Genetic Background Modulates the Phenotype of a Mouse Model of DYT1 Dystonia

DYT1 dystonia is a debilitating neurological disease characterized by involuntary twisting movements. The disease is caused by an in-frame deletion (GAG, “ΔE”) mutation in the TOR1A gene that encodes the torsinA protein. Intriguingly, only 30% of mutation carriers exhibit motor symptoms despite the fact that functional brain imaging studies show abnormal brain metabolism in all carriers. Because genetic modifiers may be a determinant of this reduced penetrance, we examined the genetic contribution of three different inbred strains of mice on the DYT1 mutation in animals that are homozygous (Tor1aΔE/ΔE) or heterozygous (Tor1aΔE/+; disease state) for the disease-causing ΔE mutation. We find that the DBA/2J, C57BL/6J, and CD1-ICR contribution of genes significantly alter lifespan in Tor1aΔE/ΔE mice, which die during the first few days of life on the 129S6/SvEvTac (129) background. The C57BL/6J (B6) strain significantly decreases life expectancy of Tor1aΔE/ΔE animals but, like 129S6/SvEvTac Tor1aΔE/+ mice, congenic C57BL/6J Tor1aΔE/+ mice do not exhibit any motor abnormalities. In contrast, the DBA/2J (D2) strain significantly increases life expectancy. This effect was not present in congenic DBA/2J Tor1aΔE/ΔE mice, indicating that the extended lifespan of F2 129/D2 mice was due to a combination of homozygous and heterozygous allelic effects. Our observations suggest that genetic modifiers may alter the penetrance of the ΔE mutation, and that mapping these modifiers may provide fresh insight into the torsinA molecular pathway

Targeting matriptase in breast cancer abrogates tumour progression via impairment of stromal-epithelial growth factor signalling.

Matriptase is an epithelia-specific membrane-anchored serine protease that has received considerable attention in recent years because of its consistent dysregulation in human epithelial tumours, including breast cancer. Mice with reduced levels of matriptase display a significant delay in oncogene-induced mammary tumour formation and blunted tumour growth. The abated tumour growth is associated with a decrease in cancer cell proliferation. Here we demonstrate by genetic deletion and silencing that the proliferation impairment in matriptase-deficient breast cancer cells is caused by their inability to initiate activation of the c-Met signalling pathway in response to fibroblast-secreted pro-HGF. Similarly, inhibition of matriptase catalytic activity using a selective small-molecule inhibitor abrogates the activation of c-Met, Gab1 and AKT, in response to pro-HGF, which functionally leads to attenuated proliferation in breast carcinoma cells. We conclude that matriptase is critically involved in breast cancer progression and represents a potential therapeutic target in breast cancer

Matriptase regulates c-Met mediated proliferation and invasion in inflammatory breast cancer.

The poor prognosis for patients with inflammatory breast cancer (IBC) compared to patients with other types of breast cancers emphasizes the need to better understand the molecular underpinnings of this disease with the goal of developing effective targeted therapeutics. Dysregulation of matriptase expression, an epithelial-specific member of the type II transmembrane serine protease family, has been demonstrated in many different cancer types. To date, no studies have assessed the expression and potential pro-oncogenic role of matriptase in IBC. We examined the functional relationship between matriptase and the HGF/c-MET signaling pathway in the IBC cell lines SUM149 and SUM190, and in IBC patient samples. Matriptase and c-Met proteins are localized on the surface membrane of IBC cells and their expression is strongly correlated in infiltrating cancer cells and in the cancer cells of lymphatic emboli in patient samples. Abrogation of matriptase expression by silencing with RNAi or inhibition of matriptase proteolytic activity with a synthetic inhibitor impairs the conversion of inactive pro-HGF to active HGF and subsequent c-Met-mediated signaling, leading to efficient impairment of proliferation and invasion of IBC cells. These data show the potential of matriptase inhibitors as a novel targeted therapy for IBC, and lay the groundwork for the development and testing of such drugs

Neuronal Nuclear Membrane Budding Occurs during a Developmental Window Modulated by Torsin Paralogs

DYT1 dystonia is a neurodevelopmental disease that manifests during a discrete period of childhood. The disease is caused by impaired function of torsinA, a protein linked to nuclear membrane budding. The relationship of NE budding to neural development and CNS function is unclear, however, obscuring its potential role in dystonia pathogenesis. We find NE budding begins and resolves during a discrete neurodevelopmental window in torsinA null neurons in vivo. The developmental resolution of NE budding corresponds to increased torsinB protein, while ablating torsinB from torsinA null neurons prevents budding resolution and causes lethal neural dysfunction. Developmental changes in torsinB also correlate with NE bud formation in differentiating DYT1 embryonic stem cells, and overexpression of torsinA or torsinB rescues NE bud formation in this system. These findings identify a torsinA neurodevelopmental window that is essential for normal CNS function and have important implications for dystonia pathogenesis and therapeutics

B6·<i>Tor1a^ΔE/+</i> mice do not have baseline motor abnormalities.

