused to either N or C termini of Kv1.3, stably expressed in Jurkat T cells, and biotinylated proteins in proximity to Kv1.3 were enriched and quantified by mass spectrometry. Results: We identified over 1, 800 Kv1.3 interactors including known interactors (beta-integrins, Stat1), although the majority were novel. We found that the N-terminus of Kv1.3 preferentially interacts with protein synthesis and protein trafficking machinery, while the C-terminus interacts with immune signaling and cell junction proteins. T-cell Kv1.3 interactors we found consisted of 335 cell surface proteins, including T-cell receptor complex, mitochondrial, calcium and cytokine-mediated signaling pathway, and lymphocyte migration proteins. 178 Kv1.3 interactors in T-cells also represent genetic risk factors for T cell-mediated autoimmunity, including STIM1, which was further validated using co-immunoprecipitation. Conclusion: Our studies revealed novel proteins and molecular pathways that interact with Kv1.3 channels in adaptive (T-cell) and innate (microglia) immune cells, providing a foundation for understanding how Kv1.3 channels may regulate immune mechanisms in autoimmune and neurological diseases.
ADME/PK Insights of Crocetin: A Molecule Having an Unusual Chemical Structure with Druglike Features Diksha Manhas, Sumit Dhiman, Harpreet Kour, Dilpreet Kour, Kuhu Sharma, Priya Wazir, Bhavna Vij, Ajay Kumar, Sanghapal D. Sawant, Zabeer Ahmed, Utpal Nandi ACS Omega, 2024 High Resolution Image Download MS PowerPoint Slide Crocetin is a promising phyto-based molecule to treat Alzheimer’s disease (AD). The chemical structure of crocetin is incongruent with various standard structural features of CNS drugs. As poor pharmacokinetic behavior is the major hurdle for any candidate to become a drug, we elucidated its druggable characteristics by implementing in silico, in vitro, and in vivo approaches, as limited ADME/PK information is available. Results demonstrate several attributes of crocetin based on rules of drug-likeness, lipophilicity, p K a, P-gp inhibitory activity, plasma stability, RBC partitioning, metabolic stability, CYP inhibitory action, blood-brain barrier (BBB) permeability, oral bioavailability, and pharmacokinetic interaction with marketed anti-Alzheimer’s drugs (memantine, donepezil, galantamine, and rivastigmine). However, aqueous solubility, chemical stability, plasma protein binding, and P-gp induction are some concerns associated with this molecule that should be taken into consideration during its further development. Overall results indicate favorable ADME/PK behavior and potential druggable candidature of crocetin.
EIDD-1931 Treatment Tweaks CYP3A4 and CYP2C8 in Arthritic Rats to Expedite Drug Interaction: Implication in Oral Therapy of Molnupiravir Mahir Bhardwaj, Dilpreet Kour, Garima Rai, Srija Bhattacharya, Diksha Manhas, Bhavna Vij, Ajay Kumar, Debaraj Mukherjee, Zabeer Ahmed, Sumit G. Gandhi, Utpal Nandi ACS Omega, 2024 EIDD-1931 is the active form of molnupiravir, an orally effective drug approved by the United States Food and Drug Administration (USFDA) against COVID-19. Pharmacokinetic alteration can cause untoward drug interaction (drug-drug/disease-drug), but hardly any information is known about this recently approved drug. Therefore, we first investigated the impact of the arthritis state on the oral pharmacokinetics of EIDD-1931 using a widely accepted complete Freund's adjuvant (CFA)-induced rat model of rheumatoid arthritis (RA) after ascertaining the disease occurrence by paw swelling measurement and X-ray examination. Comparative oral pharmacokinetic assessment of EIDD-1931 (normal state vs arthritis state) showed that overall plasma exposure was augmented (1.7-fold) with reduced clearance (0.54-fold), suggesting its likelihood of dose adjustment in arthritis conditions. In order to elucidate the effect of EIDD-1931 treatment at a therapeutic regime (normal state vs arthritis state) on USFDA-recommended panel of cytochrome P450 (CYP) enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) for drug interaction using the same disease model, we monitored protein and mRNA expressions (rat homologs) in liver tissue by western blotting (WB) and real time-polymerase chain reaction (RT-PCR), respectively. Results reveal that EIDD-1931 treatment could strongly influence CYP3A4 and CYP2C8 among experimental proteins/mRNAs. Although CYP2C8 regulation upon EIDD-1931 treatment resembles similar behavior under the arthritis state, results dictate a potentially reverse phenomenon for CYP3A4. Moreover, the lack of any CYP inhibitory effect by EIDD-1931 in human/rat liver microsomes (HLM/RLM) helps to ascertain EIDD-1931 treatment-mediated disease-drug interaction and the possibility of drug-drug interaction with disease-modifying antirheumatic drugs (DMARDs) upon coadministration. As elevated proinflammatory cytokine levels are prevalent in RA and nuclear factor-kappa B (NF-kB) and nuclear receptors control CYP expressions, further studies should focus on understanding the regulation of affected CYPs to subside unexpected drug interaction.
