Rac Binding Research Articles

F\xf6rster resonance energy transfer biosensors for fluorescence and time-gated luminescence analysis of rac1 activity

Genetically encoded, Förster resonance energy transfer (FRET) biosensors enable live-cell optical imaging of signaling molecules. Small conformational changes often limit the dynamic range of biosensors that combine fluorescent proteins (FPs) and sensing domains into a single polypeptide. To address this, we developed FRET and lanthanide-based FRET (LRET) biosensors of Rac1 activation with two key features that enhance sensitivity and dynamic range. For one, alpha helical linker domains separate FRET partners and ensure a large conformational change and FRET increase when activated Rac1 at the biosensor C-terminus interacts with an amino-terminal Rac binding domain. Incorporation of a luminescent Tb(III) complex with long (~ ms) excited state lifetime as a LRET donor enabled time-gated luminescence measurements of Rac1 activity in cell lysates. The LRET dynamic range increased with ER/K linker length up to 1100% and enabled robust detection of Rac1 inhibition in 96-well plates. The ER/K linkers had a less pronounced, but still significant, effect on conventional FRET biosensors (with FP donors and acceptors), and we were able to dynamically image Rac1 activation at cell edges using fluorescence microscopy. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.

Novel rapid immunohistochemistry using an alternating current electric field identifies Rac and Cdc42 activation in human colon cancer FFPE tissues

It is important to determine the activation status of Rac and Cdc42 in cancer tissues for the prediction of metastasis and patient prognosis. However, it has been impossible to detect their spatial activation on formalin-fixed paraffin embedded (FFPE) surgical specimens thus far. Here, we established a novel detection technique for activated Rac/Cdc42 in human colon cancer FFPE tissues by using a p21-activated kinase (PAK)-Rac binding domain (RBD) detection probe fused with glutathione S-transferase (GST), designated GST-PAK-RBD, and novel rapid-immunohistochemistry (R-IHC) systems using noncontact alterating-current electric field mixing, although there is a technical limitation in that it may not distinguish between Rac members and Cdc42. In 50 cases of colon cancer, various activation patterns of Rac/Cdc42 were observed, which were designated plasma membrane, cytoplasm, mixed pattern, and polarized distribution. The activity was striking in the invasive fronts of tumors and significantly correlated with tumor invasion properties evaluated by TNM classification. Of note, in tissue microarray (TMA) samples, 29 of 33 cases demonstrated higher Rac1/Cdc42 activity in the tumor area than the corresponding normal mucosa. In addition, positive correlations were detected between Rac/Cdc42 activity and clinicopathological factors such as venous and lymphatic vessel invasion. These results suggest that understanding Rac and Cdc42 activations in cancer tissues would be valuable as an option for molecular therapy as personalized medicine.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by invivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

The biological function of naturally occurring DOCK2 inhibitor in tissue-specific immune evasion

Abstract Study objection DOCK2 is a Rac-specific guanine nucleotide exchange factor (GEF) predominantly expressed in hematopoietic cells. DOCK2 is essential for migration and activation of leukocytes, and its mutations in humans cause severe immunodeficiency, thereby indicating that DOCK2 plays a key role in immune surveillance. Interestingly, however, we found that a naturally occurring DOCK2 inhibitor exists in our body and named this molecule DOCK2 inhibitory metabolite (D2IM). In this study, we aimed to reveal biological functions of D2IM. Method The specificity of D2IM was examined with Rac GEF assay and binding assay. T cells and neutrophils from WT mouse were treated with D2IM and subjected to Rac activation assay and chemotaxis assay. D2IM production in various tissues was monitored with mass spectrometry. In addition, influence of the lack of CS on ultraviolet- and antigen-induced ocular inflammationwas analyzed using KO mice lacking D2IM synthase. Result CS inhibited the Rac GEF activity of murine DOCK2 protein in vitro with a IC50 value of 2.0 mM. When leukocytes were treated with D2IM, chemoattractant-induced Rac activation was severely impaired, resulting in a marked reduction of migratory response of lymphocytes and neutrophils. D2IM most abundantly existed in the Harderian gland, which produces lipids to form the oily layer of the tear film, and its production was completely lost in mice lacking D2IM synthase. We found that the lack of D2IM augmented ultraviolet- and antigen-induced ocular inflammation in mice, which was suppressed by administration of eye drops containing D2IM. Conclusion D2IM is a naturally occurring DOCK2 inhibitor that contributes to create immune evasive microenvironment in the eye.

