Zhong Z, Ilieva H, Hallagan L, et al. Activated protein C therapy slows ALS-like disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglial cells

Center for Neurodegenerative and Vascular Brain Disorders and Department of Neurological Surgery, University of Rochester Medical Center, Rochester, New York 14642, USA.
The Journal of clinical investigation (Impact Factor: 13.22). 11/2009; 119(11):3437-49. DOI: 10.1172/JCI38476
Source: PubMed


Activated protein C (APC) is a signaling protease with anticoagulant activity. Here, we have used mice expressing a mutation in superoxide dismutase-1 (SOD1) that is linked to amyotrophic lateral sclerosis (ALS) to show that administration of APC or APC analogs with reduced anticoagulant activity after disease onset slows disease progression and extends survival. A proteolytically inactive form of APC with reduced anticoagulant activity provided no benefit. APC crossed the blood-spinal cord barrier in mice via endothelial protein C receptor. When administered after disease onset, APC eliminated leakage of hemoglobin-derived products across the blood-spinal cord barrier and delayed microglial activation. In microvessels, motor neurons, and microglial cells from SOD1-mutant mice and in cultured neuronal cells, APC transcriptionally downregulated SOD1. Inhibition of SOD1 synthesis in neuronal cells by APC required protease-activated receptor-1 (PAR1) and PAR3, which inhibited nuclear transport of the Sp1 transcription factor. Diminished mutant SOD1 synthesis by selective gene excision within endothelial cells did not alter disease progression, which suggests that diminished mutant SOD1 synthesis in other cells, including motor neurons and microglia, caused the APC-mediated slowing of disease. The delayed disease progression in mice after APC administration suggests that this approach may be of benefit to patients with familial, and possibly sporadic, ALS.

