Activated Protein C Battles ALS on Multiple Fronts

Activated protein C delivers a one-two-three punch to disease in a mouse model of amyotrophic lateral sclerosis (ALS), reducing microglial activation, sealing the leaky blood-spinal cord barrier, and dampening expression of the disease-linked enzyme superoxide dismutase 1 (SOD1). Researchers report in the November Journal of Clinical Investigation that APC injections extended lifespan by nearly a month in mice carrying a mutant version of human SOD1. Joint first authors Zhihui Zhong of the University of Rochester Medical Center in New York and Hristelina Ilieva of the University of California, San Diego, carried out the work in the laboratories of Berislav Zlokovic in Rochester and Don Cleveland in San Diego. APC is commonly known as an anticoagulant, but mutants unable to interact with the blood-clotting cascade were also protective, suggesting a potential therapy without the threat of bleeding as a side effect.

Multitasking Protein
ALS is only the latest in a long line of conditions where APC treatment is of interest. The reason that it is always a candidate is because, fundamentally, what it does is cut down the inflammation and protect the cells from injury, said Charles Esmon of the Oklahoma Medical Research Foundation in Oklahoma City, who wrote a commentary accompanying the ALS paper with co-author Jonathan Glass of the Emory University School of Medicine in Atlanta, Georgia. Beyond its role in regulating blood coagulation, APC’s cytoprotective effects include blocking inflammation and apoptosis, stabilizing endothelial tissue, and affecting gene expression levels (reviewed in Mosnier et al., 2007).

Scientists have explored the protein’s effects on conditions including multiple sclerosis (Han et al., 2008), inflammatory bowel disease (Scaldaferri et al., 2007), and cancer (van Sluis et al., 2009). It is already in use for the treatment of sepsis (Bernard et al., 2001) and currently under trial for stroke (Guo et al., 2004). Zlokovic was inspired to try APC for ALS when a friend died of the disease.

APC, a protease, checks the blood-clotting pathway by cleaving and inactivating factors Va and VIIIa in plasma. In its cytoprotective role, it binds the G-protein-coupled receptor PAR1, leading to downstream intracellular actions such as suppression of NF-κB and reduction of inflammation (Joyce et al., 2001). It also traverses the blood-brain barrier via interaction with endothelial protein C receptor (EPCR; Deane et al., 2009).

Slowing Disease
Zhong and colleagues divided 60 mice expressing human SOD1-G93A into five treatment groups: saline, wild-type APC, or one of three APC mutants. 3K3A-APC and 5A-APC each contain, respectively, three or five alanine substitutions in the protease domain that diminish factor Va binding without affecting the PAR1 or EPCR interactions (Mosnier et al., 2004; Mosnier et al., 2007). 3K3A-APC has less than a third of normal anticoagulant activity, and 5A-APC’s anticoagulant activity is less than ten percent of normal. Finally, S360A-APC is proteolytically inactive for both pathways, unable to bind factor Va or PAR1 (Cheng et al., 2003).

The researchers began treatment one week after the mice showed the weight loss associated with disease onset, around 77 days of age. Daily APC injections, at 40 micrograms/kilogram for WT-APC and 3K3A-APC and 100 micrograms/kilogram for 5A-APC, S360A-APC, and WT-APC, continued until death. Mice receiving a low dose of WT-APC or 3K3A-APC gained an average of 10-13 percent increase in lifespan. 5A-APC mice gained even more, with an increase in lifespan from 122 to 150 days, a 25 percent boost. S360A-APC did not affect disease course, showing that the proteolytic activity, but not anticoagulant activity, of APC is required to impact motor neuron disease.

The increased lifespan is impressive given the severity of disease in the SOD1-G93A mouse, wrote Sverine Boille of INSERM and the Brain and Spinal Cord Institute in Paris, France, in an e-mail to ARF (see full comment below). In addition, Boille noted that the researchers saw a positive effect with treatment started after symptom onset, as is likely to happen in the clinic.

Silencing SOD1
APC influences expression of a variety of genes (Riewald and Ruf, 2005). Accordingly, the researchers looked for an influence on SOD1 mRNA in SOD1-G93A animals. Levels of mRNA for both the SOD1 transgene and the endogenous gene dropped by 40 percent in spinal cord motor neurons of 5A-APC-treated mice compared with saline-treated or S360A-APC-treated animals. SOD1 protein levels in motor neurons the lumbar spinal cords of 5A-APC-treated mice were half that of saline-treated mice. SOD1 mRNA and protein levels were similarly affected by APC treatment in a neuroblastoma cell line carrying SOD1-G85R, confirming APC’s influence over SOD1 expression in neurons.

