Development of inducible transgenic mouse models for melanoma

Despite many studies on pathology and aetiology during the past decades, the molecular mechanism(s) of melanoma development remains largely unknown. Therefore the purpose of this project was to establish a transgenic mouse model able to investigate the molecular mechanism(s) of melanoma aetiology mediated by N-Ras and PTEN genes. To achieve this, an inducible gene switch approach was employed exploiting the Cre/loxP recombinase system. This approach has the advantage of avoiding embryonic lethality and helps to minimise disease(s) in other tissue(s) that may interfere with animal viability. It was envisaged that this inducible system would establish a model that accurately mimic the development of melanoma in humans. A disadvantage being that the tyrosinase-based promoter was only responsive to the inducer when melanocytes were proliferating. Initially, regulator vectors were created by sub-cloning Cre under the control of a melanocyte-specific promoter either enhanced tyrosinase (EICre) or tyrosinase related protein 2 (Trp2Cre). A Cre responsive, target N-Raslys61 transgene was also cloned, where expression was induced by Cre ablation of ‘Stop' cassette (cmv.stop.N-Raslys61) together with a report target transgene (cmv.stop.EGFP) to aid in expression characterisation. The functional activity and gene-switch specificity of these constructs were subsequently confirmed employing co-transfection of regulator and report target into B16 melanoma cells, but to confirm their activity in primary melanocytes, melanocyte culture conditions had to be defined for optimum growth and transfection as there is no optimized commercial medium available for murine melanocyte culture unlike for human melanocyte. In this study therefore, murine primary melanocyte culture method (50/50) has been defined, which exploited keratinocytes for initial melanocytes growth support as a feed layer. The other advantages of this 50/50 medium were that pigmented cells grew without spontaneous transformation and gave the higher transfection efficiency compared to media exploited by other groups. Using primary melanocytes cultured in 50/50 medium, transgene construction and identification of regulator expression were performed by RT-PCR in vitro prior to in vivo analysis thereby avoiding unnecessary breeding. When concerns had arisen regarding an unexpected lack of melanoma phenotype in vivo particularly in addition of PTEN loss, this culture protocol supplied a successful test of oncogenic potential of N-Raslys61 and PTEN loss in vitro, where N-Raslys61 expression transformed melanocytes and PTEN loss promoted N-Raslys61 to give more aggressive cells. However a functional redundancy was identified, as transformed colonies were not immortalized and eventually senesced, possibly due to their opposing gene functions being on the same signalling pathway; i.e. PTEN fails to provide additional genetic aberrant pathway(s) for the cross-talk with Ras signalling necessary to form malignant tumours. The in vivo experiments commenced by crossing transgenic expressers of EICre regulator with target cmv.stop.N-Raslys61, to generate bigenic EICre/N-Ras mice. Treatment with Ru486 initially apparently failed to exhibit an abnormal phenotype, despite confirmation of N-Raslys61 expression following hair plucking to initiate the hair cycle and anagen melanocytes proliferation, therefore PTENflx/flx mutation was introduced. Unexpectedly, a similar result was obtained following treatment of EICre/PTENflx/flx/N-Raslys61 and EICre/PTENflx/flx in the test of whether PTEN functional loss promoted N-Raslys61 tumourigenesis. However with time, at 12-15 months (systemic) Ru486 treatment, phenotypes of enlarged eyes and harderian gland adenomas were obtained in N-Raslys61 expressing mice, whilst PTEN loss did not produce additional melanocytic phenotype. This confirmation of in vivo activity prompted a more careful analysis of treated mouse skin that discovered the appearance of white hair at treated sites which gave a subtle grey appearance to the coat colour compared to age matched untreated littermates or non-transgenic controls. Subsequent analysis found that melanocyte apoptosis was induced by N-Raslys61 mediated by caspsase-3, and this may explain the lack of melanomas. This new finding implied the existence of a cell defence system to protect mice from oncogenic expression, as a general feature or to overcome specific mutations that have the potential to induce melanoma. Furthermore, the same apoptotic pathway mediated by caspase-3 was mounted against PTEN functional loss. This implied a potential surveillance mechanism to compensate for PTEN function loss and also verified in vivo, the functional redundancy in melanocytes between these two genes observed in vitro, as it may be that until the appropriate anti-apoptotic pathway overcomes this sentinel mechanism, PTEN loss synergism with N-Raslys61 is insufficient for melanoma tumourigenesis. Due to the lack of melanoma, given the well characterized effects of the microenvironment in melanoma development, this study assessed the consequences of keratinocytes disruption. This was achieved employing a keratinocyte-specific K14Cre regulator transgenic line, expressed in proliferative basal cells, hair follicles and stem cells. In Ru486-treated tetragenic compound K14Cre/EICre/cmv.stop.N-Raslys61/PTENflx/flx mice pigmented papillomas were produced. This identified a melanocyte survival loop generated by microenvironment disruption that enabled anagen melanocytes to escape apoptosis during papillomagenesis. Furthermore, the mechanism involved elevated Kit/SCF expression in papillomas. The co-localisation of Kit and TRP-2 positive melanocytes in papilloma basal layers revealed that a Kit/SCF paracrine survival loop resulted in melanocyte survival. These results clearly demonstrated melanocyte cooperation with its immediate microenvironment consistent with the requirement for proliferative keratinocyte support of primary murine melanocyte cultures. Furthermore, these pigmented papillomas, may represent a model relevant to development of human seborrheic keratoses, which are pigmented benign lesions similar to papilloma, and neither nevi nor melanoma (4-6). These murine data suggest that these lesions may arise where papilloma formation occurs alongside anagen and the Kit/SCF paracrine survival loop creates an environment in papillomas sufficient to incorporate the survival and proliferation of anagen melanocytes, although the further studies are necessary to confirm this. To date, most transgenic melanoma models employ H-Ras, with only recent development of a relevant N-Ras model where constitutive, but not inducible, N-Raslys61 expression throughout embryogenesis eventually gave a hyperplastic melanocyte phenotype which is consistent with the report of N-Ras mutation common in congenital nevi but less in acquired nevi (9-13). As with H-Ras models, it appears that the CDKN2A locus deficiency is necessary for melanoma aetiology. In this study, unlike CDKN2A, PTEN loss failed to promote N-Ras melanoma tumourigenesis. This is possibly due to regulating the same signalling pathways, creating a functional redundancy, and the same susceptibility to apoptosis from newly identified potential compensatory surveillance systems. These results show the necessity of cross-talk between multiple genetic pathways to achieve malignant tumour formation and also the advantage of an inducible gene-switch approach to identify useful compensatory systems by allowing addition/deletion of many different interesting genes. Taking the insights from this study further, logically the introduction of p16/p19 deficient mice and/or other melanocyte/melanoma development related genes (specifically not on Ras signalling pathway, e.g. MC1R pathways) would provide an up-to-date, superior mouse model able to mimic molecular aetiology of human melanoma to investigate the functions and mechanisms of other genes such as MITF, B-Raf, MC1R etc involved in the development of human melanoma