Gentiacaulein inhibits glucose transport to induce PRKAA1-mediated autophagy to clear amyloid beta and associated inflammation in primary astrocytes Ankita Sharma, Sukhleen Kaur, Abubakar Wani, Dilpreet Kour, Mehboob Ali, Syed Mudassir Ali, Lakhvinder Singh, Abhishek Gour, Utpal Nandi, Manish Datt, Parduman Raj Sharma, Conrad C Weihl, Gurdarshan Singh, Ajay Kumar Autophagy Reports, 2024 Being present in substantial numbers, astrocytes play an indispensable role in maintaining homeostasis in the brain. However, their positive or negative involvement in pathological conditions in the brain has not been explored much. In recent years, an emerging thought of targeting astrocytes for the resolution of neurodegenerative diseases has gained momentum. In this study, we have attempted to explore the likelihood of targeting astrocytes by using a natural compound, gentiacaulein (GENT), for clearance of amyloid-β (Aβ) through autophagy and amelioration of neuroinflammation associated with Aβ. We found that GENT treatment of astrocytes hampered the transport of glucose across the cell membrane, which resulted in a reduction in ATP production. With increased treatment time, AMP: ATP ratio was increased significantly, which caused the induction of PRKAA1-mediated autophagy. We further show that increased autophagy considerably enhanced the clearance of amyloid-β by astrocytes. GENT reduced the Aβ mediated inflammation by inhibiting the nuclear translocation of NF-κB and decreased the release of inflammatory cytokines TNF-α and IL-6. The role of PRKAA1 in GENT-induced autophagy and anti-inflammatory activity was confirmed when its knockdown reversed these effects. Our data suggest that targeting astrocytes can be a good strategy to prevent/treat Alzheimer’s disease.
Ellagic Acid Exerts Dual Action to Curb the Pathophysiological Manifestations of Sickle Cell Disease and Attenuate the Hydroxyurea-Induced Myelosuppression in Berkeley Mice Abhishek Gour, Dilpreet Kour, Ramajayan Pandian, Mahir Bhardwaj, Sanghapal D. Sawant, Ajay Kumar, Utpal Nandi ACS Pharmacology and Translational Science, 2023 The use of adjuvant therapy is an attractive approach to manage sickle cell disease (SCD) symptomatically. The present study aimed to investigate the potential of ellagic acid as an adjuvant therapy with hydroxyurea (HU), a key drug for SCD with myelosuppressive toxic effects. A panel of experiments was performed using SCD patient's blood (ex vivo) and transgenic mice model of SCD (in vivo). Ellagic acid exhibited the following beneficial pharmacological actions: (a) potent anti-sickling, polymerization inhibitory, and inherent non-hemolytic activity; (b) pronounced action to abrogate HU-induced neutropenia and to improve key hematological parameters during SCD (RBC, Hb, platelet levels); (c) considerable action to foster vascular tone (L-proline); (d) marked attenuating effect against oxidative stress (nitrotyrosine, hypoxanthine, MDA, GSH); (e) substantial inhibitory role against inflammation (analgesic activity and regulation of hemin, TNF-α, IL-1β, NF-κB/IκBα); (f) remarkable outcome of declining vaso-occlusive crisis (P-selectin, ERK1/2); (g) notable shielding deed against elevated biochemical marker for organ toxicity (creatinine); (h) noticeably prevented histopathological alterations of the spleen. Additionally, the pharmacokinetic study results of HU in the presence and absence of ellagic acid using a mouse model demonstrate that ellagic acid could be safely co-administered with HU. Overall findings suggest that ellagic acid is a promising candidate for adjuvant therapy in SCD based on its own significant ability against SCD and potentiating capability of HU action via targeting improvement at the various stages of pathophysiological complications during SCD and minimizing HU-induced toxicological manifestations.
