IU Indianapolis ScholarWorks :: Browsing by Author "Monje, Paula V."

Browsing by Author "Monje, Paula V."

Now showing 1 - 9 of 9

Endogenous Glycoprotein GPM6a Is Involved in Neurite Outgrowth in Rat Dorsal Root Ganglion Neurons
(MDPI, 2023-03-25) Aparicio, Gabriela I.; León, Antonella; Gutiérrez Fuster, Rocío; Ravenscraft, Baylen; Monje, Paula V.; Scorticati, Camila; Neurological Surgery, School of Medicine
The peripheral nervous system (PNS) has a unique ability for self-repair. Dorsal root ganglion (DRG) neurons regulate the expression of different molecules, such as neurotrophins and their receptors, to promote axon regeneration after injury. However, the molecular players driving axonal regrowth need to be better defined. The membrane glycoprotein GPM6a has been described to contribute to neuronal development and structural plasticity in central-nervous-system neurons. Recent evidence indicates that GPM6a interacts with molecules from the PNS, although its role in DRG neurons remains unknown. Here, we characterized the expression of GPM6a in embryonic and adult DRGs by combining analysis of public RNA-seq datasets with immunochemical approaches utilizing cultures of rat DRG explants and dissociated neuronal cells. M6a was detected on the cell surfaces of DRG neurons throughout development. Moreover, GPM6a was required for DRG neurite elongation in vitro. In summary, we provide evidence on GPM6a being present in DRG neurons for the first time. Data from our functional experiments support the idea that GPM6a could contribute to axon regeneration in the PNS.
Heregulin Activity Assays for Residual Testing of Cell Therapy Products
(BMC, 2021-11-12) Monje, Paula V.; Bacallao, Ketty; Aparicio, Gabriela I.; Lalwani, Anil; Neurological Surgery, School of Medicine
Background: Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a β1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. Results: Western blotting was used to measure the activity of three different β1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of β1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and reproducibility to detect β1-heregulin activity in the nanomolar range. Results from these assays showed that the culture medium from transplantable hSCs elicited no detectable activation of the aforementioned kinases in independent rounds of testing, indicating that the implemented measures can ensure that the final hSC product is devoid of bioactive β1-heregulin molecules prior to transplantation. Conclusions: These assays may be valuable to detect impurities such as undefined soluble factors or factors for which other biochemical or biological assays are not yet available. Our workflow can be modified as necessary to determine the presence of ErbB/HER, ERK, and Akt activators other than β1-heregulin using native samples, such as fresh isolates from cell- or tissue extracts in addition to culture medium.
Human Schwann Cell Transplantation for Spinal Cord Injury: Prospects and Challenges in Translational Medicine
(Frontiers Media, 2021-06-18) Monje, Paula V.; Deng, Lingxiao; Xu, Xiao-Ming; Neurological Surgery, School of Medicine
The benefits of transplanting cultured Schwann cells (SCs) for the treatment of spinal cord injury (SCI) have been systematically investigated in experimental animals since the early 1990s. Importantly, human SC (hSC) transplantation for SCI has advanced to clinical testing and safety has been established via clinical trials conducted in the USA and abroad. However, multiple barriers must be overcome to enable accessible and effective treatments for SCI patients. This review presents available information on hSC transplantation for SCI with the intention to uncover gaps in our knowledge and discuss areas for future development. To this end, we introduce the historical progression of the work that supports existing and prospective clinical initiatives and explain the reasons for the choice of hSCs while also addressing their limitations as cell therapy products. A search of the relevant literature revealed that rat SCs have served as a preclinical model of reference since the onset of investigations, and that hSC transplants are relatively understudied, possibly due to the sophisticated resources and expertise needed for the traditional processing of hSC cultures from human nerves. In turn, we reason that additional experimentation and a reexamination of the available data are needed to understand the therapeutic value of hSC transplants taking into consideration that the manufacturing of the hSCs themselves may require further development for extended uses in basic research and clinical settings.
Magnetic separation of peripheral nerve-resident cells underscores key molecular features of human Schwann cells and fibroblasts: an immunochemical and transcriptomics approach
(Nature Publishing Group, 2020-10-28) Peng, Kaiwen; Sant, David; Andersen, Natalia; Silvera, Risset; Camarena, Vladimir; Piñero, Gonzalo; Graham, Regina; Khan, Aisha; Xu, Xiao-Ming; Wang, Gaofeng; Monje, Paula V.; Neurological Surgery, School of Medicine
Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth. Fibroblasts are an ill-defined population consisting of highly proliferative cells that, contrary to human SCs, do not undergo senescence in culture. We initiated this study by performing an exhaustive immunological and functional characterization of adult nerve-derived human SCs and fibroblasts to reveal their properties and optimize a protocol of magnetic-activated cell sorting (MACS) to separate them effectively both as viable and biologically competent cells. We next used immunofluorescence microscopy imaging, flow cytometry analysis and next generation RNA sequencing (RNA-seq) to unambiguously characterize the post-MACS cell products. High resolution transcriptome profiling revealed the identity of key lineage-specific transcripts and the clearly distinct neural crest and mesenchymal origin of human SCs and fibroblasts, respectively. Our analysis underscored a progenitor- or stem cell-like molecular phenotype in SCs and fibroblasts and the heterogeneity of the fibroblast populations. In addition, pathway analysis of RNA-seq data highlighted putative bidirectional networks of fibroblast-to-SC signaling that predict a complementary, yet seemingly independent contribution of SCs and fibroblasts to nerve regeneration. In sum, combining MACS with immunochemical and transcriptomics approaches provides an ideal workflow to exhaustively assess the identity, the stage of differentiation and functional features of highly purified cells from human peripheral nerve tissues.
Merlin-Deficient Schwann Cells Are More Susceptible to Radiation Injury than Normal Schwann Cells In Vitro
(Thieme, 2021-01-19) Cohen, Erin; Pena, Stefanie; Mei, Christine; Bracho, Olena; Marples, Brian; Elsayyad, Nagy; Goncalves, Stefania; Ivan, Michael; Monje, Paula V.; Liu, Xue-Zhong; Fernandez-Valle, Cristina; Telischi, Fred; Dinh, Christine T.; Neurological Surgery, School of Medicine
Objectives: Vestibular schwannomas (VS) are intracranial tumors, which are caused by NF2 gene mutations that lead to loss of merlin protein. A treatment for VS is stereotactic radiosurgery, a form of radiation. To better understand the radiobiology of VS and radiation toxicity to adjacent structures, our main objectives were (1) investigate effects of single fraction (SF) radiation on viability, cytotoxicity, and apoptosis in normal Schwann cells (SCs) and merlin-deficient Schwann cells (MD-SCs) in vitro, and (2) analyze expression of double strand DNA breaks (γ-H2AX) and DNA repair protein Rad51 following irradiation. Study Design: This is a basic science study. Setting: This study is conducted in a research laboratory. Participants: Patients did not participate in this study. Main Outcome Measures: In irradiated normal SCs and MD-SCs (0–18 Gy), we measured (1) viability, cytotoxicity, and apoptosis using cell-based assays, and (2) percentage of cells with γ-H2AX and Rad51 on immunofluorescence. Results: A high percentage of irradiated MD-SCs expressed γ-H2AX, which may explain the dose-dependent losses in viability in rodent and human cell lines. In comparison, the viabilities of normal SCs were only compromised at higher doses of radiation (>12 Gy, human SCs), which may be related to less Rad51 repair. There were no further reductions in viability in human MD-SCs beyond 9 Gy, suggesting that <9 Gy may be insufficient to initiate maximal tumor control. Conclusion: The MD-SCs are more susceptible to radiation than normal SCs, in part through differential expression of γ-H2AX and Rad51. Understanding the radiobiology of MD-SCs and normal SCs is important for optimizing radiation protocols to maximize tumor control while limiting radiation toxicity in VS patients.
Oscillatory cAMP signaling rapidly alters H3K4 methylation
(Life Science Alliance, 2020-01) Huff, Tyler C.; Camarena, Vladimir; Sant, David W.; Wilkes, Zachary; Booven, Derek Van; Aron, Allegra T.; Muir, Ryan K.; Renslo, Adam R.; Chang, Christopher J.; Monje, Paula V.; Wang, Gaofeng; Neurological Surgery, School of Medicine
receptors (GPCRs) alter H3K4 methylation via oscillatory intracellular cAMP. Activation of Gs-coupled receptors caused a rapid decrease of H3K4me3 by elevating cAMP, whereas stimulation of Gi-coupled receptors increased H3K4me3 by diminishing cAMP. H3K4me3 gradually recovered towards baseline levels after the removal of GPCR ligands, indicating that H3K4me3 oscillates in tandem with GPCR activation. cAMP increased intracellular labile Fe(II), the cofactor for histone demethylases, through a non-canonical cAMP target—Rap guanine nucleotide exchange factor-2 (RapGEF2), which subsequently enhanced endosome acidification and Fe(II) release from the endosome via vacuolar H+-ATPase assembly. Removing Fe(III) from the media blocked intracellular Fe(II) elevation after stimulation of Gs-coupled receptors. Iron chelators and inhibition of KDM5 demethylases abolished cAMP-mediated H3K4me3 demethylation. Taken together, these results suggest a novel function of cAMP signaling in modulating histone demethylation through labile Fe(II).
Schwann Cell Cultures: Biology, Technology and Therapeutics
(MDPI, 2020-08-06) Monje, Paula V.; Neurological Surgery, School of Medicine
