The Neuroinflammatory Response in ALS: The Roles of Microglia and T Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by upper and lower motoneuron death. Mutations in the gene for superoxide dismutase 1 (SOD1) cause a familial form of ALS and have been used to develop transgenic mice which overexpress human mutant SOD1 (mSOD) and these mice exhibit a motoneuron disease which is pathologically and phenotypically similar to ALS. Neuroinflammation is a pathological hallmark of many neurodegenerative diseases including ALS and is typified by the activation and proliferation of microglia and the infiltration of T cells into the brain and spinal cord. Although the neuroinflammatory response has been considered a consequence of neuronal dysfunction and death, evidence indicates that manipulation of this response can alter disease progression. Previously viewed as deleterious to neuronal survival, recent reports suggest a trophic role for activated microglia in the mSOD mouse during the early stages of disease that is dependent on instructive signals from infiltrating T cells. However, at advanced stages of disease, activated microglia acquire increased neurotoxic potential, warranting further investigation into factors capable of skewing microglial activation towards a neurotrophic phenotype as a means of therapeutic intervention in ALS

Myelosuppressive Conditioning Using Busulfan Enables Bone Marrow Cell Accumulation in the Spinal Cord of a Mouse Model of Amyotrophic Lateral Sclerosis

Myeloablative preconditioning using irradiation is the most commonly used technique to generate rodents having chimeric bone marrow, employed for the study of bone marrow-derived cell accumulation in the healthy and diseased central nervous system. However, irradiation has been shown to alter the blood-brain barrier, potentially creating confounding artefacts. To better study the potential of bone marrow-derived cells to function as treatment vehicles for neurodegenerative diseases alternative preconditioning regimens must be developed. We treated transgenic mice that over-express human mutant superoxide dismutase 1, a model of amyotrophic lateral sclerosis, with busulfan to determine whether this commonly used chemotherapeutic leads to stable chimerism and promotes the entry of bone marrow-derived cells into spinal cord. Intraperitoneal treatment with busulfan at 60 mg/kg or 80 mg/kg followed by intravenous injection of green fluorescent protein-expressing bone marrow resulted in sustained levels of chimerism (~80%). Bone marrow-derived cells accumulated in the lumbar spinal cord of diseased mice at advanced stages of pathology at both doses, with limited numbers of bone marrow derived cells observed in the spinal cords of similarly treated, age-matched controls; the majority of bone marrow-derived cells in spinal cord immunolabelled for macrophage antigens. Comparatively, significantly greater numbers of bone marrow-derived cells were observed in lumbar spinal cord following irradiative myeloablation. These results demonstrate bone marrow-derived cell accumulation in diseased spinal cord is possible without irradiative preconditioning

Levetiracetam versus phenytoin for second-line treatment of paediatric convulsive status epilepticus (EcLiPSE): a multicentre, open-label, randomised trial

Background Phenytoin is the recommended second-line intravenous anticonvulsant for treatment of paediatric convulsive status epilepticus in the UK; however, some evidence suggests that levetiracetam could be an effective and safer alternative. This trial compared the efficacy and safety of phenytoin and levetiracetam for second-line management of paediatric convulsive status epilepticus.Methods This open-label, randomised clinical trial was undertaken at 30 UK emergency departments at secondary and tertiary care centres. Participants aged 6 months to under 18 years, with convulsive status epilepticus requiring second-line treatment, were randomly assigned (1:1) using a computer-generated randomisation schedule to receive levetiracetam (40 mg/kg over 5 min) or phenytoin (20 mg/kg over at least 20 min), stratified by centre. The primary outcome was time from randomisation to cessation of convulsive status epilepticus, analysed in the modified intention-to-treat population (excluding those who did not require second-line treatment after randomisation and those who did not provide consent). This trial is registered with ISRCTN, number ISRCTN22567894.Findings Between July 17, 2015, and April 7, 2018, 1432 patients were assessed for eligibility. After exclusion of ineligible patients, 404 patients were randomly assigned. After exclusion of those who did not require second-line treatment and those who did not consent, 286 randomised participants were treated and had available data: 152 allocated to levetiracetam, and 134 to phenytoin. Convulsive status epilepticus was terminated in 106 (70%) children in the levetiracetam group and in 86 (64%) in the phenytoin group. Median time from randomisation to cessation of convulsive status epilepticus was 35 min (IQR 20 to not assessable) in the levetiracetam group and 45 min (24 to not assessable) in the phenytoin group (hazard ratio 1·20, 95% CI 0·91–1·60; p=0·20). One participant who received levetiracetam followed by phenytoin died as a result of catastrophic cerebral oedema unrelated to either treatment. One participant who received phenytoin had serious adverse reactions related to study treatment (hypotension considered to be immediately life-threatening [a serious adverse reaction] and increased focal seizures and decreased consciousness considered to be medically significant [a suspected unexpected serious adverse reaction]). Interpretation Although levetiracetam was not significantly superior to phenytoin, the results, together with previously reported safety profiles and comparative ease of administration of levetiracetam, suggest it could be an appropriate alternative to phenytoin as the first-choice, second-line anticonvulsant in the treatment of paediatric convulsive status epilepticus

Investigations into the accumulation of hematopoietic cells in the spinal cord in a murine model of motor neuron disease

