Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

Analysis of Mutants Suggests Kamin Blocking in C. elegans is Due to Interference with Memory Recall Rather than Storage.

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Analysis of Mutants Suggests Kamin Blocking in C. elegans is Due to Interference with Memory Recall Rather than Storage.

Sci Rep. 2019 Feb 20;9(1):2371

Authors: Merritt DM, Melkis JG, Kwok B, Tran C, van der Kooy D

Abstract
Higher-order conditioning phenomena, including context conditioning and blocking, occur when conditioning to one set of stimuli interacts with conditioning to a second set of stimuli to modulate the strength of the resultant memories. Here we analyze higher-order conditioning in the nematode worm Caenorhabditis elegans, demonstrating for the first time the presence of blocking in this animal, and dissociating it from context conditioning. We present an initial genetic dissection of these phenomena in a model benzaldehyde/NH4Cl aversive learning system, and suggest that blocking may involve an alteration of memory retrieval rather than storage. These findings offer a fundamentally different explanation for blocking than traditional explanations, and position C. elegans as a powerful model organism for the study of higher order conditioning.

PMID: 30787354 [PubMed - in process]



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Yeast Two-Hybrid Analysis for Ubiquitin-Variant Inhibitors of Human Deubiquitinases.

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Yeast Two-Hybrid Analysis for Ubiquitin-Variant Inhibitors of Human Deubiquitinases.

J Mol Biol. 2019 Feb 11;:

Authors: Pascoe N, Seetharaman A, Teyra J, Manczyk N, Satori MA, Tjandra D, Makhnevych T, Schwerdtfeger C, Brasher BB, Moffat J, Costanzo M, Boone C, Sicheri F, Sidhu S

Abstract
We applied a Yeast-Two-Hybrid (Y2H) analysis to screen for ubiquitin variant (UbV) inhibitors of a human deubiquitinase (DUB), ubiquitin-specific protease 2 (USP2). The Y2H screen used USP2 as the bait, and a prey library consisting of UbVs randomized at four specific positions, which were known to interact with USP2 from phage display analysis. The screen yielded numerous UbVs that bound to USP2 both as a Y2H interaction in vivo and as purified proteins in vitro. The Y2H-derived UbVs inhibited the catalytic activity of USP2 in vitro with nanomolar-range potencies, and they bound and inhibited USP2 in human cells. Mutational and structural analysis showed that potent and selective inhibition could be achieved by just two substitutions in a UbV, which exhibited improved hydrophobic and hydrophilic contacts compared to the wild-type ubiquitin interaction with USP2. Our results establish Y2H as an effective platform for the development of UbV inhibitors of DUBs in vivo, providing an alternative strategy for the analysis of DUBs that are recalcitrant to phage display and other in vitro methods.

PMID: 30763569 [PubMed - as supplied by publisher]



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Transplantation of Directly Reprogrammed Human Neural Precursor Cells Following Stroke Promotes Synaptogenesis and Functional Recovery.

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Transplantation of Directly Reprogrammed Human Neural Precursor Cells Following Stroke Promotes Synaptogenesis and Functional Recovery.

Transl Stroke Res. 2019 Feb 12;:

Authors: Vonderwalde I, Azimi A, Rolvink G, Ahlfors JE, Shoichet MS, Morshead CM

Abstract
Stroke is one of the leading causes of long-term disability. Cell transplantation is a promising strategy to treat stroke. We explored the efficacy of directly reprogrammed human neural precursor cell (drNPC) transplants to promote functional recovery in a model of focal ischemic stroke in the mouse sensorimotor cortex. We show that drNPCs express neural precursor cell markers and are neurally committed at the time of transplantation. Mice that received drNPC transplants recovered motor function, irrespective of transplant vehicle or recipient sex, and with no correlation to lesion volume or glial scarring. The majority of drNPCs found in vivo, at the time of functional recovery, remained undifferentiated. Notably, no correlation between functional recovery and long-term xenograft survival was observed, indicating that drNPCs provide therapeutic benefits beyond their survival. Furthermore, increased synaptophysin expression in transplanted brains suggests that drNPCs promote neuroplasticity through enhanced synaptogenesis. Our findings provide insight into the mechanistic underpinnings of drNPC-mediated recovery for stroke and support the notion that drNPCs may have clinical applications for stroke therapy.

