Characterizing microglial gene expression in a model of secondary progressive multiple sclerosis.

Multiple sclerosis (MS) is the most common inflammatory, demyelinating and neurodegenerative disease of the central nervous system in young adults. Chronic-relapsing experimental autoimmune encephalomyelitis (crEAE) in Biozzi ABH mice is an experimental model of MS. This crEAE model is characterized by an acute phase with severe neurological disability, followed by remission of disease, relapse of neurological disease and remission that eventually results in a chronic progressive phase that mimics the secondary progressive phase (SPEAE) of MS. In both MS and SPEAE, the role of microglia is poorly defined. We used a crEAE model to characterize microglia in the different phases of crEAE phases using morphometric and RNA sequencing analyses. At the initial, acute inflammation phase, microglia acquired a pro-inflammatory phenotype. At the remission phase, expression of standard immune activation genes was decreased while expression of genes associated with lipid metabolism and tissue remodeling were increased. Chronic phase microglia partially regain inflammatory gene sets and increase expression of genes associated with proliferation. Together, the data presented here indicate that microglia obtain different features at different stages of crEAE and a particularly mixed phenotype in the chronic stage. Understanding the properties of microglia that are present at the chronic phase of EAE will help to understand the role of microglia in secondary progressive MS, to better aid the development of therapies for this phase of the disease.

Big conductance calcium-activated potassium channel openers control spasticity without sedation.

BACKGROUND AND PURPOSE: Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti-metabolite approach to identify drugs that target spasticity. EXPERIMENTAL APPROACH: Following the initial chemistry, a variety of biochemical, pharmacological and electrophysiological approaches, using isolated cells, tissue-based assays and in vivo animal models, were used to demonstrate the activity, efficacy, pharmacokinetics and mechanism of action of VSN16R. Toxicological and safety studies were performed in animals and humans. KEY RESULTS: VSN16R had nanomolar activity in tissue-based, functional assays and dose-dependently inhibited spasticity in a mouse experimental encephalomyelitis model of MS. This effect occurred with over 1000-fold therapeutic window, without affecting normal muscle tone. Efficacy was achieved at plasma levels that are feasible and safe in humans. VSN16R did not bind to known CB1 /CB2 /GPPR55 cannabinoid-related receptors in receptor-based assays but acted on a vascular cannabinoid target. This was identified as the major neuronal form of the big conductance, calcium-activated potassium (BKCa ) channel. Drug-induced opening of neuronal BKCa channels induced membrane hyperpolarization, limiting excessive neural-excitability and controlling spasticity. CONCLUSIONS AND IMPLICATIONS: We identified the neuronal form of the BKCa channel as the target for VSN16R and demonstrated that its activation alleviates neuronal excitability and spasticity in an experimental model of MS, revealing a novel mechanism to control spasticity. VSN16R is a potential, safe and selective ligand for controlling neural hyper-excitability in spasticity.

Imidazol-1-ylethylindazole voltage-gated sodium channel ligands are neuroprotective during optic neuritis in a mouse model of multiple sclerosis.

A series of imidazol-1-ylethylindazole sodium channel ligands were developed and optimized for sodium channel inhibition and in vitro neuroprotective activity. The molecules exhibited displacement of a radiolabeled sodium channel ligand and selectivity for blockade of the inactivated state of cloned neuronal Nav channels. Metabolically stable analogue 6 was able to protect retinal ganglion cells during optic neuritis in a mouse model of multiple sclerosis.

Control of spasticity in a multiple sclerosis model using central nervous system-excluded CB1 cannabinoid receptor agonists.

The purpose of this study was the generation of central nervous system (CNS)-excluded cannabinoid receptor agonists to test the hypothesis that inhibition of spasticity, due to CNS autoimmunity, could be controlled by affecting neurotransmission within the periphery. Procedures included identification of chemicals and modeling to predict the mode of exclusion; induction and control of spasticity in the ABH mouse model of multiple sclerosis; conditional deletion of CB1 receptor in peripheral nerves; side-effect profiling to demonstrate the mechanism of CNS-exclusion via drug pumps; genome-wide association study in N2(129×ABH) backcross to map polymorphic cannabinoid drug pump; and sequencing and detection of cannabinoid drug-pump activity in human brain endothelial cell lines. Three drugs (CT3, SAB378 and SAD448) were identified that control spasticity via action on the peripheral nerve CB1 receptor. These were peripherally restricted via drug pumps that limit the CNS side effects (hypothermia) of cannabinoids to increase the therapeutic window. A cannabinoid drug pump is polymorphic and functionally lacking in many laboratory (C57BL/6, 129, CD-1) mice used for transgenesis, pharmacology, and toxicology studies. This phenotype was mapped and controlled by 1-3 genetic loci. ABCC1 within a cluster showing linkage is a cannabinoid CNS-drug pump. Global and conditional CB1 receptor-knockout mice were used as controls. In summary, CNS-excluded CB1 receptor agonists are a novel class of therapeutic agent for spasticity.

