Genevieve Gowing

Bone Marrow-Derived Microglia Play a Critical Role in Restricting Senile Plaque Formation in Alzheimer's Disease

Microglia are the immune cells of the brain. Here we show a massive infiltration of highly ramified and elongated microglia within the core of amyloid plaques in transgenic mouse models of Alzheimers disease (AD). Many of these cells originate from the bone marrow, and the beta-amyloid-40 and -42 isoforms are able to trigger this chemoattraction. These newly recruited cells also exhibit a specific immune reaction to both exogenous and endogenous beta-amyloid in the brain. Creation of a new AD transgenic mouse that expresses the thymidine kinase protein under the control of the CD11b promoter allowed us to show that blood-derived microglia and not their resident counterparts have the ability to eliminate amyloid deposits by a cell-specific phagocytic mechanism. These bone marrow-derived microglia are thus very efficient in restricting amyloid deposits. Therapeutic strategies aiming to improve their recruitment could potentially lead to a new powerful tool for the elimination of toxic senile plaques.

Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain.

Here we report in vivo evidence of a neuroprotective role of proliferating microglial cells in cerebral ischemia. Using transgenic mice expressing a mutant thymidine kinase form of herpes simplex virus driven by myeloid-specific CD11b promoter and ganciclovir treatment as a tool, we selectively ablated proliferating (Mac-2 positive) microglia after transient middle cerebral artery occlusion. The series of experiments using green fluorescent protein-chimeric mice demonstrated that within the first 72 h after ischemic injury, the Mac-2 marker [unlike Iba1 (ionized calcium-binding adapter molecule 1)] was preferentially expressed by the resident microglia. Selective ablation of proliferating resident microglia was associated with a marked alteration in the temporal dynamics of proinflammatory cytokine expression, a significant increase in the size of infarction associated with a 2.7-fold increase in the number of apoptotic cells, predominantly neurons, and a 1.8-fold decrease in the levels of IGF-1. A double-immunofluorescence analysis revealed a ∼100% colocalization between IGF-1 positive cells and Mac-2, a marker of activated/proliferating resident microglia. Conversely, stimulation of microglial proliferation after cerebral ischemia by M-CSF (macrophage colony stimulating factor) resulted in a 1.9-fold increase in IGF-1 levels and a significant increase of Mac2+cells. Our findings suggest that a postischemic proliferation of the resident microglial cells may serve as an important modulator of a brain inflammatory response. More importantly, our results revealed a marked neuroprotective potential of proliferating microglia serving as an endogenous pool of neurotrophic molecules such as IGF-1, which may open new therapeutic avenues in the treatment of stroke and other neurological disorders.

Exacerbation of Motor Neuron Disease by Chronic Stimulation of Innate Immunity in a Mouse Model of Amyotrophic Lateral Sclerosis

Innate immunity is a specific and organized immunological program engaged by peripheral organs and the CNS to maintain homeostasis after stress and injury. In neurodegenerative disorders, its putative deregulation, featured by inflammation and activation of glial cells resulting from inherited mutations or viral/bacterial infections, likely contributes to neuronal death. However, it remains unclear to what extent environmental factors and innate immunity cooperate to modulate the interactions between the neuronal and non-neuronal elements in the perturbed CNS. In the present study, we addressed the effects of acute and chronic administration of lipopolysaccharide (LPS), a Gram-negative bacterial wall component, in a genetic model of neurodegeneration. Transgenic mice expressing a mutant form of the superoxide dismutase 1 (SOD1G37R) linked to familial amyotrophic lateral sclerosis were challenged intraperitoneally with a single nontoxic or repeated injections of LPS (1 mg/kg). At different ages, SOD1G37R mice responded normally to acute endotoxemia. Remarkably, only a chronic challenge with LPS in presymptomatic 6-month-old SOD1G37R mice exacerbated disease progression by 3 weeks and motor axon degeneration. Closely associated with the severity of disease is the stronger and restricted upregulation of the receptor of innate immunity Toll-like receptor 2 and proinflammatory cytokines in degenerating regions of the ventral spinal cord and efferent fiber tracts of the brain from the LPS-treated SOD1G37R mice. This robust immune response was not accompanied by the establishment of acquired immunity. Our results provide solid evidence that environmental factors and innate immunity can cooperate to influence the course of disease of an inherited neuropathology.

Requirement of myeloid cells for axon regeneration.

The role of CD11b+ myeloid cells in axonal regeneration was assessed using axonal injury models and CD11b-TKmt-30 mice expressing a mutated HSV-1 thymidine kinase (TK) gene regulated by the myeloid-specific CD11b promoter. Continuous delivery of ganciclovir at a sciatic nerve lesion site greatly decreased the number of granulocytes/inflammatory monocytes and macrophages in the distal stump of CD11b-TKmt-30 mice. Axonal regeneration and locomotor function recovery were severely compromised in ganciclovir-treated CD11b-TKmt-30 mice. This was caused by an unsuitable growth environment rather than an altered regeneration capacity of neurons. In absence of CD11b+ cells, the clearance of inhibitory myelin debris was prevented, neurotrophin synthesis was abolished, and blood vessel formation/maintenance was severely compromised in the sciatic nerve distal stump. Spinal cord-injured axons also failed to regenerate through peripheral nerve grafts in the absence of CD11b+ cells. Therefore, myeloid cells support axonal regeneration and functional recovery by creating a growth-permissive milieu for injured axons.

