Additive amelioration of ALS by co-targeting independent pathogenic mechanisms.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which glia are central mediators of motor neuron (MN) death. Since multiple cell types are involved in disease pathogenesis, the objective of this study was to determine the benefit of co-targeting independent pathogenic mechanisms in a familial ALS mouse model. METHODS: Recently, our laboratory identified that ALS microglia induce MN death in an NF-κB-dependent mechanism. We also demonstrated that a single, post-natal, intravenous injection of adeno-associated viral vector serotype 9 encoding a shRNA against mutant SOD1 is able to traverse the blood-brain barrier of ALS mice and reduce SOD1-expression in astrocytes and MNs. Reducing mutant SOD1 in MNs and astrocytes led to a robust increase in survival. To evaluate the benefit of co-targeting multiple cell types in ALS, we combined microglial NF-κB suppression with SOD1 reduction in astrocytes and MNs. RESULTS: Targeting both astrocytes and microglia resulted in an additive increase in survival and motor function by delaying both onset and progression. Strikingly, targeting all three cell types (astrocytes, motor neurons [MNs], and microglia) resulted in an additive increase in lifespan and motor function, with maximum survival reaching 204 days, 67 days longer than the mean survival of untreated control animals. INTERPRETATION: Our data suggest that a combinatorial approach co-targeting different pathogenic mechanisms in independent cell types is a beneficial therapeutic strategy for ALS

Defective axonal transport in motor neuron disease

Several recent studies have highlighted the role of axonal transport in the pathogenesis of motor neuron diseases. Mutations in genes that control microtubule regulation and dynamics have been shown to cause motor neuron degeneration in mice and in a form of human motor neuron disease. In addition, mutations in the molecular motors dynein and kinesins and several proteins associated with the membranes of intracellular vesicles that undergo transport cause motor neuron degeneration in humans and mice. Paradoxically, evidence from studies on the legs at odd angles (Loa) mouse and a transgenic mouse model for human motor neuron disease suggest that partial limitation of the function of dynein may in fact lead to improved axonal transport in the transgenic mouse, leading to delayed disease onset and increased life span

Strategy for Treating Motor Neuron Diseases Using a Fusion Protein of Botulinum Toxin Binding Domain and Streptavidin for Viral Vector Access: Work in Progress

Although advances in understanding of the pathogenesis of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) have suggested attractive treatment strategies, delivery of agents to motor neurons embedded within the spinal cord is problematic. We have designed a strategy based on the specificity of botulinum toxin, to direct entry of viral vectors carrying candidate therapeutic genes into motor neurons. We have engineered and expressed fusion proteins consisting of the binding domain of botulinum toxin type A fused to streptavidin (SAv). This fusion protein will direct biotinylated viral vectors carrying therapeutic genes into motor nerve terminals where they can enter the acidified endosomal compartments, be released and undergo retrograde transport, to deliver the genes to motor neurons. Both ends of the fusion proteins are shown to be functionally intact. The binding domain end binds to mammalian nerve terminals at neuromuscular junctions, ganglioside GT1b (a target of botulinum toxin), and a variety of neuronal cells including primary chick embryo motor neurons, N2A neuroblastoma cells, NG108-15 cells, but not to NG CR72 cells, which lack complex gangliosides. The streptavidin end binds to biotin, and to a biotinylated Alexa 488 fluorescent tag. Further studies are in progress to evaluate the delivery of genes to motor neurons in vivo, by the use of biotinylated viral vectors

In Vivo Imaging Reveals Distinct Inflammatory Activity of CNS Microglia versus PNS Macrophages in a Mouse Model for ALS

Mutations in the enzyme superoxide dismutase-1 (SOD1) cause hereditary variants of the fatal motor neuronal disease Amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous: neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring non-neuronal cells. In vivo imaging by two-photon laser-scanning microscopy was used to compare the role of microglia/macrophage-related neuroinflammation in the CNS and PNS using ALS-linked transgenic SOD1G93A mice. These mice contained labeled projection neurons and labeled microglia/macrophages. In the affected lateral spinal cord (in contrast to non-affected dorsal columns), different phases of microglia-mediated inflammation were observed: highly reactive microglial cells in preclinical stages (in 60-day-old mice the reaction to axonal transection was ∼180% of control) and morphologically transformed microglia that have lost their function of tissue surveillance and injury-directed response in clinical stages (reaction to axonal transection was lower than 50% of control). Furthermore, unlike CNS microglia, macrophages of the PNS lack any substantial morphological reaction while preclinical degeneration of peripheral motor axons and neuromuscular junctions was observed. We present in vivo evidence for a different inflammatory activity of microglia and macrophages: an aberrant neuroinflammatory response of microglia in the CNS and an apparently mainly neurodegenerative process in the PNS