Next, the researchers delved into the pathway between APC and SOD1. PAR1 and PAR3, already known to be involved in APC cytoprotective signaling (Guo et al., 2004 34804), were natural candidates. Zhang and colleagues confirmed their involvement by adding antibodies to PAR1 and PAR3 to neuroblastoma cultures, where the antibodies blocked APC’s effect on SOD1.

PAR1 activation leads to phosphorylation of the transcription factor Sp1, preventing Sp1 from moving to the nucleus and binding DNA. To analyze the impact of APC on Sp1, the researchers used confocal microscopy to localize the Sp1 in neuroblastoma cells expressing SOD1-G85R. Quantification of Sp1 signal intensity confirmed that Sp1’s nuclear localization dropped by 60 percent compared to untreated control cells.

The results suggest a pathway whereby APC, with the assistance of EPCR, crosses the blood-brain barrier to reach cells such as neurons and microglia. It likely binds PAR3 and PAR1, which in turn leads to phosphorylation of Sp1. Phosphorylated Sp1 is barred from the nucleus, presumably preventing it from activating transcription of SOD1.

Beyond SOD1
That is a very powerful interaction in this familial mutant, Zlokovic said of the SOD1 suppression. But what of sporadic ALS, which accounts for 90 percent of cases, and the additional 8 percent of cases that are familial, but not caused by SOD1 mutations The authors point out that aberrant SOD1 has been linked to sporadic ALS, as well (Gruzman et al., 2007 and see ARF related news story). In addition, the researchers also observed evidence of APC’s general neuroprotective activities—such as blocking inflammation and stabilizing the blood-brain-barrier—in the treated mice.

Microglia have been implicated in causing ALS pathology (Boille et al., 2006), and inflammation plays a role in the disease (for review, see McGeer and McGeer, 2002). In saline-treated SOD1-G93A mice, spinal cord microglial levels increased 12-fold by four weeks after disease onset, and 20-fold three weeks later, compared to wild-type animals. APC dampened this response: low-dose injections of WT-APC or 5A-APC kept microglial populations comparable to wild-type populations at four weeks, and increased to only tenfold at seven weeks. The increase in inflammatory markers such as monocyte chemoattractant protein 1 (MCP-1) seen in SOD1-G93A mice was also reduced in APC-treated animals.

In addition, APC reversed the degeneration of the blood-spinal cord barrier seen early on in mSOD1 mice (see ARF related news story on Zhong et al., 2008; Garbuzova-Davis et al., 2007; Garbuzova-Davis et al., 2007). IgG leakage into the spinal cord, high in SOD1-G93A mice, was reduced in animals treated with APC. Similarly, Prussian blue staining for hemosiderin showed that microhemorrhaging due to the SOD1 mutation was reduced in APC-treated mice. We think we have effects on the blood-brain barrier which are completely independent of SOD1, Zlokovic said.

Potential Therapies
In summary, APC fights off motor neuron disease in SOD1-G93A mice on several fronts. It is really a molecule with myriad effects, Zlokovic said. It diminishes expression of the damaging SOD1 molecule, prevents inflammation caused by microglia, and boosts the blood-spinal cord barrier. Notably, it does it all without the domains necessary to affect coagulation, although its proteolytic activity is required.

If what is true in mice is also true in manthen that is an exciting approach to an otherwise very troublesome disease, Esmon said. The if is an important caveat; treatments successful in mice have repeatedly failed to help people with the disease (see ARF Live Discussion). In addition, Boille cautioned that downregulating SOD1, an important scavenger of reactive oxygen species throughout the body might have unwanted side effects. Nothing is known about the effect of downregulating SOD1 in humans, she wrote. Downregulating the expression of scavenging proteins might have a detrimental impact. Zlokovic is currently focusing much of his attention on the clinical trial for APC in stroke, but estimates that he might try it on ALS within four or five years.

Beyond just ALS, EPCR’s ability to shuttle APC across the blood-brain barrier may offer a tantalizing mechanism to transfer a variety of APC-carried drugs into the central nervous system, Esmon suggested. However, he noted that it is not yet known if APC crosses the blood-brain barrier in people as it does in mice, and these kinds of mechanisms often differ between species.

Zhong Z, Ilieva H, Hallagan L, Bell R, Singh I, Paquette N, Thiyagarajan M, Deane R, Fernandez JA, Lane S, Zlokovic AB, Liu T, Griffin JH, Chow N, Castellino FJ, Stojanovic K, Cleveland DW, Zlokovic BV. Activated protein C therapy slows ALS-like disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglia cells. J Clin Invest. 2009 Nov;119(11):3437-49. Abstract

Esmon CT, Glass JD. The APCs of neuroprotection. J Clin Invest. 2009 Nov;119(11):3205-7. Abstract

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disease-als SOD1 topic-preclinical
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