The fine-tuning of gene expression is critical for all cellular processes; aberrations in this activity can lead to pathology, and conversely, resilience. As their role in coordinating organismal ...responses to both internal and external factors have increasingly come into focus, small non-coding RNAs have emerged as an essential component to disease etiology. Using Systemic RNA interference Defective (SID) mutants of the nematode Caenorhabditis elegans, deficient in gene silencing, we examined the potential consequences of dysfunctional epigenomic regulation in the context of Parkinson's disease (PD). Specifically, the loss of either the sid-1 or sid-3 genes, which encode a dsRNA transporter and an endocytic regulatory non-receptor tyrosine kinase, respectively, conferred neuroprotection to dopaminergic (DA) neurons in an established transgenic C. elegans strain wherein overexpression of human α-synuclein (α-syn) from a chromosomally integrated multicopy transgene causes neurodegeneration. We further show that knockout of a specific microRNA, mir-2, attenuates α-syn neurotoxicity; suggesting that the native targets of mir-2-dependent gene silencing represent putative neuroprotective modulators. In support of this, we demonstrated that RNAi knockdown of multiple mir-2 targets enhanced α-syn-induced DA neurodegeneration. Moreover, we demonstrate that mir-2 overexpression originating in the intestine can induce neurodegeneration of DA neurons, an effect that was reversed by pharmacological inhibition of SID-3 activity. Interestingly, sid-1 mutants retained mir-2-induced enhancement of neurodegeneration. Transcriptomic analysis of α-syn animals with and without a sid-1 mutation revealed 27 differentially expressed genes with human orthologs related to a variety of diseases, including PD. Among these was pgp-8, encoding a P-glycoprotein-related ABC transporter. Notably, sid-1; pgp-8 double mutants abolished the neurodegeneration resulting from intestinal mir-2 overexpression. This research positions known regulators of small RNA-dependent gene silencing within a framework that facilitates mechanistic evaluation of epigenetic responses to exogenous and endogenous factors influencing DA neurodegeneration, revealing a path toward new targets for therapeutic intervention of PD.
We previously reported that eribulin mesylate (eribulin), a tubulin‐binding drug (TBD), could remodel tumor vasculature (i.e. increase tumor vessels and perfusion) in human breast cancer xenograft ...models. However, the role of this vascular remodeling in antitumor effects is not fully understood. Here, we investigated the effects of eribulin‐induced vascular remodeling on antitumor activities in multiple human cancer xenograft models. Microvessel densities (MVD) were evaluated by immunohistochemistry (CD31 staining), and antitumor effects were examined in 10 human cancer xenograft models. Eribulin significantly increased MVD compared to the controls in six out of 10 models with a correlation between enhanced MVD levels and antitumor effects (R2 = 0.54). Because of increased MVD, we next used radiolabeled liposomes to examine whether eribulin treatment would result in increased tumoral accumulation levels of these macromolecules and, indeed, we found that eribulin, unlike vinorelbine (another TBD) enhanced them. As eribulin increased accumulation of radiolabeled liposomes, we postulated that this treatment might enhance the antitumor effect of Doxil (a liposomal anticancer agent) and facilitate recruitment of immune cells into the tumor. As expected, eribulin enhanced antitumor activity of Doxil in a post‐erlotinib treatment H1650 (PE‐H1650) xenograft model. Furthermore, infiltrating CD11b‐positive immune cells were significantly increased in multiple eribulin‐treated xenografted tumors, and natural killer (NK) cell depletion reduced the antitumor effects of eribulin. These findings suggest a contribution of the immune cells for antitumor activities of eribulin. Taken together, our results suggest that vascular remodeling induced by eribulin acts as a microenvironment modulator and, consequently, this alteration enhanced the antitumor effects of eribulin.
Eribulin, a nontaxane synthetic microtubule dynamic inhibitor, was approved by FDA and used for cancer treatment. Previously, we reported that eribulin induced vascular remodeling in the tumors by using preclinical mouse and rat xenograft models through increasing microvessel density, which consequently enhanced perfusion in the tumors (Funahashi et al., Cancer Sci., 105, 2014, 1334). Despite these advances, during eribulin treatment, a role of vascular remodeling for anti‐tumor effects is not fully elucidated. In this report, we further expanded our research by investigating pharmacological action of eribulin during the cancer treatment and found eribulin might function as a tumor microenvironment modulator in addition to its classic cytotoxic effect through vascular remodeling and contribute to effective cancer treatment.