Transgenic mice over-expressing human mutant superoxide dismutase 1 (mSOD) develop motoneuron loss resembling amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Bone marrow (BM) chimeric mice created by myeloablating and transplanting mice with green fluorescent protein (GFP)-labelled BM were used to study the recruitment of BM-derived cells (BMDCs) into spinal cords of mSOD and control mice. Accumulation of GFP+ cells in mSOD spinal cord paralleled disease progression and significantly greater numbers of GFP+ cells were observed in mSOD spinal cord at symptomatic and disease end-stages compared to controls. GFP+ BMDCs expressed the macrophage markers CD11b and F4/80, which are also expressed by microglia. GFP+ BDMCs constituted 10-20% of total CD11b/F4/80 + cells within spinal cord, indicating expansion of microglia within mSOD spinal cord is primarily through proliferation of resident microglia. Analysis of morphology and proximity of BMDCs to blood vessels revealed that only a fraction of BMDCs acquire the stellate morphology and Iba1+ immunophenotype characteristic of parenchymal microglia, and the majority of BMDCs remained in close proximity to blood vessels. Mice transplanted with BM from donors expressing GFP in only CX3CR1+ cells demonstrated that this population of cells accumulates within control and mSOD spinal cord. To determine whether myelosuppressive regimens alternative to irradiation potentiate BMDC accumulation in mSOD and control spinal cord, mice were treated with the chemotherapeutic Busulfex (BU) and transplanted with GFP+ BM. GFP+ cells were observed in spinal cords of mSOD and control mice. Cytotoxic T-cells were also observed in control and mSOD spinal cord, suggesting the dose of BU used in this study has neurotoxic and neuroinflammatory effects. The differential accumulation of CX3CR1+ BM cells and cells derived from definitive hematopoiesis was analyzed by transplanting irradiated mSOD and control mice with the CX3CR1+/GFP fraction of BM cells along with red fluorescent protein (RFP) c-Kit+Lin-Sca1+ cells. Analysis of spinal cord at disease end-stage revealed CX3CR1+/GFP and RFP+ cells in the spinal cords of mSOD and controls, indicating that circulating cells from the CX3CR1+/GFP BM fraction and cells derived from definitive haematopoiesis are accumulate in spinal cord

The neuroinflammatory response in ALS: the roles of microglia and T cells. Neurol Res Int 2012:803701

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by upper and lower motoneuron death. Mutations in the gene for superoxide dismutase 1 (SOD1) cause a familial form of ALS and have been used to develop transgenic mice which overexpress human mutant SOD1 (mSOD) and these mice exhibit a motoneuron disease which is pathologically and phenotypically similar to ALS. Neuroinflammation is a pathological hallmark of many neurodegenerative diseases including ALS and is typified by the activation and proliferation of microglia and the infiltration of T cells into the brain and spinal cord. Although the neuroinflammatory response has been considered a consequence of neuronal dysfunction and death, evidence indicates that manipulation of this response can alter disease progression. Previously viewed as deleterious to neuronal survival, recent reports suggest a trophic role for activated microglia in the mSOD mouse during the early stages of disease that is dependent on instructive signals from infiltrating T cells. However, at advanced stages of disease, activated microglia acquire increased neurotoxic potential, warranting further investigation into factors capable of skewing microglial activation towards a neurotrophic phenotype as a means of therapeutic intervention in ALS

The role of microglia in human disease: therapeutic tool or target?

Microglia have long been the focus of much attention due to their strong proliferative response (microgliosis) to essentially any kind of damage to the CNS. More recently, we reached the realization that these cells play specific roles in determining progression and outcomes of essentially all CNS disease. Thus, microglia has ceased to be viewed as an accessory to underlying pathologies and has now taken center stage as a therapeutic target. Here, we review how our understanding of microglia’s involvement in promoting or limiting the pathogenesis of diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, multiple sclerosis, X-linked adrenoleukodystrophy (X-ALD) and lysosomal storage diseases (LSD) has changed over time. While strategies to suppress the deleterious and promote the virtuous functions of microglia will undoubtedly be forthcoming, replacement of these cells has already proven its usefulness in a clinical setting. Over the past few years, we have reached the realization that microglia have a developmental origin that is distinct from that of bone marrow-derived myelomonocytic cells. Nevertheless, microglia can be replaced, in specific situations, by the progeny of hematopoietic stem cells (HSCs), pointing to a strategy to engineer the CNS environment through the transplantation of modified HSCs. Thus, microglia replacement has been successfully exploited to deliver therapeutics to the CNS in human diseases such as X-ALD and LSD. With this outlook in mind, we will discuss the evidence existing so far for microglial involvement in the pathogenesis and the therapy of specific CNS disease

Short-term analysis of PBC reconstitution by donor cells using doses of 60, 80 or 100 mg/kg BU and GFP+ accumulation in control spinal cord.

<p>(<b>A</b>) Left: FACS plot of negative control (blue peak) and GFP+ control (red peak) blood. Centre: An example of a FACS plot of blood collected at 3 weeks post-BM transplant following treatment with 100 mg/kg BU. Roughly 48% of PBCs were GFP+. Right: Immunolabeling of blood for myeloid markers (CD11b-APC, Gr1-APC) indicate high level of donor chimerism in this blood cell population by 3 weeks post-transplant. (<b>B</b>) Levels of PBC and lymphoid chimerism increased over the 4-week observation period. Levels of chimerism in circulating myeloid cells increased at a rate higher than that of lymphoid cells. (<b>C</b>) By 4 weeks post-transplant, GFP+ cells were observed in the lumbar spinal cords of BM chimeric mice. Significantly greater numbers of GFP+ cells were observed in the spinal cords of mice treated with 80 or 100 mg/kg BU compared to mice treated with 60 mg/kg. Scale bar = 50 µm. (<b>D</b>) The majority of GFP+ cells in lumbar spinal cord exhibited a rod-shaped morphology and were associated with blood vessels immunolabelled with antibody to CD31. Scale bar = 50 µm.</p