Huntington disease (HD) is an inherited neurological disorder caused by a polyglutamine expansion in the protein huntingtin and is characterized by selective neurodegeneration that preferentially occurs in striatal medium spiny neurons. Because the medium spiny neurons are innervated abundantly by glutamatergic axons from cortical neurons, the preferential degeneration in the striatal neurons supports the glutamate excitotoxicity theory for HD pathogenesis. Thus, glutamate uptake by glia may be particularly important for preventing glutamate excitotoxicity in HD. Although mutant huntingtin is expressed ubiquitously in various types of cells, it accumulates and forms aggregates in fewer glial cells than in neuronal cells. It remains largely unknown whether and how mutant huntingtin in glia can contribute to the neurological symptoms of HD. We generated transgenic mice that express N-terminal mutant huntingtin in astrocytes, a major type of glial cell that remove extracellular glutamate in the brain. Although transgenic mutant huntingtin in astrocytes is expressed below the endogenous level, it can cause age-dependent neurological phenotypes in transgenic mice. Mice expressing mutant huntingtin show body weight loss, have motor function deficits, and die earlier than wild-type or control transgenic mice. We also found that mutant huntingtin in astrocytes decreases the expression of glutamate transporter by increasing its binding to Sp1 and reducing the association of Sp1 with the promoter of glutamate transporter. These results imply an important role for glial mutant huntingtin in HD pathology and suggest possibilities for treatment.excitotoxicity ͉ glia ͉ neurodegeneration ͉ polyglutamine ͉ glutamate I n Huntington disease (HD), selective neuronal loss occurs preferentially in medium spiny neurons of the striatum and then extends to other brain regions as the disease progresses (1). Because medium spiny neurons are innervated by glutamatergic axons from cortical neurons (2), they are particularly vulnerable to glutamate excitotoxicity, a possible pathogenic mechanism for the preferential neurodegeneration seen in the striatum of HD patients (3). In support of this theory, excitotoxicity of the NMDA receptor, an ionotropic receptor for glutamate, is now associated with HD in various animal models (4, 5).The majority of cells in the brain are glia that support the survival of neuronal cells. Astrocytes are the major type of glia and express glutamate transporters that uptake extracellular glutamate to prevent glutamate neurotoxicity (6-8). Although mutant huntingtin (htt) is expressed in glial cells in the brains of HD mice and patients (9, 10), whether and how mutant htt in glia contributes to neuropathology in vivo remains unknown. Because glial cells can be therapeutic targets, establishing a transgenic mouse model expressing mutant htt specifically in glia can help develop treatment for HD.Current HD mouse models have limitations for studying glial htt contribution, because transgenic htt in these HD mice is e...

Human telomerase hTERC RNA serves as a template for the catalytic hTERT protein to synthesize telomere repeats at chromosome ends. We have recently shown that some patients with bone marrow failure syndromes are heterozygous carriers for hTERC or hTERT mutations. These sequence variations usually lead to a compromised telomerase function by haploinsufficiency. Here, we provide functional characterization of an additional 8 dis- IntroductionTelomerase is a specialized reverse transcriptase (RT) that adds long, repetitive stretches of simple telomeric DNA sequence (ie, TTAGGG in the vertebrates) onto chromosomal termini. 1 This cellular RT protein (TERT) copies a short stretch of nucleotides located within the template region of an integral RNA component (TERC) into telomeric DNA repeats. 1 Vertebrate TERCs are believed to adopt a complex, folded secondary structure 2 as depicted for human TERC (hTERC) in Figure 1A. We have recently conducted extensive site-directed mutagenesis analysis of hTERC to show that much of the structure folded as predicted. 3 As is true of many biologically active RNA molecules, most of the internally base-paired regions of hTERC can be extensively mutated without loss of function, provided that the normal base-pairing pattern is preserved. 3 However, at certain locations, especially of the single-stranded template region that is copied into telomeric DNA, specific RNA base sequences have been shown to be required for biologic activity. 4 Telomerase catalytic proteins (TERTs) from evolutionary distant organisms share a conserved structural organization that can be divided into 3 functional domains ( Figure 1C). 5 The telomerasespecific domains exist at both the N and C termini of TERTs that are not present in any of the viral RTs. 6 The N-terminal region is required to participate in enzymatic function, 7,8 in assembly of the protein with its integral hTERC RNA component,7,9 and in the homodimerization of the protein (ie, hTERT protein-protein interaction), 7,9,10 whereas the C-terminal domain is required for telomerase-specific activity other than its catalytic function 11,12 as well as in the telomeric nucleotide addition processivity process. [13][14][15] The functional RT domain with the universally conserved RT motifs is almost centrally located in the protein primary sequence ( Figure 1C). The fact that mutations of key residues that are known to affect its conventional RT catalytic activity also negatively influence telomerase activity strongly argues that telomerase RT domain is the catalytic domain of the enzyme complex. 13,[15][16][17][18] Inherited mutations in both hTERC RNA and hTERT protein underlie rare bone marrow failure syndromes, autosomal dominant dyskeratosis congenita (DC) and acquired aplastic anemia (AA). [19][20][21] DC is characterized by abnormal skin pigmentation, nail dystrophy, and oral leukoplakia and is often complicated by life-threatening bone marrow failure and immunodeficiency. 22 Lymphocytes from patients show decreased hTERC expression, decreased t...