The effects of age and ganglioside composition on the rate of motor nerve terminal regeneration following antibody-mediated injury in mice

Gangliosides are glycosphingolipids highly enriched in neural plasma membranes, where they mediate a diverse range of functions and can act as targets for auto-antibodies present in human immune-mediated neuropathy sera. The ensuing autoimmune injury results in axonal and motor nerve terminal (mNT) degeneration. Both aging and ganglioside-deficiency have been linked to impaired axonal regeneration. To assess the effects of age and ganglioside expression on mNT regeneration in an autoimmune injury paradigm, anti-ganglioside antibodies and complement were applied to young adult and aged mice wildtype (WT) mice, mice deficient in either b- and c-series (GD3sKO) or mice deficient in all complex gangliosides (GM2sKO). The extent of mNT injury and regeneration was assessed immediately or after 5 days, respectively. Depending on ganglioside expression and antibody-specificity, either a selective mNT injury or a combined injury of mNTs and neuromuscular glial cells was elicited. Immediately after induction of the injury, between 1.5% and 11.8% of neuromuscular junctions (NMJs) in the young adult groups exhibited healthy mNTs. Five days later, most NMJs, regardless of age and strain, had recovered their mNTs. No significant differences could be observed between young and aged WT and GM2sKO mice; aged GD3sKO showed a mildly impaired rate of mNT regeneration when compared with their younger counterparts. Comparable rates were observed between all strains in the young and the aged mice. In summary, the rate of mNT regeneration following anti-ganglioside antibody and complement-mediated injury does not differ majorly between young adult and aged mice irrespective of the expression of particular gangliosides

