About

James H. Eberwine, Ph.D., is the Elmer Holmes Bobst Professor of Pharmacology at the University of Pennsylvania School of Medicine. He is a member of multiple institutes within the university, including the Institute of Neurological Sciences, the Abramson Cancer Center, the Center for Bioinformatics, the Institute for Medicine and Engineering, the Genomics Institute, and the Center for Cell Engineering. Dr. Eberwine is also a Co-Director of the PENN Program in Single Cell Biology and the Penn Center for Subcellular Genomics. His research focuses on understanding the molecular basis of neuronal functioning, with an emphasis on neuronal adaptation processes such as tetanic potentiation, glucocorticoid-induced, age-induced, and opiate-induced adaptation. His laboratory employs molecular biology techniques, single-cell genetics, cDNA cloning, in situ hybridization, in situ transcription, mRNA amplification, and expression profiling to analyze gene expression at the single-cell level. Dr. Eberwine's work involves analyzing the mRNA and protein complement of individual cells, as well as mRNA movement and translation within single cells, to generate molecular and bioprocess fingerprints of various cell types and disease states. He has contributed to the understanding of subcellular localization of mRNAs in neurons, demonstrating that multiple mRNAs are localized in neuronal dendrites and providing formal proof of local mRNA translation in dendrites. His research has shown that the intracellular sites of mRNA localization and translation can be altered by synaptic stimulation, highlighting the dynamic nature of neuronal protein synthesis. His insights into neuronal cell biology emphasize the complexities of cellular function and the importance of local translation in neuronal activity.

Research topics

• Biology
• Computational biology
• Cell biology
• Neuroscience
• Molecular biology

Selected publications

• Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-06-13

  preprintOpen accessSenior authorCorresponding

  Background: Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting 'normal' mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, 2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA. Results: 1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. Conclusion: This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.

  PublisherOA PDFDOI

• "HIGH-THROUGHPUT AND MULTIPLEX ANALYSIS OF SINGLE-CELL, SINGLE-MITOCHONDRIAL DNA MUTATION USING HYDROGEL DROPLET MICROFLUIDICS AND ROLLING CIRCLE AMPLIFICATION"

  2024-10-07

  articleOpen access

  While understanding mitochondrial DNA (mtDNA) heteroplasmy and intercellular heterogeneity requires singlecell, single-mtDNA analysis, existing methods restrict studies to small cell populations [1,2].Water-in-oil droplet microfluidics improved the throughput of single cell analysis >100,000 cells, but it has been challenging for subcellular organelle analysis as their hierarchy in single-cell level should be resolved by multistep bioassays [3].Hydrogel droplet microfluidics has great potential for sub-cellular molecule analysis as they allow single-cell compartmentalization even after multiple times of buffer exchange steps.Here, we present a high-throughput, multiplex method for single-cell, single-mtDNA mutation analysis.(Figure 1).mtDNA from individual cells were isolated within porous structure of agarose beads after the agarose gelation and cell lysis.A microfluidic droplet generator was used to form a dense porous structure enough to retain mtDNA from a single cell, and the retention ratio of mtDNA was evaluated ~95% by a qPCR (Figure 2A,B).To mitigate the hindered throughput of agarose droplet generation due to its high viscosity, we incorporated parallelized microfluidic droplet generators which achieved throughput >70,000 drops/min (Figure 2C).mtDNA from single cells within agarose beads were analyzed by using a rolling circle amplification (RCA) (Figure 3A).RCA can localize amplified signal as a few hundred nm to a few m punctate, which are large enough to be retained in agarose bead.First, padlock probe was annealed to a specific region of mtDNA after the denaturation using 60% DMSO.Subsequently, padlock probe was ligated to form a circular DNA and primer was hybridized on the circle DNA.After RCA reaction, RCA products, large punctate were generated within agarose beads and visualized by fluorescent detection probes.Based on this method, Cross-contamination of mtDNA between beads during a sample preparation was validated by using mouse and human cells (Figure 3B).There was minimal cross-contamination by noting that agarose beads have either ATTO565 or ATTO647 fluorescence signal, which was confirmed by a flow cytometer (Figure 3C,D).Agarose beads that had encapsulated single cell was ensured by SYBR green gDNA stain.Fluorescence signal of RCA products generated from mtDNA showed significant difference between SYBR green positive and negative beads.Then we evaluated multiplex assay on single-mtDNA from single cell to analyze large area deletion by targeting D-loop and 4,977 deletion regions (Figure 4A).RCA products labelled with different fluorescence were distributed throughout a single agarose bead (Figure 4B,C).Different fluorescence profile of each bead analyzed by imaging and flow cytometry, which revealed heteroplasmy and intercellular heterogeneity of mtDNA 4,977 deletion (Figure 4D,E).Furthermore, padlock probe-based RCA assay was applied for mtDNA single nucleotide variation (SNV) analysis in single-cell level (Figure 5A).By locating SNV region at the end of padlock probe, SNV of mtDNA can be detected by RCA products labelled with different fluorescent detection probes.It was shown that more RCA products were generated from padlock probes targeting wild type in both single-plex and multiplex assay (Figure 5B).The proposed method will open a new avenue for advancing our understanding of the biological role of mtDNA heteroplasmy.