PMID: 30747366 [PubMed - as supplied by publisher]



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CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks.

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CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks.

Elife. 2019 Feb 12;8:

Authors: Deneault E, Faheem M, White SH, Rodrigues DC, Sun S, Wei W, Piekna A, Thompson T, Howe JL, Chalil L, Kwan V, Walker S, Pasceri P, Roth FP, Yuen RK, Singh KK, Ellis J, Scherer SW

Abstract
Induced pluripotent stem cell (iPSC)-derived neurons are increasingly used to model Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of penetrant and weaker polygenic risk variants to ASD, 'isogenic' iPSC-derived neurons are critical. We developed a set of procedures to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD. Heterozygous de novo and rare-inherited presumed-damaging variants were characterized in ASD risk genes/loci. Combinations of putative etiologic variants (GLI3/KIF21A or EHMT2/UBE2I) in separate families were modeled. We used a multi-electrode array, with patch-clamp recordings, to determine a reproducible synaptic phenotype in 25% of the individuals with ASD (other relevant data on the remaining lines was collected). Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. The biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research.
Editorial note: This article has been through an editorial process in which authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

PMID: 30747104 [PubMed - in process]



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Identification and Functional Testing of Novel Interacting Protein Partners for the Stress Sensors Wsc1p and Mid2p of Saccharomyces cerevisiae.

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Identification and Functional Testing of Novel Interacting Protein Partners for the Stress Sensors Wsc1p and Mid2p of Saccharomyces cerevisiae.

G3 (Bethesda). 2019 04 09;9(4):1085-1102

Authors: Santiago-Cartagena E, González-Crespo S, Vélez V, Martínez N, Snider J, Jessulat M, Aoki H, Minic Z, Akamine P, Mejías I, Pérez LM, Rymond BC, Babu M, Stagljar I, Rodríguez-Medina JR

Abstract
Wsc1p and Mid2p are transmembrane signaling proteins of cell wall stress in the budding yeast Saccharomyces cerevisiae When an environmental stress compromises cell wall integrity, they activate a cell response through the Cell Wall Integrity (CWI) pathway. Studies have shown that the cytoplasmic domain of Wsc1p initiates the CWI signaling cascade by interacting with Rom2p, a Rho1-GDP-GTP exchange factor. Binding of Rom2p to the cytoplasmic tail of Wsc1p requires dephosphorylation of specific serine residues but the mechanism by which the sensor is dephosphorylated and how it subsequently interacts with Rom2p remains unclear. We hypothesize that Wsc1p and Mid2p must be physically associated with interacting proteins other than Rom2p that facilitate its interaction and regulate the activation of CWI pathway. To address this, a cDNA plasmid library of yeast proteins was expressed in bait strains bearing membrane yeast two-hybrid (MYTH) reporter modules of Wsc1p and Mid2p, and their interacting preys were recovered and sequenced. 14 previously unreported interactors were confirmed for Wsc1p and 29 for Mid2p The interactors' functionality were assessed by cell growth assays and CWI pathway activation by western blot analysis of Slt2p/Mpk1p phosphorylation in null mutants of each interactor under defined stress conditions. The susceptibility of these strains to different stresses were tested against antifungal agents and chemicals. This study reports important novel protein interactions of Wsc1p and Mid2p that are associated with the cellular response to oxidative stress induced by Hydrogen Peroxide and cell wall stress induced by Caspofungin.