Lesional-targeting of neuroprotection to the inflammatory penumbra in experimental multiple sclerosis.

Progressive multiple sclerosis is associated with metabolic failure of the axon and excitotoxicity that leads to chronic neurodegeneration. Global sodium-channel blockade causes side effects that can limit its use for neuroprotection in multiple sclerosis. Through selective targeting of drugs to lesions we aimed to improve the potential therapeutic window for treatment. This was assessed in the relapsing-progressive experimental autoimmune encephalomyelitis ABH mouse model of multiple sclerosis using conventional sodium channel blockers and a novel central nervous system-excluded sodium channel blocker (CFM6104) that was synthesized with properties that selectively target the inflammatory penumbra in experimental autoimmune encephalomyelitis lesions. Carbamazepine and oxcarbazepine were not immunosuppressive in lymphocyte-driven autoimmunity, but slowed the accumulation of disability in experimental autoimmune encephalomyelitis when administered during periods of the inflammatory penumbra after active lesion formation, and was shown to limit the development of neurodegeneration during optic neuritis in myelin-specific T cell receptor transgenic mice. CFM6104 was shown to be a state-selective, sodium channel blocker and a fluorescent p-glycoprotein substrate that was traceable. This compound was >90% excluded from the central nervous system in normal mice, but entered the central nervous system during the inflammatory phase in experimental autoimmune encephalomyelitis mice. This occurs after the focal and selective downregulation of endothelial p-glycoprotein at the blood-brain barrier that occurs in both experimental autoimmune encephalomyelitis and multiple sclerosis lesions. CFM6104 significantly slowed down the accumulation of disability and nerve loss in experimental autoimmune encephalomyelitis. Therapeutic-targeting of drugs to lesions may reduce the potential side effect profile of neuroprotective agents that can influence neurotransmission. This class of agents inhibit microglial activity and neural sodium loading, which are both thought to contribute to progressive neurodegeneration in multiple sclerosis and possibly other neurodegenerative diseases.

Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-ß.

OBJECTIVES: Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-ß with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes. METHODS: Mature cytokines cloned downstream of LAP and a MMP cleavage site were expressed in 293T cells and assessed for latency and biological activity by Western blotting and bioassay. RESULTS: We demonstrate here that fusion proteins of TGF-ß, erythropoietin, IL-1ra, IL-10, IL-4, BMP-7, IGF1 and IL-17 were rendered latent by fusion to LAP, requiring cleavage to become active in respective bioassays. As further proof of principle, we also show that delivery of engineered TGF-ß can inhibit experimental autoimmune encephalomyelitis and that this approach can be used to efficiently deliver cytokines to the brain and spinal cord in mice with this disease. CONCLUSIONS: The latent cytokine approach can be successfully applied to a range of molecules, including cytokines of different molecular structure and mass, growth factors and a cytokine antagonist.

Neurodegeneration progresses despite complete elimination of clinical relapses in a mouse model of multiple sclerosis.

BACKGROUND: [corrected] Multiple Sclerosis has two clinical phases reflecting distinct but inter-related pathological processes: focal inflammation drives the relapse-remitting stage and neurodegeneration represents the principal substrate of secondary progression. In contrast to the increasing number of effective anti-inflammatory disease modifying treatments for relapse-remitting disease, the absence of therapies for progressive disease represents a major unmet clinical need. This raises the unanswered question of whether elimination of clinical relapses will prevent subsequent progression and if so how early in the disease course should treatment be initiated. Experimental autoimmune encephalomyelitis in the Biozzi ABH mouse recapitulates the clinical and pathological features of multiple sclerosis including relapse-remitting episodes with inflammatory mediated demyelination and progressive disability with neurodegeneration. To address the relationship between inflammation and neurodegeneration we used an auto-immune tolerance strategy to eliminate clinical relapses in EAE in a manner analogous to the clinical effect of disease modifying treatments. RESULTS: By arresting clinical relapses in EAE at two distinct stages, early and late disease, we demonstrate that halting immune driven demyelination even after the first major clinical event is insufficient to prevent long-term neurodegeneration and associated gliosis. Nonetheless, early intervention is partially neuroprotective, whereas later interventions are not. Furthermore early tolerisation is also associated with increased remyelination. CONCLUSIONS: These findings are consistent with both a partial uncoupling of inflammation and neurodegeneration and that the regenerative response of remyelination is negatively correlated with inflammation. These findings strongly support the need for early combinatorial treatment of immunomodulatory therapies and neuroprotective treatments to prevent long-term neurodegeneration in multiple sclerosis.

Practical guide to the induction of relapsing progressive experimental autoimmune encephalomyelitis in the Biozzi ABH mouse.