  PublisherDOI

• High‐Throughput Single‐Cell, Single‐Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics

  Angewandte Chemie International Edition · 2024-03-12 · 13 citations

  articleOpen access

  There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single-mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

  PublisherOA PDFDOI

• High‐Throughput Single‐Cell, Single‐Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics

  Angewandte Chemie · 2024-03-12 · 4 citations

  article

  Abstract There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single‐cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single‐cell genomic, RNA, and protein analysis, their application to sub‐cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose‐based droplet microfluidic approach for single‐cell, single‐mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi‐step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single‐mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

  PublisherDOI

• Single cell phototransfection of mRNAs encoding SARS-CoV2 spike and nucleocapsid into human astrocytes results in RNA dependent translation interference

  Frontiers in Drug Delivery · 2024-03-05 · 1 citations

  articleOpen accessSenior authorCorresponding

  Multi-RNA co-transfection is starting to be employed to stimulate immune responses to SARS-CoV-2 viral infection. While there are good reasons to utilize such an approach, there is little background on whether there are synergistic RNA-dependent cellular effects. To address this issue, we use transcriptome-induced phenotype remodeling (TIPeR) via phototransfection to assess whether mRNAs encoding the Spike and Nucleocapsid proteins of SARS-CoV-2 virus into single human astrocytes (an endogenous human cell host for the virus) and mouse 3T3 cells (often used in high-throughput therapeutic screens) synergistically impact host cell biologies. An RNA concentration-dependent expression was observed where an increase of RNA by less than 2-fold results in reduced expression of each individual RNAs. Further, a dominant inhibitory effect of Nucleocapsid RNA upon Spike RNA translation was detected that is distinct from codon-mediated epistasis. Knowledge of the cellular consequences of multi-RNA transfection will aid in selecting RNA concentrations that will maximize antigen presentation on host cell surface with the goal of eliciting a robust immune response. Further, application of this single cell stoichiometrically tunable RNA functional genomics approach to the study of SARS-CoV-2 biology promises to provide details of the cellular sequalae that arise upon infection in anticipation of providing novel targets for inhibition of viral replication and propagation for therapeutic intervention.

  PublisherOA PDFDOI

• High-throughput single-cell, single-mitochondrial DNA assay using hydrogel droplet microfluidics

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-01-31

  preprintOpen access

  There is growing interest in understanding the biological implications of single cell heterogeneity and intracellular heteroplasmy of mtDNA, but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95% mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

  PublisherOA PDFDOI

• Single-mitochondrion sequencing uncovers distinct mutational patterns and heteroplasmy landscape in mouse astrocytes and neurons

  BMC Biology · 2024-07-29 · 4 citations

  articleOpen accessSenior author

  BACKGROUND: Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting "normal" mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes, we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes. In this study, we amplified mt-genomes from 1645 single mitochondria isolated from mouse single astrocytes and neurons to (1) determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, (2) assess differences in mtDNA SNVs between neurons and astrocytes, and (3) study co-segregation of variants in the mouse mtDNA. RESULTS: (1) The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. (2) Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G > A and 9419:C > T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. (3) Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. CONCLUSIONS: This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.