PMID: 30733383 [PubMed - indexed for MEDLINE]



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Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

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Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

Mol Cell. 2019 03 07;73(5):900-914.e9

Authors: Gallo D, Kim T, Szakal B, Saayman X, Narula A, Park Y, Branzei D, Zhang Z, Brown GW

Abstract
Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

PMID: 30733119 [PubMed - indexed for MEDLINE]



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On-demand serum-free media formulations for human hematopoietic cell expansion using a high dimensional search algorithm.

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On-demand serum-free media formulations for human hematopoietic cell expansion using a high dimensional search algorithm.

Commun Biol. 2019;2:48

Authors: Kim MM, Audet J

Abstract
Substitution of serum and other clinically incompatible reagents is requisite for controlling product quality in a therapeutic cell manufacturing process. However, substitution with chemically defined compounds creates a complex, large-scale optimization problem due to the large number of possible factors and dose levels, making conventional process optimization methods ineffective. We present a framework for high-dimensional optimization of serum-free formulations for the expansion of human hematopoietic cells. Our model-free approach utilizes evolutionary computing principles to drive an experiment-based feedback control platform. We validate this method by optimizing serum-free formulations for first, TF-1 cells and second, primary T-cells. For each cell type, we successfully identify a set of serum-free formulations that support cell expansions similar to the serum-containing conditions commonly used to culture these cells, by experimentally testing less than 1 × 10-5 % of the total search space. We also demonstrate how this iterative search process can provide insights into factor interactions that contribute to supporting cell expansion.

PMID: 30729186 [PubMed]



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Modelling of photoreceptor donor-host interaction following transplantation reveals a role for Crx, Müller glia and Rho/ROCK signalling in neurite outgrowth.

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Modelling of photoreceptor donor-host interaction following transplantation reveals a role for Crx, Müller glia and Rho/ROCK signalling in neurite outgrowth.

Stem Cells. 2019 Feb 04;:

Authors: Tsai ELS, Ortin-Martinez A, Gurdita A, Comanita L, Yan N, Smiley S, Delplace V, Shoichet MS, Nickerson PE, Wallace VA

Abstract
The goal of photoreceptor transplantation is to establish functional synaptic connectivity between donor cells and second order neurons in the host retina. There is, however, limited evidence of donor-host photoreceptor connectivity post-transplant. In this report, we investigated the effect of the host retinal environment on donor photoreceptor neurite outgrowth in vivo and identify a neurite outgrowth-promoting effect of host Crx(-/-) retinas following transplantation of purified GFP-expressing photoreceptors. To investigate the non-cell autonomous factors that influence donor cell neurite outgrowth in vitro, we established a donor-host co-culture system using postnatal retinal aggregates. Retinal cell aggregation is sensitive to several factors, including plate coating substrate, cell density, and the presence of Müller glia. Donor photoreceptors exhibit motility in aggregate cultures and can engraft into established aggregate structures. The neurite outgrowth-promoting phenotype observed in Crx(-/-) recipients in vivo is recapitulated in donor-host aggregate co-cultures, demonstrating the utility of this surrogate in vitro approach. The removal of Müller glia from host aggregates reduced donor cell neurite outgrowth, identifying a role for this cell type in donor-host signalling. Although disruption of chondroitin sulfate proteoglycans in aggregates had no effect on the neurite outgrowth of donor photoreceptors, disruption of Rho/ROCK signalling enhanced outgrowth. Collectively, these data show a novel role of Crx, Müller glia and Rho/ROCK signalling in controlling neurite outgrowth and provide an accessible in vitro model that can be used to screen for factors that regulate donor-host connectivity. SIGNIFICANCE STATEMENT: Poor photoreceptor connectivity in transplants is an unresolved issue. Here, we show that photoreceptor neurite extension is regulated by Rho/ROCK signaling. This advance could be used in future work to enhance the connectivity of transplanted photoreceptors. © AlphaMed Press 2019.

PMID: 30715780 [PubMed - as supplied by publisher]



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Exogenous Neural Precursor Cell Transplantation Results in Structural and Functional Recovery in a Hypoxic-Ischemic Hemiplegic Mouse Model.