Solution structures and biophysical analysis of full-length group A PAKs reveal they are monomeric and auto-inhibited in cis.

The group A p21-activated kinases (PAKs) exist in an auto-inhibited form until activated by GTPase binding and auto-phosphorylation. In the auto-inhibited form, a regulatory domain binds to the kinase domain (KD) blocking the binding of substrates, and CDC42 or Rac binding to the regulatory domain relieves this auto-inhibition allowing auto-phosphorylation on the KD activation loop. We have determined the crystal structure of the PAK3 catalytic domain and by small angle X-ray scattering, the solution-phase structures of full-length inactive PAK1 and PAK3. The structures reveal a compact but elongated molecular shape that demonstrates that, together with multiple independent biophysical measurements and in contrast with previous assumptions, group A PAKs are monomeric both before and after activation, consistent with an activation mechanism of cis-auto-inhibition and initial cis-auto-phosphorylation, followed by transient dimerisation to allow trans-auto-phosphorylation for full activation, yielding a monomeric active PAK protein.

Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo

SummaryCell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.

Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Nonnredundant Functions in Vivo

Cell migration often involves the formation of sheet‐like lamellipodia, generated by actin filament branching through Arp2/3 complex. In these structures, the latter is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family. Recent structural studies defined two independent Rac1 binding sites on WRC within the Sra‐1/PIR121 subunit of the pentameric WRC, but the functions of these sites in vivo have remained unknown. Here we used CRISPR/Cas9‐mediated gene disruption of Sra‐1 and its paralogue PIR121 in murine B16‐F1 cells combined with Sra‐1 mutant rescue to dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra‐1. We found that the A site, positioned adjacent to the binding region of WAVE‐WCA mediating actin‐ and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site towards the C‐terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium amoebas. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants , we conclude these residues are important for Rac/D site interaction. Finally, lamellipodia formation was observable even after disruption of both Rac interaction sites in constitutively active WRC, showing Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but not mandatory for WRC accumulation in the lamellipodium.

Coronin7 regulates WASP and SCAR through CRIB mediated interaction with Rac proteins.

Coronin7 (CRN7) stabilizes F-actin and is a regulator of processes associated with the actin cytoskeleton. Its loss leads to defects in phagocytosis, motility and development. It harbors a CRIB (Cdc42- and Rac-interactive binding) domain in each of its WD repeat domains which bind to Rac GTPases preferably in their GDP-loaded forms. Expression of wild type CRN7 in CRN7 deficient cells rescued these defects, whereas proteins with mutations in the CRIB motifs which were associated with altered Rac binding were effective to varying degrees. The presence of one functional CRIB was sufficient to reestablish phagocytosis, cell motility and development. Furthermore, by molecular modeling and mutational analysis we identified the contact regions between CRN7 and the GTPases. We also identified WASP, SCAR and PAKa as downstream effectors in phagocytosis, development and cell surface adhesion, respectively, since ectopic expression rescued these functions.

Probabilistic classification learning with corrective feedback is associated with in vivo striatal dopamine release in the ventral striatum, while learning without feedback is not.

The basal ganglia (BG) mediate certain types of procedural learning, such as probabilistic classification learning on the ‘weather prediction task’ (WPT). Patients with Parkinson's disease (PD), who have BG dysfunction, are impaired at WPT‐learning, but it remains unclear what component of the WPT is important for learning to occur. We tested the hypothesis that learning through processing of corrective feedback is the essential component and is associated with release of striatal dopamine. We employed two WPT paradigms, either involving learning via processing of corrective feedback (FB) or in a paired associate manner (PA). To test the prediction that learning on the FB but not PA paradigm would be associated with dopamine release in the striatum, we used serial 11C‐raclopride (RAC) positron emission tomography (PET), to investigate striatal dopamine release during FB and PA WPT‐learning in healthy individuals. Two groups, FB, (n = 7) and PA (n = 8), underwent RAC PET twice, once while performing the WPT and once during a control task. Based on a region‐of‐interest approach, striatal RAC‐binding potentials reduced by 13–17% in the right ventral striatum when performing the FB compared to control task, indicating release of synaptic dopamine. In contrast, right ventral striatal RAC binding non‐significantly increased by 9% during the PA task. While differences between the FB and PA versions of the WPT in effort and decision‐making is also relevant, we conclude striatal dopamine is released during FB‐based WPT‐learning, implicating the striatum and its dopamine connections in mediating learning with FB. Hum Brain Mapp 35:5106–5115, 2014. © 2014 The Authors. Human Brain Mapping Published by Wiley Periodicals, Inc.