<p>Male B6·<i>Tor1a^ΔE/+</i>and B6·<i>Tor1a^+/+</i> animals were monitored for gross motor abnormalities. Horizontal activity in the open field for 60 min (5 min per point) sessions at 6 mos (A), 9 mos (D), and 12 mos of age (G) does not differ between genotypes, rm-ANOVA, (F[11,187] = 1.266, p = 0.25 at 6 mos; F[4.67,107.5] = 1.88, p = 0.11 at 9 mos; F[5.02,115.52] = 1.56, p = 0.18 at 12 mos). Total distance traveled and total rearing over 60 min are shown as bargraphs (B, C, E, F, H, I). Each bar represents the mean of total activity over one hour. Assessment of total horizontal distance traveled and total rearing by student's T-test, also found no difference between genotypes at any of the observed ages total distance: t<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Cookson1" target="_blank">[17]</a> = 1.40 ; p = 0.18, at 6 mos; t(23) = 0.84, p = 0.92, at 9 mos; t<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Risch1" target="_blank">[23]</a> = 0.65, p = 0.52, at 12 mos (B, E, H), and for total rearing: t<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Cookson1" target="_blank">[17]</a> = 0.62, p = 0.54, at 6 mos; t<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Risch1" target="_blank">[23]</a> = −0.90, p = 0.38, at 9 mos; t<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Risch1" target="_blank">[23]</a> = −0.90, p = 0.38 at 12 mos (C, F, I). (J) One year old B6·<i>Tor1a^ΔE/+</i>and B6·<i>Tor1a^+/+</i> mice learn at the same rate during the three consecutive training days on the accelerating rotarod, rm-ANOVA, significant main effect of training day F[2, 46] = 72.06, p = 0.00 but do not perform differently (no interaction between training day and genotype: F[1.57, 46)] = 1.25, p = 0.29). (K) Both groups perform the same on the testing day (3 fixed speeds, 4 trials each), rm-ANOVA: no interaction between speed and genotype (F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Risch1" target="_blank">[23]</a> = 0.91 (p = 0.35). (L) Seven month old B6·<i>Tor1a^ΔE/+</i>and B6·<i>Tor1a^+/+</i> mice perform similarly on the balance beam. Latency to cross the 5 mm square beam is shown for 4 consecutive days (2 trials/day), rm-ANOVA: main effect of day: F[2.83, 63] = 10.12 (p = 0.00), no interaction between day and genotype, F[3,63] = 0.83 (p = 0.48). (M) The number of footslips is shown for the last day of testing and no difference is found, T[20.85] = 1.16; (p = 0.26).</p

Mouse background modulates lifespan of <i>Tor1a^ΔE/ΔE</i> mice.

<p>A. Kaplan-Meier survival curves of <i>Tor1a^ΔE/ΔE</i> mice. D2, B6 and CD1-ICR genes modulate lifespan in <i>Tor1a^ΔE/ΔE</i> mice compared to original 129 background (Chi-square test used to generate p values: p<0.0001 for 129 versus 129/D2, 129/C57; p<0.05 for 129 versus 129/CD1). B. CLUSTALW2 multiple sequence alignment of genomic DNA from D2, B6, and 129 wildtype mice and Refseq demonstrates that all three strains have aspartic acid (D) at position 217 (*) of the mouse torsinA protein; figure depicts base pairs which correspond to the amino acid coding sequence 212–221 (AERITDVALD) C. Long-lived F2 129/D2 <i>Tor1a^ΔE/ΔE</i> mouse and <i>Tor1a^+/+</i> littermate. 129/D2 <i>Tor1a^ΔE/ΔE</i> mouse is smaller than control at postnatal day 14 (a.), poorly groomed with partially closed eyes (b.), exhibits improper limb placement (c.), and demonstrates hindlimb clasping on tail suspension tests (d., e.).</p

B6·<i>Tor1a^ΔE/+</i> mice do not respond differently to pharmacological challenges in the open-field.