Differential binding patterns of anti-sulfatide antibodies to glial membranes

Sulfatide is a major glycosphingolipid in myelin and a target for autoantibodies in autoimmune neuropathies. However neuropathy disease models have not been widely established, in part because currently available monoclonal antibodies to sulfatide may not represent the diversity of anti-sulfatide antibody binding patterns found in neuropathy patients. We sought to address this issue by generating and characterising a panel of new anti-sulfatide monoclonal antibodies. These antibodies have sulfatide reactivity distinct from existing antibodies in assays and in binding to peripheral nerve tissues and can be used to provide insights into the pathophysiological roles of anti-sulfatide antibodies in demyelinating neuropathies

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy

Introduction Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. Results and Conclusions In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy

Neuronally expressed a-series gangliosides are sufficient to prevent the lethal age-dependent phenotype in GM3-only expressing mice

Gangliosides are expressed on plasma membranes throughout the body and enriched in the nervous system. A critical role for complex a‐ and b‐series gangliosides in central and peripheral nervous system ageing has been established through transgenic manipulation of enzymes in ganglioside biosynthesis. Disrupting GalNAc‐transferase (GalNAc‐T), thus eliminating all a‐ and b‐series complex gangliosides (with consequent over‐expression of GM3 and GD3) leads to an age‐dependent neurodegeneration. Mice that express only GM3 ganglioside (double knockout produced by crossing GalNAc‐T‐/‐ and GD3 synthase‐/‐ mice, Dbl KO) display markedly accelerated neurodegeneration with reduced survival. Degenerating axons and disrupted to the node of Ranvier architecture are key features of complex ganglioside‐deficient mice. Previously, we have shown that reintroduction of both a‐ and b‐series gangliosides into neurons on a global GalNAcT ‐/‐ background is sufficient to rescue this age‐dependent neurodegenerative phenotype. To determine the relative roles of a‐ and b‐series gangliosides in this rescue paradigm, we herein reintroduced GalNAc‐T into neurons of Dbl KO mice, thereby reconstituting a‐series but not b‐series complex gangliosides. We assessed survival, axon degeneration, axo‐glial integrity, inflammatory markers, and lipid‐raft formation in these Rescue mice compared to wild type and Dbl KO mice. We found that this neuronal reconstitution of a‐series complex gangliosides abrogated the adult lethal phenotype in Dbl KO mice, and partially attenuated the neurodegenerative features. This suggests that whilst neuronal expression of a‐series gangliosides is critical for survival during ageing, it is not entirely sufficient to restore complete nervous system integrity in the absence of either b‐series or glial a‐series gangliosides

Complement inhibition prevents glial nodal membrane injury in a GM1 antibody-mediated mouse model