Dimerization of DOCK2 Is Essential for DOCK2-Mediated Rac Activation and Lymphocyte Migration

The migratory properties of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain the Dbl homology domain typically found in guanine nucleotide exchange factors (GEFs), DOCK2 mediates the GTP-GDP exchange reaction for Rac via its DOCK homology region (DHR)-2 (also known as CZH2 or Docker) domain. DOCK2 DHR-2 domain is composed of three lobes, and Rac binding site and catalytic center are generated entirely from lobes B and C. On the other hand, lobe A has been implicated in dimer formation, yet its physiological significance remains unknown. Here, we report that lobe A-mediated DOCK2 dimerization is crucial for Rac activation and lymphocyte migration. We found that unlike wild-type DOCK2, DOCK2 mutant lacking lobe A failed to restore motility and polarity when expressed in thymoma cells and primary T cells lacking endogenous expression of DOCK2. Similar results were obtained with the DOCK2 point mutant having a defect in dimerization. Deletion of lobe A from the DHR-2 domain did not affect Rac GEF activity in vitro. However, fluorescence resonance energy transfer analyses revealed that lobe A is required for DOCK2 to activate Rac effectively during cell migration. Our results thus indicate that DOCK2 dimerization is functionally important under the physiological condition where only limited amounts of DOCK2 and Rac are localized to the plasma membrane.

Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation

Small GTPases of the Rho family have been implicated in important cellular processes such as cell migration and adhesion, protein secretion, and/or gene transcription. In the lymphoid system, these GTPases participate in the signaling cascades that are activated after engagement of antigen receptors. However, little is known about the role that Rho GTPases play in IL-2-mediated responses. Here, we show that IL-2 induces Rac1 activation in Kit 225 T cells. We identified by mass spectrometry the muscle isoform of glycogen phosphorylase (PYGM) as a novel Rac1 effector molecule in IL-2-stimulated cells. The interaction between the active form of Rac1 (Rac1-GTP) and PYGM was established directly through a domain comprising amino acids 191-270 of PYGM that exhibits significant homology with the Rac binding domain of PAK1. The integrity of this region was crucial for PYGM activation. Importantly, IL-2-dependent cellular proliferation was inhibited upon blocking both the activation of Rac1 and the activity of PYGM. These results reveal a new role for Rac1 in cell signaling, showing that this GTPase triggers T cell proliferation upon IL-2 stimulation by associating with PYGM and modulating its enzymatic activity.

Abstract B55: Mutation of PlexinB1 in prostate cancer

Abstract Semaphorins are secreted proteins that have a role in chemotaxis and are implicated in cancer. Plexins are membrane-bound receptors for semaphorins. We have found a high frequency of mutations in the plexinB1 gene and overexpression of the protein in prostate tumours (Wong et al., 2007; PNAS 104: 19040-5) indicating a role for plexinB1 in the pathogenesis of prostate cancer. The mutations of plexinB1 found in prostate tumours are functionally significant, increasing cell motility, invasive capacity, adhesion and inhibiting cell collapse. The mutations affect signalling pathways downstream of plexinB1, inhibiting Rac, Rnd, and R-Ras binding to plexinB1 and R-RasGAP activity. The mutations have the net effect of increasing cell motility by the loss of inhibitory pathways. We have now found that knockdown of endogenous plexinB1 in the prostate cancer cell line LNCaP inhibits anchorage-independent growth, while stimulation with semaphorin4D, the ligand for plexinB1, increases the migration and invasive capacity of LNCaP cells. These effects are reversed by expression of shRNA to plexinB1. Stimulation with semaphorin4D also results in ErbB2 and Akt activation. The opposite effects occur in the prostate cancer cell line PC3 in response to semaphorin4D. The opposite response of LNCaP and PC3 may be due to high levels of ErbB2 or c-Met expressed by the two cell lines respectively. These findings provide evidence for a subset of prostate tumours in which plexinB1 acts as a tumour-enhancing gene by activating ErbB2 and in which mutation of plexinB1 results in loss of cancer-inhibitory pathways. Citation Format: Magali Williamson, John Masters. Mutation of PlexinB1 in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B55.

