SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy

Abstract

Hyperglycemia-induced oxidative stress is an inciting event in the development of diabetic complications including diabetic neuropathy. Our observations of significant oxidative stress and morphological abnormalities in mitochondria led us to examine manganese superoxide dismutase (SOD2), the enzyme responsible for mitochondrial detoxification of oxygen radicals. We demonstrate that overexpression of SOD2 decreases superoxide (O(2)(-)) in cultured primary dorsal root ganglion (DRG) neurons and subsequently blocks caspase-3 activation and cellular injury. Underexpression of SOD2 in dissociated DRG cultures from adult SOD2(+/-) mice results in increased levels of O2-, activation of caspase-3 cleavage and decreased neurite outgrowth under basal conditions that are exacerbated by hyperglycemia. These profound changes in sensory neurons led us to explore the effects of decreased SOD2 on the development of diabetic neuropathy (DN) in mice. DN was assessed in SOD2(+/-) C57BL/6J mice and their SOD2(+/+) littermates following streptozotocin (STZ) treatment. These animals, while hyperglycemic, do not display any signs of DN. DN was observed in the C57BL/6Jdb/db mouse, and decreased expression of SOD2 in these animals increased DN. Our data suggest that SOD2 activity is an important cellular modifier of neuronal oxidative defense against hyperglycemic injury.

Figure 1. SOD2 Protects Sensory Neurons from O₂^•− Mediated Damage

A) Adenoviral infection specifically increases SOD1 or SOD2 in DRG neurons. The graph shows SOD activity in lysates from Ad.OMC (a comparison mitochondrial protein), Ad.SOD1, or Ad.SOD2-infected DRG neurons. Ad.SOD2 specifically upregulated the activity of SOD2 but not SOD1. *p<0.01 compared to Ad.OMC-infected DRG neurons. B) Western blots of lysates of (1) Ad.SOD1-infected, (2) Ad.SOD2-infected, or (G) Ad.GFP-infected DRG neurons that are probed for SOD1 or SOD2. C) Oxidation of non-fluorescent CM-H₂DCFDA to green fluorescent DCF indicates an increase in oxidative stress, particularly H₂O₂ generation following treatment with 20 mM added glucose that is blocked in SOD2 transfected cells. *p<0.01 compared with untreated control; **p<0.01 compared with Ad.GFP/glucose. D) SOD2, but not GFP overexpression, prevents 20 mM glucose-induced caspase-3 activation. ***p < 0.001 compared to untreated control.

Figure 2. Decreased Expression of SOD2 Increases Glucose-Mediated O₂^•− and Apoptosis

DRG neurons from adult SOD2^+/+ and SOD2^+/− mice were exposed to 20 mM added glucose. A) O₂^•− generation was measured using an in vitro Amplex Red oxidation reaction at 5 h (Vincent et al., 2005a). **p < 0.001 compared to SOD2^+/+ in basal glucose; +p < 0.01 compared to SOD2^+/+ in basal glucose; *p < 0.05 compared to SOD^+/− in basal glucose. B) DRG neurons were loaded with TMRM (50 nM), then mitochondrial membrane potential assessed through the increase in red fluorescence. In the presence of 20 mM glucose, hyperpolarization was greater and depolarization occurred earlier in SOD2^+/− compared to SOD2^+/+ neurons. C) Caspase-3 activation was determined by counting the percent of DRG neurons labeled with a fluorescent caspase-3 substrate (CaspaTag). Glucose-induced caspase-3 activation was increased in both the SOD2^+/−and SOD^+/+ cultures, compared to basal glucose, ***p < 0.001. D) Neurites were measured in DRG neurons 12 h after plating in control or hyperglycemia (20 mM added glucose) media. Mean neurite length was shorter in 20 mM added glucose than basal glucose, ***p < 0.001 for SOD2^+/+ and ###p < 0.05 for SOD2^+/−.

Figure 3. Measures of Nerve Function and Antioxidant Potential in SOD2^+/+ and SOD2^+/− Mice on a C57BL/6 Background

A) Tail flick (TF) latencies are measured at 12 and 24 weeks post induction of diabetes. B) Sciatic Motor Nerve Conduction Velocity (SMNCV) was assessed at 12 and 24 weeks. C) Total Radical Antioxidant Potential (TRAP) was measured in DRG after 24 weeks. No differences were found between the four experimental groups. n=10 for each experimental group.

Figure 4. Measures of Nerve Function and Antioxidant Potential in SOD2^+/+ and SOD2^+/− Mice on a db+ or db/db Background

Nerve function and antioxidant potential was assessed using tail flick latency (TF) at 8 and 12 weeks (A), Sciatic Motor Nerve Conduction Velocity (SMNCV) at 12 and 24 weeks (B), and DRG TRAP at 24 weeks (C). In A, TF latencies are significantly longer in db/db, *p < 0.001. In B, a significant difference in SMNCV is only detected at 24 weeks between the db⁺ and db/db SOD2^+/−mice (*p < 0.001). In C, TRAP was significantly lower in db/db mice compared with db⁺ (⁺p<0.05). n=10 for each experimental group.

Figure 5. Measures of IENFD in SOD2^+/+ and SOD2^+/− Mice on a C57BL/6 db+ or db/db Background

Representative SOD2^+/+ db+ (A) and SOD2^+/−db/db (B) images are shown to illustrate the flattened confocal mouse footpad sections processed for PGP 9.5 immunofluorescence. The white line in A identifies the division between dermis and epidermis and the arrows indicate IENFD that cross this division. Fewer fibers are evident in the diabetic footpad (B) compared with the non-diabetic control (A). Bar = 50 μm. In C, IENFD expressed as percent of total epidermal area. *p < 0.01 between SOD2^+/+ db+ and SOD2^+/+ db/db, and between SOD2^+/−db⁺ compared to SOD2^+/− db/db. +p < 0.05 between SOD2^+/+ db/db compared to SOD2^+/− db/db. n=3 for each experimental group.