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Exogenous Neural Precursor Cell Transplantation Results in Structural and Functional Recovery in a Hypoxic-Ischemic Hemiplegic Mouse Model.

eNeuro. 2018 Sep-Oct;5(5):

Authors: Rumajogee P, Altamentova S, Li L, Li J, Wang J, Kuurstra A, Khazaei M, Beldick S, Menon RS, van der Kooy D, Fehlings MG

Abstract
Cerebral palsy (CP) is a common pediatric neurodevelopmental disorder, frequently resulting in motor and developmental deficits and often accompanied by cognitive impairments. A regular pathobiological hallmark of CP is oligodendrocyte maturation impairment resulting in white matter (WM) injury and reduced axonal myelination. Regeneration therapies based on cell replacement are currently limited, but neural precursor cells (NPCs), as cellular support for myelination, represent a promising regeneration strategy to treat CP, although the transplantation parameters (e.g., timing, dosage, mechanism) remain to be determined. We optimized a hemiplegic mouse model of neonatal hypoxia-ischemia that mirrors the pathobiological hallmarks of CP and transplanted NPCs into the corpus callosum (CC), a major white matter structure impacted in CP patients. The NPCs survived, engrafted, and differentiated morphologically in male and female mice. Histology and MRI showed repair of lesioned structures. Furthermore, electrophysiology revealed functional myelination of the CC (e.g., restoration of conduction velocity), while cylinder and CatWalk tests demonstrated motor recovery of the affected forelimb. Endogenous oligodendrocytes, recruited in the CC following transplantation of exogenous NPCs, are the principal actors in this recovery process. The lack of differentiation of the transplanted NPCs is consistent with enhanced recovery due to an indirect mechanism, such as a trophic and/or "bio-bridge" support mediated by endogenous oligodendrocytes. Our work establishes that transplantation of NPCs represents a viable therapeutic strategy for CP treatment, and that the enhanced recovery is mediated by endogenous oligodendrocytes. This will further our understanding and contribute to the improvement of cellular therapeutic strategies.

PMID: 30713997 [PubMed - indexed for MEDLINE]



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Blockade of TGF-β signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models.

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Blockade of TGF-β signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models.

Oncoimmunology. 2019;8(2):e1539613

Authors: Newsted D, Banerjee S, Watt K, Nersesian S, Truesdell P, Blazer LL, Cardarelli L, Adams JJ, Sidhu SS, Craig AW

Abstract
Epithelial ovarian cancer (EOC) is a leading cause of cancer-related death in women. EOC is often diagnosed at late stages, with peritoneal metastases and ascites production. Current surgery and platinum-based chemotherapy regimes fail to prevent recurrence in most patients. High levels of Transforming growth factor-β (TGF-β) within ascites has been linked to poor prognosis. TGF-β signaling promotes epithelial-mesenchymal transition (EMT) in EOC tumor cells, and immune suppression within the tumor microenvironment, with both contributing to chemotherapy resistance and metastasis. The goal of this study was to develop specific synthetic inhibitory antibodies to the Type II TGF-β receptor (TGFBR2), and test these antibodies in EOC cell and tumor models. Following screening of a phage-displayed synthetic antigen-binding fragment (Fab) library with the extracellular domain of TGFBR2, we identified a lead inhibitory Fab that suppressed TGF-β signaling in mouse and human EOC cell lines. Affinity maturation of the lead inhibitory Fab resulted in several derivative Fabs with increased affinity for TGFBR2 and efficacy as suppressors of TGF-β signaling, EMT and EOC cell invasion. In EOC xenograft and syngeneic tumor models, blockade of TGFBR2 with our lead antibodies led to improved chemotherapy response. This correlated with reversal of EMT and immune exclusion in these tumor models with TGFBR2 blockade. Together, these results describe new inhibitors of the TGF-β pathway that improve antitumor immunity, and response to chemotherapy in preclinical EOC models.

PMID: 30713798 [PubMed]



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