Schwann cell (SC) cultures from experimental animals and human donors can be prepared using nearly any type of nerve at any stage of maturation to render stage- and patient-specific populations. Methods to isolate, purify, expand in number, and differentiate SCs from adult, postnatal and embryonic sources are efficient and reproducible as these have resulted from accumulated refinements introduced over many decades of work. Albeit some exceptions, SCs can be passaged extensively while maintaining their normal proliferation and differentiation controls. Due to their lineage commitment and strong resistance to tumorigenic transformation, SCs are safe for use in therapeutic approaches in the peripheral and central nervous systems. This review summarizes the evolution of work that led to the robust technologies used today in SC culturing along with the main features of the primary and expanded SCs that make them irreplaceable models to understand SC biology in health and disease. Traditional and emerging approaches in SC culture are discussed in light of their prospective applications. Lastly, some basic assumptions in vitro SC models are identified in an attempt to uncover the combined value of old and new trends in culture protocols and the cellular products that are derived.
Using a Transection Paradigm to Enhance the Repair Mechanisms of an Investigational Human Cell Therapy
(Sage, 2022) Chau, Monica J.; Quintero, Jorge E.; Monje, Paula V.; Voss, Stephen Randal; Welleford, Andrew S.; Gerhardt, Greg A.; van Horne, Craig G.; Neurological Surgery, School of Medicine
One promising strategy in cell therapies for Parkinson's disease (PD) is to harness a patient's own cells to provide neuroprotection in areas of the brain affected by neurodegeneration. No treatment exists to replace cells in the brain. Thus, our goal has been to support sick neurons and slow neurodegeneration by transplanting living repair tissue from the peripheral nervous system into the substantia nigra of those with PD. Our group has pioneered the transplantation of transection-activated sural nerve fascicles into the brain of human subjects with PD. Our experience in sural nerve transplantation has supported the safety and feasibility of this approach. As part of a paradigm to assess the reparative properties of human sural nerve following a transection injury, we collected nerve tissue approximately 2 weeks after sural nerve transection for immunoassays from 15 participants, and collected samples from two additional participants for single nuclei RNA sequencing. We quantified the expression of key neuroprotective and select anti-apoptotic genes along with their corresponding protein levels using immunoassays. The single nuclei data clustered into 10 distinctive groups defined on the basis of previously published cell type-specific genes. Transection-induced reparative peripheral nerve tissue showed RNA expression of neuroprotective factors and anti-apoptotic factors across multiple cell types after nerve injury induction. Key proteins of interest (BDNF, GDNF, beta-NGF, PDGFB, and VEGF) were upregulated in reparative tissue. These results provide insight on this repair tissue's utility as a neuroprotective cell therapy.
Vitamin C regulates Schwann cell myelination by promoting DNA demethylation of pro-myelinating genes
(Wiley, 2021-06) Huff, Tyler C.; Sant, David W.; Camarena, Vladimir; Van Booven, Derek; Andrade, Nadja S.; Mustafi, Sushmita; Monje, Paula V.; Wang, Gaofeng; Neurological Surgery, School of Medicine
Ascorbic acid (vitamin C) is critical for Schwann cells to myelinate peripheral nerve axons during development and remyelination after injury. However, its exact mechanism remains elusive. Vitamin C is a dietary nutrient that was recently discovered to promote active DNA demethylation. Schwann cell myelination is characterized by global DNA demethylation in vivo and may therefore be regulated by vitamin C. We found that vitamin C induces a massive transcriptomic shift (n = 3,848 genes) in primary cultured Schwann cells while simultaneously producing a global increase in genomic 5-hydroxymethylcytosine (5hmC), a DNA demethylation intermediate which regulates transcription. Vitamin C up-regulates 10 pro-myelinating genes which exhibit elevated 5hmC content in both the promoter and gene body regions of these loci following treatment. Using a mouse model of human vitamin C metabolism, we found that maternal dietary vitamin C deficiency causes peripheral nerve hypomyelination throughout early development in resulting offspring. Additionally, dietary vitamin C intake regulates the expression of myelin-related proteins such as periaxin (PRX) and myelin basic protein (MBP) during development and remyelination after injury in mice. Taken together, these results suggest that vitamin C cooperatively promotes myelination through 1) increased DNA demethylation and transcription of pro-myelinating genes, and 2) its known role in stabilizing collagen helices to form the basal lamina that is necessary for myelination.

Browsing by Author "Monje, Paula V."

Results Per Page

Sort Options