David B. Wang

Mimicking aspects of frontotemporal lobar degeneration and Lou Gehrig's disease in rats via TDP-43 overexpression.

Since the discovery of neuropathological lesions made of TDP-43 and ubiquitin proteins in cases of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), there is a burst of effort on finding related familial mutations and developing animal models. We used an adeno-associated virus (AAV) vector for human TDP-43 expression targeted to the substantia nigra (SN) of rats. Though TDP-43 was expressed mainly in neuronal nuclei as expected, it was also expressed in the cytoplasm, and dotted along the plasma membrane of neurons. Cytoplasmic staining was both diffuse and granular, indicative of preinclusion lesions, over 4 weeks. Ubiquitin deposited in the cytoplasm, specifically in the TDP-43 group, and staining for microglia was increased dose-dependently by 1-2 logs in the TDP-43 group, while neurons were selectively obliterated. Neuronal death induced by TDP-43 was pyknotic and apoptotic. TDP-43 gene transfer caused loss of dopaminergic neurons in the SN and their axons in the striatum. Behavioral motor dysfunction resulted after TDP-43 gene transfer that was vector dose-dependent and progressive over time. The cytoplasmic expression, ubiquitination, and neurodegeneration mimicked features of the TDP-43 diseases, and the gliosis, apoptosis, and motor impairment may also be relevant to TDP-43 disease forms involving nigrostriatal degeneration.

Expansive Gene Transfer in the Rat CNS Rapidly Produces Amyotrophic Lateral Sclerosis Relevant Sequelae When TDP-43 is Overexpressed

Improved spread of transduction in the central nervous system (CNS) was achieved from intravenous administration of adeno-associated virus serotype-9 (AAV9) to neonatal rats. Spinal lower motor neuron transduction efficiency was estimated to be 78% using the highest vector dose tested at a 12-week interval. The widespread expression could aid studying diseases that affect both the spinal cord and brain, such as amyotrophic lateral sclerosis (ALS). The protein most relevant to neuropathology in ALS is transactive response DNA-binding protein 43 (TDP-43). When expressed in rats, human wild-type TDP-43 rapidly produced symptoms germane to ALS including paralysis of the hindlimbs and muscle wasting, and mortality over 4 weeks that did not occur in controls. The hindlimb atrophy and weakness was evidenced by assessments of rotarod, rearing, overall locomotion, muscle mass, and histology. The muscle wasting suggested denervation, but there was only 14% loss of motor neurons in the TDP-43 rats. Tissues were negative for ubiquitinated, cytoplasmic TDP-43 pathology, suggesting that altering TDP-43s nuclear function was sufficient to cause the disease state. Other relevant pathology in the rats included microgliosis and degenerating neuronal perikarya positive for phospho-neurofilament. The expression pattern encompassed the distribution of neuropathology of ALS, and could provide a rapid, relevant screening assay for TDP-43 variants and other disease-related proteins.

Pronounced microgliosis and neurodegeneration in aged rats after tau gene transfer

Microtubule-associated protein tau gene transfer to the substantia nigra of rats using the adeno-associated virus (AAV) vector previously led to neuropathology and neurodegeneration in young rats. In this study, we compared equal tau gene transfer in either 3 or 20-month-old rats, in order to test the hypothesis that late middle-aged rats are more susceptible to neurodegeneration. Two intervals and two vector doses of the tau vector probed for age-related differences in the initial sensitivity to low-level tau expression. Gene transfer efficiency was similar for both ages, but the tau vector caused more dopaminergic cell loss and a greater behavioral deficit in aged rats at specific doses and time points. Tau gene transfer caused microgliosis relative to the control vector, and to a greater extent in aged rats. The maximal microglial response occurred at 2 weeks preceding the peak dopaminergic cell loss by 8 weeks. The cellular and behavioral outcomes were more severe in the aged rats, validating the model for studies of age-related diseases.