Tumor-initiating cells (TICs) are a sub-population of cells that exhibit a robust ability to self-renew and contribute to the formation of primary tumors, the relapse of previously treated tumors, and the development of metastases. TICs have been identified in various tumors, including those of the breast, and are particularly enriched in the basal-like and claudin-low subtypes of breast cancer. The signaling pathways that contribute to the function and maintenance of TICs are under intense study. We explored the potential involvement of the NF-κB family of transcription factors in TICs in cell lines that are representative of basal-like and claudin-low breast cancer. NF-κB was found to be activated in breast cancer cells that form tumorspheres efficiently. Moreover, both canonical and non-canonical NF-κB signaling is required for these cells to self-renew in vitro and to form xenograft tumors efficiently in vivo using limiting dilutions of cells. Consistent with this, canonical and non-canonical NF-κB signaling is activated in TICs isolated from breast cancer cell lines. Experimental results indicate that NF-κB promotes the function of TICs by stimulating epithelial-to-mesenchymal transition (EMT) and by upregulating the expression of the inflammatory cytokines IL-1β and IL-6. The results suggest the use of NF-κB inhibitors for clinical therapy of certain breast cancers.

Huntington disease (HD) is caused by an expansion of the polyglutamine (polyQ) repeat (>37Q) in huntingtin (htt), and age of onset is inversely correlated with the length of the polyQ repeat. Mutant htt with expanded polyQ is ubiquitously expressed in various types of cells, including glia, but causes selective neurodegeneration. Our recent study demonstrated that expression of the N-terminal mutant htt with a large polyQ repeat (160Q) in astrocytes is sufficient to induce neurological symptoms in mice (Bradford, J., Shin, J. Y., Roberts, M., Wang, C. E., Li, X.-J., and Li, S. H. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 22480 -22485). Because glia-neuron interactions are critical for maintaining the normal function and survival of neurons in the brain and because mutant htt is more abundant in neurons than in glial cells, it is important to investigate whether glial htt can still contribute to HD pathology when mutant htt is abundantly expressed in neuronal cells. We generated transgenic mice that express mutant htt with 98Q in astrocytes. Unlike our recently generated htt-160Q transgenic mice, htt-98Q mice do not show obvious neurological phenotypes, suggesting that the length of the polyQ repeat determines the severity of glial dysfunction. However, htt-98Q mice show increased susceptibility to glutamate-induced seizure. Mice expressing mutant htt in astrocytes were mated with N171-82Q mice that express mutant htt primarily in neuronal cells. Double transgenic mice expressing mutant htt in both neuronal and glial cells display more severe neurological symptoms and earlier death than N171-82Q mice. These findings indicate a role of glial mutant htt in exacerbating HD neuropathology and underscore the importance of improving glial function in treating HD. Huntington disease (HD)3 is an inherited neurological disorder caused by a polyglutamine (polyQ) expansion in huntingtin (htt). The polyQ expansion is also the common genetic mutation for eight other neurodegenerative diseases including spinal cerebellar ataxia 1-3, 6, 7, 17. A noticeable pathology feature of these diseases is that the mutant proteins, which are ubiquitously expressed in the body and brain, cause selective neurodegeneration in distinct brain regions in each disease (1). In HD, selective neuronal loss preferentially occurs in the striatum, the deep layers of the cerebral cortex and extends to many other brain regions during the late stage of the disease (2). This phenomenon has led to extensive studies of the effects of mutant htt in neuronal cells. Several HD transgenic mouse models have been generated including those expressing mutant htt under the endogenous htt promoter (knock-in), transgenic human htt promoters (YAC, BAC, and R6/2), or the mouse neuronal prion promoter (N171-82Q) (3). Mice expressing truncated htt (R6/2 and N171-82Q) show more severe phenotypes than YAC or BAC transgenic mice that express fulllength mutant htt (3, 4), suggesting that N-terminal mutant htt is more pathogenic than full-length mutant htt. In addition to the p...

Myeloid-derived suppressor cells (MDSCs) are a major obstacle to promising forms of cancer immunotherapy, but tools to broadly limit their immunoregulatory effects remain lacking. In this study, we assessed the therapeutic effect of the humanized anti-Jagged1/2 blocking antibody CTX014 on MDSC-mediated T cell suppression in tumor-bearing mice. CTX014 decreased tumor growth, impacted the accumulation and tolerogenic activity of MDSCs in tumors, and inhibited the expression of immunosuppressive factors arginase I and iNOS. Consequently, anti-Jagged therapy overcame tumor-induced T cell tolerance, increased the infiltration of reactive CD8+ T-cells into tumors, and enhanced the efficacy of T cell-based immunotherapy. Depletion of MDSC-like cells restored tumor growth in mice treated with anti-Jagged, whereas co-injection of MDSC-like cells from anti-Jagged-treated mice with cancer cells delayed tumor growth. Jagged1/2 was induced in MDSCs by tumor-derived factors via NFkB-p65 signaling, and conditional deletion of NFkB-p65 blocked MDSC function. Collectively, our results offer a preclinical proof of concept for the use of anti-Jagged1/2 to reprogram MDSC-mediated T cell suppression in tumors, with implications to broadly improve the efficacy of cancer therapy.