The involvement of the complement pathway in Guillain–Barré syndrome pathogenesis has been demonstrated in both patient biosamples and animal models. One proposed mechanism is that anti-ganglioside antibodies mediate neural membrane injury through the activation of complement and the formation of membrane attack complex pores, thereby allowing the uncontrolled influx of ions, including calcium, intracellularly. Calcium influx activates the calcium-dependent protease calpain, leading to the cleavage of neural cytoskeletal and transmembrane proteins and contributing to subsequent functional failure. Complement inhibition has been demonstrated to provide effective protection from injury in anti-ganglioside antibody-mediated mouse models of axonal variants of Guillain–Barré syndrome; however, the role of complement in the pathogenesis of demyelinating variants has yet to be established. Thus, it is currently unknown whether complement inhibition would be an effective therapeutic for Guillain–Barré syndrome patients with injuries to the Schwann cell membrane. To address this, we recently developed a mouse model whereby the Schwann cell membrane was selectively targeted with an anti-GM1 antibody resulting in significant disruption to the axo-glial junction and cytoplasmic paranodal loops, presenting as conduction block. Herein, we utilize this Schwann cell nodal membrane injury model to determine the relevance of inhibiting complement activation. We addressed the early complement component C2 as the therapeutic target within the complement cascade by using the anti-C2 humanized monoclonal antibody, ARGX-117. This anti-C2 antibody blocks the formation of C3 convertase, specifically inhibiting the classical and lectin complement pathways and preventing the production of downstream harmful anaphylatoxins (C3a and C5a) and membrane attack complexes. Here, we demonstrate that C2 inhibition significantly attenuates injury to paranodal proteins at the node of Ranvier and improves respiratory function in ex vivo and in vivo Schwann cell nodal membrane injury models. In parallel studies, C2 inhibition also protects axonal integrity in our well-established model of acute motor axonal neuropathy mediated by both mouse and human anti-GM1 antibodies. These data demonstrate that complement inhibition prevents injury in a Schwann cell nodal membrane injury model, which is representative of neuropathies associated with anti-GM1 antibodies, including Guillain–Barré syndrome and multifocal motor neuropathy. This outcome suggests that both the motor axonal and demyelinating variants of Guillain–Barré syndrome should be included in future complement inhibition clinical trials

Neuronal Expression of GalNAc Transferase Is Sufficient to Prevent the Age-Related Neurodegenerative Phenotype of Complex Ganglioside-Deficient Mice

Gangliosides are widely expressed sialylated glycosphingolipids with multifunctional properties in different cell types and organs. In the nervous system, they are highly enriched in both glial and neuronal membranes. Mice lacking complex gangliosides attributable to targeted ablation of the B4galnt1 gene that encodes β-1,4-N-acetylegalactosaminyltransferase 1 (GalNAc–transferase; GalNAcT−/−) develop normally before exhibiting an age-dependent neurodegenerative phenotype characterized by marked behavioral abnormalities, central and peripheral axonal degeneration, reduced myelin volume, and loss of axo-glial junction integrity. The cell biological substrates underlying this neurodegeneration and the relative contribution of either glial or neuronal gangliosides to the process are unknown. To address this, we generated neuron-specific and glial-specific GalNAcT rescue mice crossed on the global GalNAcT−/− background [GalNAcT−/−-Tg(neuronal) and GalNAcT−/−-Tg(glial)] and analyzed their behavioral, morphological, and electrophysiological phenotype. Complex gangliosides, as assessed by thin-layer chromatography, mass spectrometry, GalNAcT enzyme activity, and anti-ganglioside antibody (AgAb) immunohistology, were restored in both neuronal and glial GalNAcT rescue mice. Behaviorally, GalNAcT−/−-Tg(neuronal) retained a normal “wild-type” (WT) phenotype throughout life, whereas GalNAcT−/−-Tg(glial) resembled GalNAcT−/− mice, exhibiting progressive tremor, weakness, and ataxia with aging. Quantitative electron microscopy demonstrated that GalNAcT−/− and GalNAcT−/−-Tg(glial) nerves had significantly increased rates of axon degeneration and reduced myelin volume, whereas GalNAcT−/−-Tg(neuronal) and WT appeared normal. The increased invasion of the paranode with juxtaparanodal Kv1.1, characteristically seen in GalNAcT−/− and attributed to a breakdown of the axo-glial junction, was normalized in GalNAcT−/−-Tg(neuronal) but remained present in GalNAcT−/−-Tg(glial) mice. These results indicate that neuronal rather than glial gangliosides are critical to the age-related maintenance of nervous system integrity

Dynamic coverage control design of multi-agent systems under ellipse sensing regions

summary:This paper studies the dynamic coverage control problem for cooperative region reconnaissance where a group of agents are required to reconnoitre a given region. The main challenge of this problem is that the sensing region of each agent is an ellipse. This modeling results in asymmetric(directed) interactions among agents. First, the region reconnaissance is formulated as a coverage problem, where each point in the given region should be surveyed until a preset level is achieved. Then, a coverage control law is designed that minimizes coverage performance index by finite switches between nominal control laws and perturbation control law. Finally, numerical simulations are provided to indicate the efficiency of the proposed control law