Excess Circulating Alternatively Activated Myeloid (M2) Cells Accelerate ALS Progression While Inhibiting Experimental Autoimmune Encephalomyelitis

Circulating immune cells including autoreactive T cells and monocytes have been documented as key players in maintaining, protecting and repairing the central nervous system (CNS) in health and disease. Here, we hypothesized that neurodegenerative diseases might be associated, similarly to tumors, with increased levels of circulating peripheral myeloid derived suppressor cells (MDSCs), representing a subset of suppressor cells that often expand under pathological conditions and inhibit possible recruitment of helper T cells needed for fighting off the disease.We tested this working hypothesis in amyotrophic lateral sclerosis (ALS) and its mouse model, which are characterized by a rapid progression once clinical symptoms are evident. Adaptive transfer of alternatively activated myeloid (M2) cells, which homed to the spleen and exhibited immune suppressive activity in G93A mutant superoxide dismutase-1 (mSOD1) mice at a stage before emergence of disease symptoms, resulted in earlier appearance of disease symptoms and shorter life expectancy. The same protocol mitigated the inflammation-induced disease model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), which requires circulating T cells for disease induction. Analysis of whole peripheral blood samples obtained from 28 patients suffering from sporadic ALS (sALS), revealed a two-fold increase in the percentage of circulating MDSCs (LIN(-/Low)HLA-DR(-)CD33(+)) compared to controls.Taken together, these results emphasize the distinct requirements for fighting the inflammatory neurodegenerative disease, multiple sclerosis, and the neurodegenerative disease, ALS, though both share a local inflammatory component. Moreover, the increased levels of circulating MDSCs in ALS patients indicates the operation of systemic mechanisms that might lead to an impairment of T cell reactivity needed to overcome the disease conditions within the CNS. This high level of suppressive immune cells might represent a risk factor and a novel target for therapeutic intervention in ALS at least at the early stage

Siglec-H is a microglia-specific marker that discriminates microglia from CNS-associated macrophages and CNS-infiltrating monocytes

International audienceSeveral types of myeloid cell are resident in the CNS. In the steady state, microglia are present in the CNS parenchyma, whereas macrophages reside in boundary regions of the CNS, such as perivascular spaces, the meninges and choroid plexus. In addition, monocytes infiltrate into the CNS parenchyma from circulation upon blood-brain barrier breakdown after CNS injury and inflammation. Although several markers, such as CD11b and ionized calcium-binding adapter molecule 1 (Iba1), are frequently used as microglial markers, they are also expressed by other types of myeloid cell and microglia-specific markers were not defined until recently. Previous transcriptome analyses of isolated microglia identified a transmembrane lectin, sialic acid-binding immunoglobulin-like lectin H (Siglec-H), as a molecular signature for microglia; however, this was not confirmed by histological studies in the nervous system and the reliability of Siglec-H as a microglial marker remained unclear. Here, we demonstrate that Siglec-H is an authentic marker for microglia in mice by immunohistochemistry using a Siglec-H-specific antibody. Siglec-H was expressed by parenchymal microglia from developmental stages to adulthood, and the expression was maintained in activated microglia under injury or inflammatory condition. However, Siglec-H expression was absent from CNS-associated macrophages and CNS-infiltrating monocytes, except for a minor subset of cells. We also show that the Siglech gene locus is a feasible site for specific targeting of microglia in the nervous system. In conclusion, Siglec-H is a reliable marker for microglia that will allow histological identification of microglia and microglia-specific gene manipulation in the nervous system

Adherent Monomer-Misfolded SOD1

Background: Multiple cellular functions are compromised in amyotrophic lateral sclerosis (ALS). In familial ALS (FALS) with Cu/Zn superoxide dismutase (SOD1) mutations, the mechanisms by which the mutation in SOD1 leads to such a wide range of abnormalities remains elusive. Methodology/Principal Findings: To investigate underlying cellular conditions caused by the SOD1 mutation, we explored mutant SOD1-interacting proteins in the spinal cord of symptomatic transgenic mice expressing a mutant SOD1, SOD1 Leu126delTT with a FLAG sequence (DF mice). This gene product is structurally unable to form a functional homodimer. Tissues were obtained from both DF mice and disease-free mice expressing wild-type with FLAG SOD1 (WF mice). Both FLAG-tagged SOD1 and cross-linking proteins were enriched and subjected to a shotgun proteomic analysis. We identified 34 proteins (or protein subunits) in DF preparations, while in WF preparations, interactions were detected with only 4 proteins. Conclusions/Significance: These results indicate that disease-causing mutant SOD1 likely leads to inadequate proteinprotein interactions. This could be an early and crucial process in the pathogenesis of FALS

Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive loss of motoneurons, motor weakness and death within 1–5 years after disease onset. Therapeutic options remain limited despite a substantial number of approaches that have been tested clinically. In particular, various neurotrophic factors have been investigated. Failure in these trials has been largely ascribed to problems of insufficient dosing or inability to cross the blood–brain barrier (BBB). We have recently uncovered the neurotrophic properties of the haematopoietic protein granulocyte-colony stimulating factor (G-CSF). The protein is clinically well tolerated and crosses the intact BBB. This study examined the potential role of G-CSF in motoneuron diseases. We investigated the expression of the G-CSF receptor in motoneurons and studied effects of G-CSF in a motoneuron cell line and in the SOD1(G93A) transgenic mouse model. The neurotrophic growth factor was applied both by continuous subcutaneous delivery and CNS-targeted transgenic overexpression. This study shows that given at the stage of the disease where muscle denervation is already evident, G-CSF leads to significant improvement in motor performance, delays the onset of severe motor impairment and prolongs overall survival of SOD1(G93A)tg mice. The G-CSF receptor is expressed by motoneurons and G-CSF protects cultured motoneuronal cells from apoptosis. In ALS mice, G-CSF increased survival of motoneurons and decreased muscular denervation atrophy. We conclude that G-CSF is a novel neurotrophic factor for motoneurons that is an attractive and feasible drug candidate for the treatment of ALS

Measurement of Contractile Stress Generated by Cultured Rat Muscle on Silicon Cantilevers for Toxin Detection and Muscle Performance Enhancement

Background: To date, biological components have been incorporated into MEMS devices to create cell-based sensors and assays, motors and actuators, and pumps. Bio-MEMS technologies present a unique opportunity to study fundamental biological processes at a level unrealized with previous methods. The capability to miniaturize analytical systems enables researchers to perform multiple experiments in parallel and with a high degree of control over experimental variables for high-content screening applications.Methodology/Principal Findings: We have demonstrated a biological microelectromechanical system (BioMEMS) based on silicon cantilevers and an AFM detection system for studying the physiology and kinetics of myotubes derived from embryonic rat skeletal muscle. It was shown that it is possible to interrogate and observe muscle behavior in real time, as well as selectively stimulate the contraction of myotubes with the device. Stress generation of the tissue was estimated using a modification of Stoney's equation. Calculated stress values were in excellent agreement with previously published results for cultured myotubes, but not adult skeletal muscle. Other parameters such as time to peak tension (TPT), the time to half relaxation (KRT) were compared to the literature. It was observed that the myotubes grown on the BioMEMS device, while generating stress magnitudes comparable to those previously published, exhibited slower TPT and KRT values. However, growth in an enhanced media increased these values. From these data it was concluded that the myotubes cultured on the cantilevers were of an embryonic phenotype. The system was also shown to be responsive to the application of a toxin, veratridine.Conclusions/Significance: The device demonstrated here will provide a useful foundation for studying various aspects of muscle physiology and behavior in a controlled high-throughput manner as well as be useful for biosensor and drug discovery applications

Enhanced neuronal Met signalling levels in ALS mice delay disease onset

Signalling by receptor tyrosine kinases (RTKs) coordinates basic cellular processes during development and in adulthood. Whereas aberrant RTK signalling can lead to cancer, reactivation of RTKs is often found following stress or cell damage. This has led to the common belief that RTKs can counteract degenerative processes and so strategies to exploit them for therapy have been extensively explored. An understanding of how RTK stimuli act at cellular levels is needed, however, to evaluate their mechanism of therapeutic action. In this study, we genetically explored the biological and functional significance of enhanced signalling by the Met RTK in neurons, in the context of a neurodegenerative disease. Conditional met-transgenic mice, namely Rosa26LacZ−stop−Met, have been engineered to trigger increased Met signalling in a temporal and tissue-specific regulated manner. Enhancing Met levels in neurons does not affect either motor neuron (MN) development or maintenance. In contrast, increased neuronal Met in amyotrophic lateral sclerosis (ALS) mice prolongs life span, retards MN loss, and ameliorates motor performance, by selectively delaying disease onset. Thus, our studies highlight the properties of RTKs to counteract toxic signals in a disease characterized by dysfunction of multiple cell types by acting in MNs. Moreover, they emphasize the relevance of genetically assessing the effectiveness of agents targeting neurons during ALS evolution