We have determined that treatment of mutant SOD1–expressing mice with enzymatically active APC analogs delivered after disease onset retarded progression of ALS-like disease, increased lifespan, and, more importantly, increased duration of the symptomatic phase. The enzymatic activity of APC, but not its anticoagulant activity, was critical for the beneficial effects. A primary mechanism for APC’s action is through passage across the BSCB in an EPCR-dependent manner, followed by transcriptional downregulation of mutant SOD1 in motor neurons and their non-neuronal neighbors, including microglia and cells comprising microvessels.
BSCB disruption (18
), swelling (18
), and ischemic changes (42
) are consistent features in spinal cord pathology of ALS mice (18
). Ischemia worsens motor neuron degeneration and functional outcome, as shown in mice with a mutation that eliminates hypoxia-responsive induction of the vascular endothelial growth factor A gene (42
), which develop late-onset motor neuron degeneration (43
). Endothelial cells of the microvasculature, along with pericytes and astrocytes, were essential to stabilize the BSCB, but we have also shown that APC’s slowing of disease was not mediated by reduction of mutant SOD1 within the endothelial cells, the first cells to encounter plasma-delivered APC. Indeed, mutant SOD1 damage directly within the endothelial cells was shown by selective gene excision to play little, if any, role in disease pathogenesis. Activation of microglia and astrocytes and the accompanying inflammatory response, on the other hand, play a major role in progressive BSCB opening in SOD1 mutants after disease onset (18
). Thus, we conclude that BSCB stabilization by APCs given postsymptomatically — as in the present study — will critically depend on mutant SOD1 reduction in microglia and astrocytes after APC transport across the capillary wall. Additional transgenic mouse models will be needed to resolve whether the well-described protective effects of APC on endothelium (8
) can improve capillary integrity in SOD1 mutants independent of the observed SOD1 blockade in nonendothelial cells and/or endothelia.
A convergence of evidence has led to a consensus that SOD1 mutations cause disease by acquisition of 1 or more toxic properties, rather than by loss of dismutase activity, including mutant damage to mitochondria, damage from aberrant mutant SOD1 secretion (45
), endoplasmic reticulum stress from blockage of ejection of misfolded proteins from it (46
), and hyperactivation of extracellular superoxide production by microglia (47
), as reviewed recently (48
). We would emphasize that whatever the most relevant toxicities, APC-mediated mutant SOD1 downregulation within motor neurons and microglia represents a therapeutic approach that is directly linked to disease mechanism. APC-mediated diminution of mutant SOD1 synthesis in motor neurons may contribute to the delay in disease initiation, whereas — perhaps more importantly — lowered SOD1 mutant levels within astrocytes, microglia, or peripheral macrophages are highly likely to be responsible for slowed disease progression. This conclusion would be in accordance with prior findings that selective mutant SOD1 gene excision from astrocytes (17
), from microglia and peripheral macrophage lineages (16
), or by bone marrow replacement of mutant myeloid cells with normal ones (50
) can strikingly slow disease progression despite no effect on disease onset.
The only other SOD1 gene–silencing approach previously proven to slow disease progression is antisense DNA oligonucleotide infusion (51
), but these oligonucleotides are not BBB or BSCB permeant, requiring delivery by direct CNS infusion following invasive surgery. Methods of gene silencing through retroviral delivery of transcription-mediated shRNAs have been shown to dramatically slow disease onset, but only when administered to very young SOD1G93A
animals. Even with delivery at a juvenile age, this approach was of no benefit in slowing the rate of disease progression (52
). Modest survival benefits from slowing disease onset, but without benefit in slowing progression, have also been seen with viral delivery prior to disease onset by direct injection into the spinal cord to very focally silence SOD1 (53
). Thus, the ability of APC to slow disease progression, combined with simple peripheral administration, represent unique therapeutic advantages compared with other SOD1-silencing strategies. Moreover, APC is already approved for use in adult patients. Indications include severe sepsis (54
), and a clinical trial is currently underway to assess the benefit of APC in acute ischemic stroke (http://www.clinicaltrials.gov/ct2/show/NCT00533546). What is clear from our efforts and the prior ones (5
) is that transient exposure, without continuous infusion, of APC can produce long-lasting neuroprotective effects. With the recognition that accumulation of aberrant SOD1 species has been linked to most cases of sporadic ALS (56
), strategies based on activation of the protein C cellular pathway are promising directions for treating patients with familial, and possibly sporadic, ALS.