  PublisherOA PDFDOI

• Subcellular omics: a new frontier pushing the limits of resolution, complexity and throughput

  Nature Methods · 2023-03-01 · 18 citations

  articleOpen access1st authorCorresponding
  PublisherOA PDFDOI

• CHEX-seq detects single-cell genomic single-stranded DNA with catalytical potential

  Nature Communications · 2023-11-14 · 4 citations

  articleOpen accessSenior author

  Genomic DNA (gDNA) undergoes structural interconversion between single- and double-stranded states during transcription, DNA repair and replication, which is critical for cellular homeostasis. We describe "CHEX-seq" which identifies the single-stranded DNA (ssDNA) in situ in individual cells. CHEX-seq uses 3'-terminal blocked, light-activatable probes to prime the copying of ssDNA into complementary DNA that is sequenced, thereby reporting the genome-wide single-stranded chromatin landscape. CHEX-seq is benchmarked in human K562 cells, and its utilities are demonstrated in cultures of mouse and human brain cells as well as immunostained spatially localized neurons in brain sections. The amount of ssDNA is dynamically regulated in response to perturbation. CHEX-seq also identifies single-stranded regions of mitochondrial DNA in single cells. Surprisingly, CHEX-seq identifies single-stranded loci in mouse and human gDNA that catalyze porphyrin metalation in vitro, suggesting a catalytic activity for genomic ssDNA. We posit that endogenous DNA enzymatic activity is a function of genomic ssDNA.

  PublisherOA PDFDOI

• Astrocyte ethanol exposure reveals persistent and defined calcium response subtypes and associated gene signatures

  Journal of Biological Chemistry · 2022-06-15 · 11 citations

  articleOpen access

  Astrocytes play a critical role in brain function, but their contribution during ethanol (EtOH) consumption remains largely understudied. In light of recent findings on the heterogeneity of astrocyte physiology and gene expression, an approach with the ability to identify subtypes and capture this heterogeneity is necessary. Here, we combined measurements of calcium signaling and gene expression to define EtOH-induced astrocyte subtypes. In the absence of a demonstrated EtOH receptor, EtOH is believed to have effects on the function of many receptors and downstream biological cascades that underlie calcium responsiveness. This mechanism of EtOH-induced calcium signaling is unknown and this study provides the first step in understanding the characteristics of cells displaying these observed responses. To characterize underlying astrocyte subtypes, we assessed the correlation between calcium signaling and astrocyte gene expression signature in response to EtOH. We found that various EtOH doses increased intracellular calcium levels in a subset of astrocytes, distinguishing three cellular response types and one nonresponsive subtype as categorized by response waveform properties. Furthermore, single-cell RNA-seq analysis of astrocytes from the different response types identified type-enriched discriminatory gene expression signatures. Combining single-cell calcium responses and gene expression analysis identified specific astrocyte subgroups among astrocyte populations defined by their response to EtOH. This result provides a basis for identifying the relationship between astrocyte susceptibility to EtOH and corresponding measurable markers of calcium signaling and gene expression, which will be useful to investigate potential subgroup-specific influences of astrocytes on the physiology and pathology of EtOH exposure in the brain.

  PublisherOA PDFDOI

Recent grants

• NIH Grant U01MH09895304

  NIH · $2.3M · 2016

• NIH Grant R37AG009900

  NIH · $3.4M · 2011

• NIH Grant R01MH058561

  NIH · $2.8M · 2008

• Center for Sub-Cellular Genomics

  NIH · $9.0M · 2018–2024

• Neuronal ciRNA characterization and impact upon channel functioning

  NIH · $3.2M · 2016–2021

Frequent coauthors

• Junhyong Kim

  California University of Pennsylvania

  57 shared

• Kevin Miyashiro

  Translational Therapeutics (United States)

  42 shared

• Jai‐Yoon Sul

  Translational Therapeutics (United States)

  37 shared

• Jennifer Spaethling

  Translational Therapeutics (United States)

  28 shared

• Stephen Fisher

  26 shared

• Peter B. Crino

  University of Maryland, College Park

  25 shared

• Bernhard Kühn

  Children's Hospital of Pittsburgh

  24 shared

• Balakrishnan Ganapathy

  Biogen (United States)

  22 shared