Biozzi ABH mice develop a reproducible, relapsing-remitting form of experimental autoimmune encephalomyelitis (EAE) that becomes secondary progressive with disease duration. The relapses observed are T-cell dependent and can be inhibited by immune tolerance induction. In contrast the progressive neurodegeneration is T cell-independent and continues despite the re-induction of immune tolerance. Here we present a practical guide to EAE induction in the ABH mouse and approaches used to control relapses such that both autoimmune-independent and autoimmune-dependent mechanisms of neurodegeneration can be explored. Disease-related weight changes are associated with blood-brain barrier dysfunction and clinical disease. A new method for detecting neurodegeneration is described along with new experimental details that will aid in the undertaking of studies in EAE in mice, with particularly emphasis on ABH mice.

Immunosuppression with FTY720 is insufficient to prevent secondary progressive neurodegeneration in experimental autoimmune encephalomyelitis.

BACKGROUND: There has been poor translation for the use of immunosuppressive agents from experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), into the treatment of MS. This may be due to the fact that most EAE studies examine prophylactic, pre-treatment regimes that prove to be therapeutically-ineffective in long-established, often progressive, MS. FTY720 (fingolimod/Gilenya) is a sphingosine-1-phosphate receptor modulator. This is a new oral agent that markedly reduces the number of relapses in people with MS, compared with currently licensed injectable agents such as the beta interferons. FTY720 has activity against lymphocytes but may also influence oligodendroglia and could therefore have the potential to influence progressive MS, by promoting remyelination. METHODS: The effect of FTY720 was assessed in relapsing-progressive EAE in mice. RESULTS: Early intervention during relapsing EAE could completely inhibit subsequent relapses, inhibited the accumulation of neurodegeneration, and facilitated motor recovery. However, when examined in secondary progressive EAE, that develops after the accumulation of deficit from relapsing disease, long-term treatment with FTY720 failed to slow deterioration when initiated late (4 months) into the disease course. CONCLUSIONS: This study indicates that early intervention with immunosuppressive agents may inhibit the generation of the neurodegenerative microenvironment, which is no longer responsive to potent immunosuppression. However, if treatment is initiated too late, progressive, neurological-disease continues unabated. This suggests that immunosuppression is insufficient to control secondary progression in animals, as has been found so far to be the case in MS, and may warrant early intervention with FTY720 for optimal treatment benefit.

Patterns of expiratory and inspiratory activation for thoracic motoneurones in the anaesthetized and the decerebrate rat.

The nervous control of expiratory muscles is less well understood than that of the inspiratory muscles, particularly in the rat. The patterns of respiratory discharges in adult rats were therefore investigated for the muscles of the caudal intercostal spaces, with hypercapnia and under either anaesthesia or decerebration. With neuromuscular blockade and artificial ventilation, efferent discharges were present for both inspiration and expiration in both external and internal intercostal nerves. This was also the case for proximal internal intercostal nerve branches that innervate only internal intercostal and subcostalis muscles. If active, this region of muscle in other species is always expiratory. Here, inspiratory bursts were almost always present. The expiratory activity appeared only gradually and intermittently, when the anaesthesia was allowed to lighten or as the pre-decerebration anaesthesia wore off. The intermittent appearance is interpreted as the coupling of a slow medullary expiratory oscillator with a faster inspiratory one. The patterns of nerve discharges, in particular the inspiratory or biphasic activation of the internal and subcostalis layers, were confirmed by observations of equivalent patterns of EMG discharges in spontaneously breathing preparations, using denervation procedures to identify which muscles generated the signals. Some motor units were recruited in both inspiratory and expiratory bursts. These patterns of activity have not previously been described and have implications both for the functional role of multiple respiratory oscillators in the adult and for the mechanical actions of the muscles of the caudal intercostal spaces, including subcostalis, which is a partly bisegmental muscle.

Electrophysiological actions of the rubrospinal tract in the anaesthetised rat.

The rubrospinal tract (RST) of the rat is widely used in studies of regeneration and plasticity, but the electrophysiology of its spinal actions has not been described. In anaesthetised rats with neuromuscular blockade, a tungsten microelectrode was located in the region of the red nucleus (RN) by combining stereotaxis with recording of antidromic potentials evoked from the contralateral spinal cord. Single stimuli through this electrode typically elicited two descending volleys in the contralateral dorsolateral funiculus (DLF) separated by about 1 ms, and one volley recorded from the ipsilateral DLF. Latencies of the ipsilateral and the early contralateral volley were similar. The activation of these volleys depended on the location of the stimulation site in or near the RN. Evidence is adduced to show that: (a) the late contralateral volley is carried by fibres of RST neurones, synaptically activated; (b) the early contralateral volley is mostly carried by RST fibres stimulated directly; (c) the ipsilateral volley is sometimes carried by RST fibres from the RN on the side contralateral to the stimulus; (d) otherwise, either early volley may derive from fibres in other tracts. Synaptic potentials related to the volleys were recorded within the cervical enlargement and their distribution plotted on cross-sections of the spinal cord. These measurements suggest that the great majority of RST terminations are on interneurones in the intermediate region contralateral to the RN. Direct synaptic actions on motoneurones are likely to be weak. Stimulation parameters appropriate for specific activation of the RST in future studies are suggested.