<p>Locomotor activity of B6·<i>Tor1a^ΔE/+</i> and B6·<i>Tor1a^+/+</i> mice was monitored for 90 minutes (5 min per point) total. After a 30 minute habituation period, mice were injected with (A) scopolamine (1.0 mg/kg) or (D) GBR12909 (5.0 mg/kg) or vehicle (indicated by arrow) and monitored for an additional 60 minutes. Scopolamine and GBR12909 stimulated locomotor activity of both genotypes but there was no interaction between genotype and drug. Scopolamine challenge, rm-ANOVA reveals no main effect of genotype and no significant interaction between time and genotype (F[2.52, 55.35] = 1.05, p = 0.37) of horizonal activity in the openfield (A). When totaled over the entire time in the openfield, there is no significant effect of genotype on total horizontal distance, t<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Bressman1" target="_blank">[22]</a> = 0.040, (p = 0.97) or total rearing t[22)] = −0.143, (p = 0.86; B–C). GBR challenge, rm-ANOVA demonstrated a significant main effect of drug, F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Martella1" target="_blank">[25]</a> = 7.39, p = 0.01, but no significant main effect of genotype (F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Martella1" target="_blank">[25]</a> = .287, p = 0.60) and no significant interaction between drug and genotype (F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Martella1" target="_blank">[25]</a> = 0.08, p = 0.78; D). Evaluation of total horizontal distance and total rearing with two-way ANOVA finds significant main effect of drug (F(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Shakkottai1" target="_blank">[20]</a> = 16.02, p = 0.00 (total horizontal distance); F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Beck1" target="_blank">[21]</a> = 0.20, p = 0.66 (total rearing)) but no interaction between drug and genotype (F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Shakkottai1" target="_blank">[20]</a> = 1.46, p = 0.24 (total horizontal distance); F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Beck1" target="_blank">[21]</a> = 0.50, p = 0.49 (total rearing; E–F). (G) Quinpirole (0.1 mg/kg) was injected at the start of the open field session and locomotor activity was monitored for 120 minutes. Quinpirole inhibited locomotor activity of both groups of mice compared to vehicle but did not elicit a significant difference between genotypes. Rm-anova demonstrated a significant main effect of drug F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Kamm1" target="_blank">[29]</a> = 13.65, p = 0.001, a significant main effect of genotype (F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Kamm1" target="_blank">[29]</a> = 0.28, p = 0.05), but no significant interaction between drug and genotype (F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Kamm1" target="_blank">[29]</a> = 0.28, p = 0.06). (H–I) Examination of total horizontal distance and total rearing with two-way ANOVA finds a significant main effect of drug (F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Ulug1" target="_blank">[30]</a> = 11.330, p = 0.002 (total horizontal distance); F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Ulug1" target="_blank">[30]</a> = 9.68, p = 0.004 (total rearing)) but no interaction between drug and genotype (F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Ulug1" target="_blank">[30]</a> = 0.74. p = 0.40 (total horizontal distance); F<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Tanabe1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032245#pone.0032245-Ulug1" target="_blank">[30]</a> = 0.20, p = 0.66 (total rearing).</p

Genotyping parameters for <i>Tor1a</i> knock-in mice.

<p>Genotyping parameters for <i>Tor1a</i> knock-in mice.</p

HATL5: a cell surface serine protease differentially expressed in epithelial cancers.

Over the last two decades, cell surface proteases belonging to the type II transmembrane serine protease (TTSP) family have emerged as important enzymes in the mammalian degradome, playing critical roles in epithelial biology, regulation of metabolic homeostasis, and cancer. Human airway trypsin-like protease 5 (HATL5) is one of the few family members that remains uncharacterized. Here we demonstrate that HATL5 is a catalytically active serine protease that is inhibited by the two Kunitz type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and 2, as well as by serpinA1. Full-length HATL5 is localized on the cell surface of cultured mammalian cells as demonstrated by confocal microscopy. HATL5 displays a relatively restricted tissue expression profile, with both transcript and protein present in the cervix, esophagus, and oral cavity. Immunohistochemical analysis revealed an expression pattern where HATL5 is localized on the cell surface of differentiated epithelial cells in the stratified squamous epithelia of all three of these tissues. Interestingly, HATL5 is significantly decreased in cervical, esophageal, and head and neck carcinomas as compared to normal tissue. Analysis of cervical and esophageal cancer tissue arrays demonstrated that the squamous epithelial cells lose their expression of HATL5 protein upon malignant transformation