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 Séverine Boillée of INSERM and the Brain and Spinal Cord Institute in Paris, France, in an e-mail to ARF (see full comment below). In addition, Boillée 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 (Boillée 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 man…then 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, Boillée 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.—Amber Dance.

References:
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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Comments on News and Primary Papers

  1. This paper from Zhong et al. assesses the effect of activated protein C (APC) therapy on ALS mice expressing hSOD1G93A. The authors show that daily i.p. injection of APC increases survival in a dose-dependent manner. The survival was increased by more than 25 days, which is impressive for this rapid progressing and broadly used ALS mouse model. In addition, the treatment was started after onset (around one week after the onset defined by weight peak), making the result even more important and significant, especially when one thinks about potential therapeutic use. Indeed, since most of the ALS cases are sporadic and of unknown origin, patients are diagnosed after symptoms begin. Therefore, a drug for ALS should have the potential of slowing down the disease after onset.

    The result of the treatment is, therefore, impressive, but even more surprising is the finding of the pathway by which APC leads to slowing of disease progression. While the drug was used to determine if reducing the blood spinal cord barrier leakage (previously observed in this model by the same Zlokovic and Cleveland group (see Zhong et al., 2008) would be of benefit, the authors found that the beneficial effect of APC was linked to the downregulation of SOD1. However, they elegantly showed, by using a Cre/Lox approach, that downregulating SOD1 expression in the endothelial cells had no effect on the disease. The effect most likely came from downregulation of SOD1 in motor neurons and microglial cells (and probably astrocytes, not tested), as they showed by using laser microdissection and cell isolation.

    APC slowed the disease progression, including motor neuron denervation and microglial activation. It would also be interesting to know if at the same disease stage microglial cells are less activated after APC treatment, leading to downregulation of mutant SOD1 in microglia and motor neurons.

    These results make APC a potential candidate for therapies, but caution should be paid to potential peripheral effects. Indeed, in transgenic mice overexpressing human SOD1, APC downregulated both the expression of the human transgene and the endogenous mouse protein. However, nothing is known about the effect of downregulating SOD1 in humans. Since SOD1 acts as a radical oxygen species (ROS) scavenger, one could argue that in any neurodegenerative disease when ROS get produced, downregulating the expression of scavenging proteins might have a detrimental impact. In addition, downregulating SOD1 in Schwann cells by the Cre/Lox approach showed an acceleration of the disease in ALS mice (Lobsiger et al., 2009). Therefore, downregulating SOD1 outside of the CNS might have a negative impact on the disease, and the balance between the positive and the potential negative effect of SOD1 downregulation would have to be evaluated.

    This work pertains to the major question about the effect of any drug tested in ALS mice. This model has been criticized in recent years because results obtained in the mice are not recapitulated in clinical trials. Several issues could, of course, be at play, the major one being that mouse biology is different from human biology. But additionally, it is important to look at the type of trial in the mouse that one would like to bring to the clinical. As previously mentioned, drugs in humans have to be used after the symptoms of ALS appear; therefore, it is very important, as in this study, to try the drug in these same conditions in the mouse. Furthermore, this new study now adds another point to be considered, that is, the potential of the drug to act directly on the expression of the mutant protein causing the disease.

    Finally, it would, of course, be very interesting to try APC in other models to see 1) the effect of downregulating SOD1 in models that do not express mutant SOD1 (for other neurodegenerative diseases or models with other mutations responsible for ALS, such as TDP43); 2) the potential participation of wild-type SOD1 aberrant species in motor neuron degeneration; and 3) the actual contribution of APC’s effect on the blood-brain barrier.

    View all comments by Severine Boillee

References

News Citations

  1. Chicago: Axonal Transport Not So Fast in Neurodegenerative Disease
  2. Research Brief: SOD1 Mutants Cause Early Vascular Changes

Webinar Citations

  1. Mice on Trial? Issues in the Design of Drug Studies

Paper Citations

  1. . The cytoprotective protein C pathway. Blood. 2007 Apr 15;109(8):3161-72. PubMed.
  2. . Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets. Nature. 2008 Feb 28;451(7182):1076-81. PubMed.
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  5. . Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med. 2001 Mar 8;344(10):699-709. PubMed.
  6. . Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron. 2004 Feb 19;41(4):563-72. PubMed.
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  11. . Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med. 2003 Mar;9(3):338-42. PubMed.
  12. . Protease-activated receptor-1 signaling by activated protein C in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J Biol Chem. 2005 May 20;280(20):19808-14. PubMed.
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  19. . 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. PubMed.
  20. . The APCs of neuroprotection. J Clin Invest. 2009 Nov;119(11):3205-7. PubMed.

External Citations

  1. currently under trial

Further Reading

Papers

  1. . Effects of activated protein C on neonatal hypoxic ischemic brain injury. Brain Res. 2008 May 19;1210:56-62. PubMed.
  2. . 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. PubMed.
  3. . The APCs of neuroprotection. J Clin Invest. 2009 Nov;119(11):3205-7. PubMed.
  4. . Mutant SOD1 G93A microglia have an inflammatory phenotype and elevated production of MCP-1. Neuroreport. 2009 Oct 28;20(16):1450-5. PubMed.
  5. . Microglia in ALS: the good, the bad, and the resting. J Neuroimmune Pharmacol. 2009 Dec;4(4):389-98. PubMed.
  6. . Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med. 2003 Mar;9(3):338-42. PubMed.
  7. . An ALS mouse model with a permeable blood-brain barrier benefits from systemic cyclosporine A treatment. J Neurochem. 2004 Feb;88(4):821-6. PubMed.

Primary Papers

  1. . 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. PubMed.
  2. . The APCs of neuroprotection. J Clin Invest. 2009 Nov;119(11):3205-7. PubMed.