Structure and control of the actin regulatory WAVE complex

Members of the Wiskott-Aldrich Syndrome Protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation by the Arp2/3 complex. The WASP relative, WAVE, regulates lamellipodia formation within a 400 kDa, hetero-pentameric WAVE Regulatory Complex (WRC). The WRC is inactive toward the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. We report the 2.3 Å crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting how these signals stimulate WRC activity toward the Arp2/3 complex. Spatial proximity of the Rac binding site and a large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.

PAW21 Rewarding visual cues increase dopamine neurotransmission in Parkinson's patients with impulse control disorders: a PET study

BackgroundCurrent evidence connects impulse control disorders (ICDs) to dopaminergic replacement therapy used for the treatment of Parkinson's disease (PD). Common ICDs include hypersexuality, pathological gambling, compulsive shopping and binge eating....

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91(phox) serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91(phox) requires the cytosolic proteins p67(phox), p47(phox), and Rac (a small GTPase). p67(phox), comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47(phox) and there interacts with Rac; these processes are prerequisite for gp91(phox) activation. Here we show that a region of p67(phox) (amino acids 190-200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67(phox) to support superoxide production by gp91(phox)-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91(phox)-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67(phox) interaction with the gp91(phox) NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67(phox) (amino acids 190-210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91(phox).

Abstract 3183: Dysregulated expression and function of Plexin-B1 in ovarian cancers

Abstract Background: Ovarian cancer has a tendency to spread within the peritoneal cavity early in the disease course. Understanding the metastatic pathways of ovarian cancer is thus of high importance for devising ovarian cancer treatment. Plexin-B1 is a cognate receptor of Semaphorin 4D (Sema4D). This study aims at investigating the role of Plexin-B1 in ovarian cancer. Materials and Methods: Expression of Plexin-B1 and its ligand in ovarian epithelial cell lines was examined by western blot and qPCR. Their expression in benign, borderline, and malignant tumors was revealed by immunohistochemistrty of paraffin embedded tissues. High-resolution melting (HRM) curve analysis was used to screen for mutation in Rac binding region of PLXNB1. Recombinant Sema4D was purified and used to treat OVCA420 cells which express high level of Plexin-B1. siRNA was used to knock-down Plexin-B1 in OVCA420. Cell migration and invasion ability was assayed by transwell migration and invasion assay. Results: Plexin-B1 was most highly overexpressed in serous ovarian cancer cell lines OVCA420, OVCA429, and SW626, whereas Sema4D was ovexpressed in OVCAR3, TOV21G, and PAI. In contrast, Plexin-B1 and Sema4D were not expressed in immortalized normal ovarian epithelial cell lines. Immunohistochemical studies also revealed high Plexin B1 expression in ovarian borderline tumors and invasive cancers while no Plexin B1 expression was detectable in benign tumor. On the other hand, downregulation of Plexin-B1 was observed in the metastatic foci compared with their corresponding primary tumors. There is also a tendency of higher Plexin-B1 expression in serous histotype than in other histotypes. A preliminary HRM screening of Plexin-B1 cDNA from 28 ovarian cancers detected no mutation in the Rac binding region. Knock-down of Plexin-B1 by siRNA caused increased migration and invasion of ovarian cancer cells. In contrast to Sema4D, treatment of ovarian cancer cells OVCA420 by follicle-stimulating hormone which enhances ovarian cancer growth, led to Plexin-B1 downregulation. Conclusion: Our study suggested that Plexin-B1 may contribute to early ovarian carcinogenesis. Its dyregulation in ovarian cancers is paradoxically associated with altered cancer cell invasion and migration. The role of Plexin B1 in ovarian cancer progression is complex. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3183.

Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations

Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Phospholipase C isozymes as effectors of Ras superfamily GTPases

The physiological effects of many extracellular stimuli are initiated through receptor-promoted activation of phospholipase C and inositol lipid signaling pathways. The historical view that phospholipase C-promoted signaling primarily occurs through activation of heterotrimeric G proteins or tyrosine kinases has expanded in recent years with the realization that at least three different mammalian phospholipase C isozymes are directly activated by members of the Ras superfamily of GTPases. Thus, Ras, Rap, Rac, and Rho GTPases all specifically regulate certain phospholipase C isozymes, and insight into the physiological significance of these signaling responses is beginning to accrue. High resolution three-dimensional structures of phospholipase C isozymes also are beginning to shed light on their mechanism of activation.

Changes in striatal dopamine D2 receptor binding in pre‐clinical Huntington’s disease

Carriers of the Huntington disease (HD) mutation develop a progressive neurodegenerative disorder after a pre-clinical phase. We examined the value of (11)C-raclopride PET (RAC) as a biomarker for pre-clinical HD pathophysiology. In a prospective cohort study with clinical and neuropsychological assessment we collected complete RAC data in 18 pre-clinical mutation carriers (HD-PMC) and 11 controls. Follow-up was 2 years. We calculated striatal RAC binding potential (BP) to measure dopamine D2 receptor availability. No HD-PMC had overt neuropsychological dysfunction. RAC-BP in putamen was abnormal in up to 44% of HD-PMC. The rate of RAC-BP decline (2.6% per year) was not significantly higher than in controls. Follow-up putaminal BP correlated weakly with predicted distance to onset of clinical HD (P = 0.034), but the rate of decline did not. Three HD-PMC developed motor abnormalities suspect for HD but did not show an increased rate of decline of putaminal BP. Many HD-PMC have striatal abnormalities but we found no clearly increased rate of D2 receptor changes around the onset of clinical HD. A longer follow-up of the present study cohort is needed to establish the value of RAC-BP in assessing the risk of clinical conversion from striatal D2 binding data.

Mtss1 Suppresses BCR-ABL Induced Cell Migration and Is Downregulated in CML Stem Cells.

Migration and adhesion properties of hematopoietic stem cells (HSC) are disrupted in chronic myeloid leukemia (CML). Egression of these cells from the bone marrow is associated with cytoskeletal changes including actin remodeling. In a microarray screen of differentially regulated genes in HSC from BCR-ABL positive transgenic mice, we found downregulation of multiple genes involved in actin-associated changes of cell structure, adhesion, and migration (i.e. intersectin-1, cortactin, Mtss1, synaptopodin, and Gem GTPase). Mtss1 was further studied since it has been described to be a binding partner of Rac, which is essential for BCR-ABL mediated transformation, and a potential tumor suppressor. Using quanitative RT-PCR, Mtss1 downregulation (6-fold) was confirmed in HSC and unfractionated bone marrow and spleen cells from SCLtTA/BCR-ABL transgenic mice after 3 weeks of BCR-ABL induction as well as in human BCR-ABL positive cell lines. Treatment of BCR-ABL positive (32D/BCR-ABL, K562, KYO-1) but not BCR-ABL negative (32D, U937) cell lines with 5μ M Imatinib led to upregulation of Mtss1 mRNA (5- to 10-fold) and protein, suggesting that downregulation of Mtss1 is dependent on BCR-ABL kinase activity. Retroviral transduction of Mtss1 into 32D/BCR-ABL cells almost completely inhibited BCR-ABL induced cell motility of individual cells seeded on murine bone marrow stromal cells in time-lapse video experiments over the course of two hours. Interestingly, we found that retroviral transduction of Mtss1 into 32D/BCR-ABL cells completely suppressed migration of these cells to extra-hematopoietic sites in vivo upon intravenous transplantation into syngeneic C3H mice. Moreover, when Mtss1-transduced cells were injected subcutaneously, the size of the tumors was significantly decreased as compared to empty vector-transduced 32D/BCR-ABL cells (p<0.05), confirming that Mtss1 may be a tumor suppressor.These results demonstrate that Mtss1 antagonizes BCR-ABL induced cell migration and is downregulated by BCR-ABL in CML stem cells, suggesting that downregulation of Mtss1 and other cytoskeletal adaptor proteins may be required for egression of CML stem cells from the bone marrow niche. Since the same Mtss1 protein domain is responsible for both Rac and actin binding, Mtss1 may interfere with Rac function and thereby inhibit the effects of Rac on migration and cytoskeletal dynamics of HSC.