Ablation of Proliferating Microglia Does Not Affect Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis Caused by Mutant Superoxide Dismutase

Microglial activation is a hallmark of all neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here, a detailed characterization of the microglial cell population within the spinal cord of a mouse model of familial ALS was performed. Using flow cytometry, we detected three distinct microglial populations within the spinal cord of mice overexpressing mutant superoxide dismutase (SOD1): mature microglial cells (CD11b+, CD45low), myeloid precursor cells (CD11b+, CD45int), and macrophages (CD11b+, CD45high). Characterization of cell proliferation within the CNS of SOD1G93A mice revealed that the expansion in microglial cell population is mainly attributable to the proliferation of myeloid precursor cells. To assess the contribution of proliferating microglia in motor neuron degeneration, we generated CD11b-TKmut-30; SOD1G93A doubly transgenic mice that allow the elimination of proliferating microglia on administration of ganciclovir. Surprisingly, a 50% reduction in reactive microglia specifically in the lumbar spinal cord of CD11b-TKmut-30; SOD1G93A doubly transgenic mice had no effect on motor neuron degeneration. This suggests that proliferating microglia-expressing mutant SOD1 are not central contributors of the neurodegenerative process in ALS caused by mutant SOD1.

The past, present and future of stem cell clinical trials for ALS

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that is characterized by progressive degeneration of motor neurons in the cortex, brainstem and spinal cord. This leads to paralysis, respiratory insufficiency and death within an average of 3 to 5 years from disease onset. While the genetics of ALS are becoming more understood in familial cases, the mechanisms underlying disease pathology remain unclear and there are no effective treatment options. Without understanding what causes ALS it is difficult to design treatments. However, in recent years stem cell transplantation has emerged as a potential new therapy for ALS patients. While motor neuron replacement remains a focus of some studies trying to treat ALS with stem cells, there is more rationale for using stem cells as support cells for dying motor neurons as they are already connected to the muscle. This could be through reducing inflammation, releasing growth factors, and other potential less understood mechanisms. Prior to moving into patients, stringent pre-clinical studies are required that have at least some rationale and efficacy in animal models and good safety profiles. However, given our poor understanding of what causes ALS and whether stem cells may ameliorate symptoms, there should be a push to determine cell safety in pre-clinical models and then a quick translation to the clinic where patient trials will show if there is any efficacy. Here, we provide a critical review of current clinical trials using either mesenchymal or neural stem cells to treat ALS patients. Pre-clinical data leading to these trials, as well as those in development are also evaluated in terms of mechanisms of action, validity of conclusions and rationale for advancing stem cell treatment strategies for this devastating disorder.

Absence of tumor necrosis factor-α does not affect motor neuron disease caused by superoxide dismutase 1 mutations

An increase in the expression of the proinflammatory cytokine tumor necrosis factor α (TNF-α) has been observed in patients with amyotrophic lateral sclerosis (ALS) and in the mice models of the disease. TNF-α is a potent activator of macrophages and microglia and, under certain conditions, can induce or exacerbate neuronal cell death. Here, we assessed the contribution of TNF-α in motor neuron disease in mice overexpressing mutant superoxide dismutase 1 (SOD1) genes linked to familial ALS. This was accomplished by the generation of mice expressing SOD1G37R or SOD1G93A mutants in the context of TNF-α gene knock out. Surprisingly, the absence of TNF-α did not affect the lifespan or the extent of motor neuron loss in SOD1 transgenic mice. These results provide compelling evidence indicating that TNF-α does not directly contribute to motor neuron degeneration caused by SOD1 mutations.

Increased Glioma Growth in Mice Depleted of Macrophages

Macrophages can promote the growth of some tumors, such as those of the breast and lung, but it is unknown whether this is true for all tumors, including those of the nervous system. On the contrary, we have previously shown that macrophages can slow the progression of malignant gliomas through a tumor necrosis factor-dependent mechanism. Here, we provide evidence suggesting that this antitumor effect could be mediated by T lymphocytes, as their number was drastically reduced in tumor necrosis factor-deficient mice and inversely correlated with glioma volume. However, this correlation was only observed in allogeneic recipients, prompting a reevaluation of the role of macrophages in a nonimmunogenic context. Using syngeneic mice expressing the herpes simplex virus thymidine kinase under the control of the CD11b promoter, we show that macrophages can exert an antitumor effect without the help of T lymphocytes. Macrophage depletion achieved by ganciclovir treatment resulted in a 33% increase in glioma volume. The antitumor effect of macrophages was not likely due to a tumoricidal activity because phagocytosis or apoptosis of glioma cells, transduced ex vivo with a lentiviral vector expressing green fluorescent protein, was rarely observed. Their antitumor effect was also not due to a destructive action on the tumor vasculature because macrophage depletion resulted in a modest reduction in vascular density. Therefore, this study suggests that macrophages can attenuate glioma growth by an unconventional mechanism. This study also validates a new transgenic model to explore the role of macrophages in cancer.

Synergistic Effects of GDNF and VEGF on Lifespan and Disease Progression in a Familial ALS Rat Model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle.

Intermittent Hypoxia and Stem Cell Implants Preserve Breathing Capacity in a Rodent Model of Amyotrophic Lateral Sclerosis

RATIONALE Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. OBJECTIVES To preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end stage. METHODS SOD1(G93A) rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. MEASUREMENTS AND MAIN RESULTS The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. CONCLUSIONS AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.