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Available from: Jose A. Fernandez, Dec 26, 2013
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    • "Probably the lack of effect of h(A)PC on reducing plaque development can be explained by the in vivo bioavailability after injection. Previously it was shown that i.p. administration of 0.1 or 0.8 mg/kg mAPC produces a transient rise in APC levels in circulation for 3 hours, with a plateau after around 20 min [32], [63]. This transient rise in (A)PC concentration in circulation, that was achieved twice a week in our model, was apparently not enough to initiate protective signaling that lasted long enough to provide long term protection against atherosclerosis development during the time span of our study. "
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    ABSTRACT: Activated protein C (APC) is a serine protease that has both anticoagulant and cytoprotective properties. The cytoprotective effects are protease activated receptor 1 (PAR-1) and endothelial protein C receptor (EPCR) dependent and likely underlie protective effects of APC in animal models of sepsis, myocardial infarction and ischemic stroke. S360A-(A)PC, a variant (A)PC that has no catalytic activity, binds EPCR and shifts pro-inflammatory signaling of the thrombin-PAR-1 complex to anti-inflammatory signaling. In this study we investigated effects of human (h)wt-PC, hS360A-PC, hwt-APC and hS360A-APC in acute (mouse model of acute myocardial ischemia/reperfusion (I/R) injury) and chronic inflammation (apoE-/- mouse model of atherosclerosis). All h(A)PC variants significantly reduced myocardial infarct area (p<0.05) following I/R injury. IL-6 levels in heart homogenates did not differ significantly between sham, placebo and treatment groups in I/R injury. None of the h(A)PC variants decreased number and size of atherosclerotic plaques in apoE-/- mice. Only hS360A-APC slightly affected phenotype of plaques. IL-6 levels in plasma were significantly (p<0.001) decreased in hwt-APC and hS360A-PC treated mice. In the last group levels of monocyte chemotactic protein 1 (MCP-1) were significantly increased (p<0.05). In this study we show that both hwt and hS360A-(A)PC protect against acute myocardial I/R injury, which implies that protection from I/R injury is independent of the proteolytic activity of APC. However, in the chronic atherosclerosis model hwt and hS360-(A)PC had only minor effects. When the dose, species and mode of (A)PC administration will be adjusted, we believe that (A)PC will have potential to influence development of chronic inflammation as occurring during atherosclerosis as well.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "We have developed a proposed sequence of events that mediate disease pathology in the SOD1G93A mouse based on the results presented here (Fig. 25). However, although there is now a growing consensus in the field that the axon and synapses are the first cellular sites of degeneration, it is not known whether NMJ denervation is initiated autonomously at that site or by pathology in the cell body, in nonneuronal cells or even in non-MNs (Bettini et al. 2007; Conforti et al. 2007; Gould and Oppenheim 2007; Zhong et al. 2008, 2009; Yoshikawa et al. 2009). The specific molecular mechanisms mediating axon/synapse loss in ALS are still largely unknown (Saxena and Caroni 2007). "
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    ABSTRACT: Pathological events are well characterized in amyotrophic lateral sclerosis (ALS) mouse models, but review of the literature fails to identify a specific initiating event that precipitates disease pathology. There is now growing consensus in the field that axon and synapses are first cellular sites of degeneration, but controversy exists over whether axon and synapse loss is initiated autonomously at those sites or by pathology in the cell body, in nonneuronal cells or even in nonmotoneurons (MNs). Previous studies have identified pathological events in the mutant superoxide dismutase 1 (SOD1) models involving spinal cord, peripheral axons, neuromuscular junctions (NMJs), or muscle; however, few studies have systematically examined pathogenesis at multiple sites in the same study. We have performed ultrastructural examination of both central and peripheral components of the neuromuscular system in the SOD1(G93A) mouse model of ALS. Twenty percent of MNs undergo degeneration by P60, but NMJ innervation in fast fatigable muscles is reduced by 40% by P30. Gait alterations and muscle weakness were also found at P30. There was no change in axonal transport prior to initial NMJ denervation. Mitochondrial morphological changes are observed at P7 and become more prominent with disease progression. At P30 there was a significant decrease in excitatory axo-dendritic and axo-somatic synapses with an increase in C-type axo-somatic synapses. Our study examined early pathology in both peripheral and central neuromuscular system. The muscle denervation is associated with functional motor deficits and begins during the first postnatal month in SOD1(G93A) mice. Physiological dysfunction and pathology in the mitochondria of synapses and MN soma and dendrites occur, and disease onset in these animals begins more than 2 months earlier than originally thought. This information may be valuable for designing preclinical trials that are more likely to impact disease onset and progression.
    Full-text · Article · Jul 2013 · Brain and Behavior
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    • "In this regard, the emergence of numerous transgenic mouse models of lower motor neuron conditions provide insight into the mechanisms underlying these pathologies (Gurney et al., 1994; Wong et al., 1995, 2002; Hsieh-Li et al., 2000; Kaspar et al., 2003; Ishiyama et al., 2004; Turner et al., 2009; Wegorzewska et al., 2009; Kimura et al., 2010; Towne et al., 2010; Xu et al., 2010; Guo et al., 2011; Riboldi et al., 2011; Pratt et al., 2013). For example, the Cu/Zn superoxide dismutase type-1 (SOD-1) mouse model was developed in order to further understand the etiology and pathogenesis of a subtype of amyotrophic lateral sclerosis (Gurney et al., 1994; Wong et al., 1995; Raoul et al., 2005; Zhong et al., 2009; Towne et al., 2010; Riboldi et al., 2011). This is also the case for the survival motor neuron 1 (SMN) knockout mouse model of spinal muscular atrophy (SMA) (Hsieh-Li et al., 2000). "
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    ABSTRACT: Lower motor neuron dysfunction is one of the most debilitating motor conditions. In this regard, transgenic mouse models of various lower motor neuron dysfunctions provide insight into the mechanisms underlying these pathologies and can also aid the development of new therapies. Viral-mediated gene therapy can take advantage of the muscle-motor neuron topographical relationship to shuttle therapeutic genes into specific populations of motor neurons in these mouse models. In this context, motor end plates (MEPs) are highly specialized regions on the skeletal musculature that offer direct access to the pre-synaptic nerve terminals, henceforth to the spinal cord motor neurons. The aim of this study was two-folded. First, it was to characterize the exact position of the MEP regions for several muscles of the mouse forelimb using acetylcholinesterase histochemistry. This MEP-muscle map was then used to guide a series of intramuscular injections of Fluoro-Gold (FG) in order to characterize the distribution of the innervating motor neurons. This analysis revealed that the MEPs are typically organized in an orthogonal fashion across the muscle fibers and extends throughout the full width of each muscle. Furthermore, targeting the full length of the MEP regions gave rise labeled motor neurons that are organized into columns spanning through more spinal cord segments than previously reported. The present analysis suggests that targeting the full width of the muscles’ MEP regions with FG increases the somatic availability of the tracer. This process ensures a greater uptake of the tracer by the pre-synaptic nerve terminals, hence maximizing the labeling in spinal cord motor neurons. This investigation should have positive implications for future studies involving the somatic delivery of therapeutic genes into motor neurons for the treatment of various motor dysfunctions.
    Full-text · Article · May 2013 · Frontiers in Neurology
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