Trogocytosis of MHC-I/Peptide Complexes Derived from Tumors and Infected Cells Enhances Dendritic Cell Cross-Priming and Promotes Adaptive T Cell Responses

The transporter associated with antigen processing (TAP) and the major histocompatibility complex class I (MHC-I), two important components of the MHC-I antigen presentation pathway, are often deficient in tumor cells. The restoration of their expression has been shown to restore the antigenicity and immunogenicity of tumor cells. However, it is unclear whether TAP and MHC-I expression in tumor cells can affect the induction phase of the T cell response. To address this issue, we expressed viral antigens in tumors that are either deficient or proficient in TAP and MHC-I expression. The relative efficiency of direct immunization or immunization through cross-presentation in promoting adaptive T cell responses was compared. The results demonstrated that stimulation of animals with TAP and MHC-I proficient tumor cells generated antigen specific T cells with greater killing activities than those of TAP and MHC-I deficient tumor cells. This discrepancy was traced to differences in the ability of dendritic cells (DCs) to access and sample different antigen reservoirs in TAP and MHC-I proficient versus deficient cells and thereby stimulate adaptive immune responses through the process of cross-presentation. In addition, our data suggest that the increased activity of T cells is caused by the enhanced DC uptake and utilization of MHC-I/peptide complexes from the proficient cells as an additional source of processed antigen. Furthermore, we demonstrate that immune-escape and metastasis are promoted in the absence of this DC ‘arming’ mechanism. Physiologically, this novel form of DC antigen sampling resembles trogocytosis, and acts to enhance T cell priming and increase the efficacy of adaptive immune responses against tumors and infectious pathogens.

Genetic strategies to study TDP-43 in rodents and to develop preclinical therapeutics for amyotrophic lateral sclerosis

The neuropathological hallmark of the majority of amyotrophic lateral sclerosis (ALS) and a class of frontotemporal lobar degeneration is ubiquitinated cytoplasmic aggregates composed of transactive response DNA binding protein 43 kDa (TDP‐43). Genetic manipulation of TDP‐43 in animal models has been used to study the protein’s role in pathogenesis. Transgenic rodents for TDP‐43 have recapitulated key aspects of ALS such as paralysis, loss of spinal motor neurons and muscle atrophy. Viral vectors are an alternate approach to express pathological proteins in animals. Use of the recombinant adeno‐associated virus vector serotype 9 has permitted widespread transgene expression throughout the central nervous system after intravenous administration. Expressing TDP‐43 in rats with this method produced a phenotype that was consistent with and similar to TDP‐43 transgenic lines. Increased levels of TDP‐43 in the nucleus are toxic to neurons and sufficient to produce ALS‐like symptoms. Animal models based on TDP‐43 will address the relationships between TDP‐43 expression levels, pathology, neuronal loss, muscle atrophy, motor function and causative mechanisms of disease. New targets that modify TDP‐43 function, or targets from previous ALS models and other models of spinal cord diseases, could be tested for efficacy in the recent rodent models of ALS based on TDP‐43. The vector approach could be an important therapeutic channel because the entire spinal cord can be affected from a one‐time peripheral administration.

Transcriptome analysis of a tau overexpression model in rats implicates an early pro-inflammatory response

Neurofibrillary tangles comprised of the microtubule-associated protein tau are pathological features of Alzheimers disease and several other neurodegenerative diseases, such as progressive supranuclear palsy. We previously overexpressed tau in the substantia nigra of rats and mimicked some of the neurodegenerative sequelae that occur in humans such as tangle formation, loss of dopamine neurons, and microgliosis. To study molecular changes involved in the tau-induced disease state, we used DNA microarrays at an early stage of the disease process. A range of adeno-associated virus (AAV9) vector doses for tau were injected in groups of rats with a survival interval of 2 weeks. Specific decreases in messages for dopamine-related genes validated the technique with respect to the dopaminergic cell loss observed. Of the mRNAs upregulated, there was a dose-dependent effect on multiple genes involved in immune response such as chemokines, interferon-inducible genes and leukocyte markers, only in the tau vector groups and not in dose-matched controls of either transgene-less empty vector or control green fluorescent protein vector. Histological staining for dopamine neurons and microglia matched the loss of dopaminergic markers and upregulation of immune response mRNAs in the microarray data, respectively. RT-PCR for selected markers confirmed the microarray results, with similar changes found by either technique. The mRNA data correlate well with previous findings, and underscore microgliosis and immune response in the degenerative process following tau overexpression.