Development of monoclonal antibody is critical for targeted drug delivery because its characteristics determine improved therapeutic efficacy and reduced side-effect. Antibody therapeutics target ...surface molecules; hence, internalization is desired for drug delivery. As an antibody-drug conjugate, a critical parameter is drug-to-antibody ratio wherein the quantity of drugs attached to the antibody influences the antibody structure, stability, and efficacy. Here, we established a cell-based immunotoxin screening system to facilitate the isolation of functional antibodies with internalization capacities, and discovered an anti-human CD71 monoclonal antibody. To overcome the limitation of drug-to-antibody ratio, we employed the encapsulation capacity of liposome, and developed anti-CD71 antibody-conjugated liposome that demonstrated antigen-antibody dependent cellular uptake when its synthesis was optimized. Furthermore, anti-CD71 antibody-conjugated liposome encapsulating doxorubicin demonstrated antigen-antibody dependent cytotoxicity. In summary, this study demonstrates the powerful pipeline to discover novel functional antibodies, and the optimal method to synthesize immunoliposomes. This versatile technology offers a rapid and direct approach to generate antibodies suitable for drug delivery modalities.
ABSTRACT Background Cytoplasmic mislocalization of TAR-DNA binding protein of 43 kDa (TDP-43) is a major hallmark of amyotrophic lateral sclerosis (ALS). TDP-43 aggregation is detected in the ...cortical and spinal motor neurons in most ALS cases; however, pathological mechanism of this mislocalized TDP-43 remains unknown. Methods We generated a tetracycline-inducible TDP-43 A315T system on a mouse artificial chromosome (MAC) vector to avoid transgene-insertional mutagenesis, established a mouse embryonic stem (ES) cell line holding this MAC vector system, and investigated whether overexpressed exogenous TDP-43 A315T was mislocalized in the cytoplasm of the ES cell-derived neurons and triggered the neurotoxic effects on these cells. Results Inducible TDP-43 A315T system was successfully loaded onto the MAC and introduced into the mouse ES cells. These ES cells could differentiate into motor neurons and interneurons. Overexpression of TDP-43 A315T by addition of doxycycline in both neurons resulted in mislocalization to cytoplasm. Mislocalized TDP-43 caused cell death of motor neurons, but not interneurons. Conclusion Vulnerability to cytoplasmic mislocalized TDP-43 is selective on neuronal types, whereas mislocalization of overexpressed TDP-43 occurs in even insusceptible neurons. This inducible gene expression system using MAC remains useful for providing critical insights into appearance of TDP-43 pathology.
Mammalian artificial chromosomes derived from native chromosomes have been applied to biomedical research and development by generating cell sources and transchromosomic (Tc) animals. Human ...artificial chromosome (HAC) is a precedent chromosomal vector which achieved generation of valuable humanized animal models for fully human antibody production and human pharmacokinetics. While humanized Tc animals created by HAC vector have attained significant contributions, there was a potential issue to be addressed regarding stability in mouse tissues, especially highly proliferating hematopoietic cells. Mouse artificial chromosome (MAC) vectors derived from native mouse chromosome 11 demonstrated improved stability, and they were utilized for humanized Tc mouse production as a standard vector. In mouse, however, stability of MAC vector derived from native mouse chromosome other than mouse chromosome 11 remains to be evaluated. To clarify the potential of mouse centromeres in the additional chromosomes, we constructed a new MAC vector from native mouse chromosome 10 to evaluate the stability in Tc mice. The new MAC vector was transmitted through germline and stably maintained in the mouse tissues without any apparent abnormalities. Through this study, the potential of additional mouse centromere was demonstrated for Tc mouse production, and new MAC is expected to be used for various applications.
Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without effective disease-modifying therapies. The accumulation of amyloid-β peptide (Aβ) is associated with AD. However, ...identifying new compounds that antagonize the underlying cellular pathologies caused by Aβ has been hindered by a lack of cellular models amenable to high-throughput chemical screening. To address this gap, we use a robust and scalable yeast model of Aβ toxicity where the Aβ peptide transits through the secretory and endocytic compartments as it does in neurons. The pathogenic Aβ 1—42 peptide forms more oligomers and is more toxic than Aβ 1—40 and genome-wide genetic screens identified genes that are known risk factors for AD. Here, we report an unbiased screen of ∼140,000 compounds for rescue of Aβ toxicity. Of ∼30 hits, several were 8-hydroxyquinolines (8-OHQs). Clioquinol (CQ), an 8-OHQ previously reported to reduce Aβ burden, restore metal homeostasis, and improve cognition in mouse AD models, was also effective and rescued the toxicity of Aβ secreted from glutamatergic neurons in Caenorhabditis elegans. In yeast, CQ dramatically reduced Aβ peptide levels in a copper-dependent manner by increasing degradation, ultimately restoring endocytic function. This mirrored its effects on copper-dependent oligomer formation in vitro, which was also reversed by CQ. This unbiased screen indicates that copper-dependent Aβ oligomer formation contributes to Aβ toxicity within the secretory/endosomal pathways where it can be targeted with selective metal binding compounds. Establishing the ability of the Aβ yeast model to identify disease-relevant compounds supports its further exploitation as a validated early discovery platform.
Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting ...capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies.
Liposomal anticancer agents can effectively deliver drugs to tumor lesions, but their therapeutic effects are enhanced in only limited number of patients. Appropriate biomarkers to identify responder ...patients to these liposomal agents will improve their treatment efficacies. We carried out pharmacological and histopathological analyses of mouse xenograft models bearing human ovarian cancers (Caov‐3, SK‐OV‐3, KURAMOCHI, and TOV‐112D) to correlate the therapeutic effects of doxorubicin‐encapsulated liposome (Doxil®) and histological characteristics linked to the enhanced permeability and retention effect. We next generated 111In‐encapsulated liposomes to examine their capacities to determine indications for Doxil® treatment by single‐photon emission computed tomography (SPECT)/CT imaging. Antitumor activities of Doxil® were drastically enhanced in Caov‐3, moderately in SK‐OV‐3, and minimally in KURAMOCHI and TOV‐112D when compared to doxorubicin. Microvessel density and vascular perfusion were high in Caov‐3 and SK‐OV‐3, indicating a close relation with the enhanced antitumor effects. Next, 111In‐encapsulated liposomes were given i.v. to the animals. Their tumor accumulation and area under the curve values over 72 h were high in Caov‐3, relatively high in SK‐OV‐3, and low in two other tumors. Importantly, as both Doxil® effects and liposomal accumulation varied in the SK‐OV‐3 group, we individually obtained SPECT/CT images of SK‐OV‐3‐bearing mouse (n = 11) before Doxil® treatment. Clear correlation between liposomal tumor accumulation and effects of Doxil® was confirmed (R2 = 0.73). Taken together, our experiments definitely verified that enhanced therapeutic effects through liposomal formulations of anticancer agents depend on tumor accumulation of liposomes. Tumor accumulation of the radiolabeled liposomes evaluated by SPECT/CT imaging is applicable to appropriately determine indications for liposomal antitumor agents.
Advantages of liposomal anti‐cancer agents include increased anti‐tumor activities through EPR effect, and reduced side effects associated with their free drugs; however, despite these clear benefits, we are unequivocally reminded that liposomal agents enhance therapeutic effects to the limited number of cancer patients. Here, we performed pharmacological and histopathological analyses of mouse xenograft models bearing human ovarian cancers to correlate therapeutic effects of Doxil and histological characteristics linked to EPR, and we subsequently generated radiolabeled liposomes to examine their capacities as biomarkers to determine indications for Doxil treatment by SPECT/CT imaging. Our preclinical study clearly indicates the correlations among pharmacological effects of Doxil, histological factors linked to EPR, tumor accumulation of the radiolabeled liposomes, as well as SPECT/CT tests of the radiolabeled liposomes, and demonstrates applicability of SPECT/CT imaging to properly decide the indications for liposomal anti‐cancer agents with significant potential towards the clinical practice.
α-Synuclein (α-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast α-syn inhibits endoplasmic reticulum ...(ER)-to-Golgi (ERrightward arrowGolgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ERrightward arrowGolgi trafficking. The homologous Rab1 rescues α-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of α-syn pathobiology. In a cell-free system with purified transport factors α-syn inhibited ERrightward arrowGolgi trafficking in an α-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of α-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with α-syn and with diverse vesicle markers, suggesting that α-syn can impair multiple trafficking steps. Other Rabs did not ameliorate α-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, α-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.
Genomic multiplication of the locus-encoding human α-synuclein (α-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson's disease (PD). Here we report the ...results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of α-syn over the course of aging in the nematode Caenorhabditis elegans. Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age- and dose-dependent α-syn-induced degeneration in the dopamine neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting ≈2% of the population over 65 years of age.
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