Isocitrate dehydrogenase 1 is downregulated during early skin tumorigenesis which can be inhibited by overexpression of manganese superoxide dismutase

Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme that converts isocitrate to alpha‐ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development are unclear. We used a skin cell transformation model and mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter TPA and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with dimethylbenz[α]anthracene/TPA also showed decreases in IDH1 expression and activity. In non‐promotable JB6 P‐cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed, TPA‐induced cell transformation. Finally, manganese superoxide dismutase overexpression suppressed tumor promoter induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha‐ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress might contribute to IDH1 inactivation, because manganese superoxide dismutase, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02317.x, 2012)

In vivo Survivors of Transformed Mouse Ovarian Surface Epithelial Cells Display Diverse Phenotypes for Gene Expression and Tumorigenicity

Ovarian cancer is the fifth most common cause of cancer death in women. Due to a lack of appropriate animal models, studies involving tumorigenicity, tumor progression and immune response at the molecular level are limited. We isolated many clones derived from thesurvivors of a transformed mouse ovarian epithelial cell line IG-10 in immune- competent mice and found that the clones displayed diverse phenotypes. Most clones were deficient in components of the MHC-I antigen presentation pathway. Soft-agarose colony assays showed different growth rates among clones. However, this did not completely correlate with each clone’s in vivo tumorigenicity regarding growth, tumor mass and ascites formation, suggesting the possibility that the clones may display contrasting intrinsic gene expression. We therefore performed two types of arrays to evaluate gene expression at transcriptional and translational levels. The results showed differences in expression of COL4α5, NOS-2, and SOCS-1 genes at the transcriptional level, MIP-2 gene at the protein level and CCL5, CXCL-10, IL-1α genes at both transcriptional and protein levels between low and high tumorigenic clones. Thus, our animal cell model together with the identified genes may provide a useful tool to study ovarian cancer immune response, tumorigenicity and tumor-host cell interactions in the tumor microenvironment.

Abstract B59: Targeting metabolic enzymes in cancer prevention: Mechanistic insight into reciprocal regulation of isoforms of pyruvate kinase and isocitrate dehydrogenase in the early stage of cancer development

Cancer cells distinguish themselves from normal cells in multiple ways. One such distinction is that cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation even in the presence of oxygen, known as the “Warburg Effect”. Although glycolysis is inefficient to produce ATP compared with oxidative phosphorylation, metabolic intermediates produced during glycolysis may provide building blocks for cancer cells. Associated with this metabolic switch is up- or down-regulation of important metabolic enzymes, such as the M1 and M2 isoforms of pyruvate kinase (PKM1; PKM2) and isocitrate dehydrogenase 1 and 2 (IDH1; IDH2). PKM2 is highly expressed and critical for proliferation of tumor cells. Our initial studies showed that both UV irradiation and tumor promoters altered cellular metabolism via upregulating the activity and expression levels of PKM2 and downregulating PKM1. However, this M1/M2 shift has not been reported in early stage of cancer development. IDH1, a cytosolic enzyme which converts isocitrate to alpha-ketoglutarate, was recently discovered to be mutated in human brain cancer and leukemia. Interestingly, wild-type IDH1 produces alpha-ketoglutarate; however, mutant IDH1 generates 2-hydroxyglutarate, a known “oncometabolite.” We have found that decreased expression and activity levels of IDH1, not IDH2 were seen both in vitro in the tumor promotable JB6 P+ cell model and in vivo using mouse skin epidermal tissues, following UV and 12–0-tetradecanoylphorbol 13-acetate (TPA) treatment. More importantly, knockdown of IDH1 enhanced whereas overexpression of IDH1 suppressed cell transformation induced by tumor promoter TPA. During tumorigenesis, oncogenic activation can also generate oxidative stress. Since metabolism is the major source for ROS (reactive oxygen species) production, metabolic changes often observed in cancer cells may also be regulated by oxidative stress. Our initial studies revealed that PKM2 was upregulated and IDH1 downregulated in promotable mouse skin epidermal JB6 P+ cells, not in non-promotable JB6 P-cells. We have observed in previous studies that MnSOD expression/activity is lower in P+ cells, compared to P-cells, thus providing evidence that PKM2 activation and IDH1 inactivation may be regulated by oxidative stress. Lastly, overexpression of manganese superoxide dismutase (MnSOD) suppressed tumor promoter-induced PKM2 activation and decreased IDH1 expression. These results suggest that oxidative stress may contribute to PKM2 activation and IDH1 inactivation during early stage tumor promotion, suggesting mediation with MnSOD, a mitochondrial antioxidant enzyme, may play a cancer preventative role in regulating cellular metabolism. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B59.