Downregulation of Sprr1a Contributes to the Pathobiology of Sickle Cell Disease

Sickle cell disease (SCD) presents with multiple comorbidities including pain and organ damage. Transcriptomic analysis of dorsal root ganglion (DRG) revealed a significant decrease in the small proline rich protein 1a (Sprr1a) in HbSS-BERK sickle mice compared to control HbAA-BERK at an early age of ~2 months (Paul et al., Nature Sci Data 2017). Sprr1a is associated with axonal regeneration and cornification, which prevent nerve damage and evaporation from the skin, exposure to infections and mechanical stress, all of which occur in SCD. HbSS-BERK mice show thinner skin, nerve damage and increased sensitivity to mechanical and thermal stimuli (Kohli et al., Blood 2010). We hypothesized that Sprr1a downregulation in sickle mice leads to cutaneous alterations, thus increasing sensitivity to noxious stimuli leading to hyperalgesia and that restoring Sprr1a expression would reduce hyperalgesia. Utilizing Sprr1a-knockout (Sprr1a-KO) mice, we examined the gain and/or loss of skin and neuronal function with Sprr1a deletion. Compared to wild-type (WT) C57BL/6 mice, Sprr1a-KO mice showed a ~30% decrease in epidermal skin thickness (p<0.001); significant increase in Evan blue leakage in the skin in response to substance P and capsaicin (p<0.001 and 0.003, respectively); 30-40% increase in calcium release from DRG neurons in response to capsaicin and menthol (p<0.05 and 0.01, respectively); and increased phospho-p38 MAPK in the spinal cords (p<0.003). Complementary to these observations, Sprr1a-KO mice evinced heightened mechanical, heat and cold sensitivity, which increased with age from 1.2 to 7.0 months of age in both male and female mice compared to C57BL/6 WT. These data suggest that loss of Sprr1a leads to skin thinning, increased permeability and activation of peripheral and central nervous system leading to mechanical and thermal hyperalgesia (pain). To identify the molecular pathways involved in different functions of Sprr1a, we performed mass-spectrometry on the DRG of WT and Sprr1a-KO mice on a LTQ Orbitrap XL mass spectrometer (Thermo Scientific) coupled to a Prominence Nano LC (Shimadzu) using the Xcalibur version 2.7.0 (Thermo Scientific) and Proteome Discoverer 2.2 software. In parallel, we genetically overexpressed Sprr1a in HbSS-BERK mice (Sprr1a-OE-SS) using the Gateway cloning system combined with PiggyBac transposon: We obtained 1 cell embryo from HbSS mice, and carried out pronuclear injection with Sprr1a cDNA. The resulting HbSS mice harboring the Sprr1a transgene were screened by appropriate genotyping of tail snips. Proteomic analysis of DRG and subsequent network analysis using the STRING database tool of the significantly modified proteins (p<0.05) revealed that their interactions with Sprr1a have never been investigated. Many of the significantly altered proteins in Sprr1a-KO are associated with SCD pathobiology, including, but not limited to, SPIC (7-fold increase, p<0.0001), associated with red blood cell (RBC) recycling and VCAM1 regulation; CD9 (22-fold increase, p<0.0001), involved in platelet activation and cell adhesion; SPTBN4 (2-fold decrease, p<0.05), neuropathy, and kininogen (3-fold decrease, p<0.001). Decreased kininogen is observed in sickle cell patients, which is further reduced during crisis (Verma et al., Res Commun Chem Pathol Pharmacol 1983). Thus, downregulation of Sprr1a may contribute to SCD pathobiology, which may in turn lead to pain. This appears to be the case, because overexpression of Sprr1a in HbSS-BERK sickle mice (Sprr1a-OE-SS) led to amelioration of mechanical and cold hyperalgesia (p<0.01 & 0.05, respectively) at 2.8 months of age compared to age/gender matched HbSS-BERK mice. This decrease was maintained up to 9.8 months of age (last period of observation) in Sprr1a-OE-SS, which was similar to the sensitivity in control HbAA-BERK mice and significantly lower than HbSS-BERK mice. Together, these data suggest a causal link between Sprr1a downregulation and pain in SCD. It is likely that Sprr1a loss contributes to complex sickle pathobiology leading to dehydration due to loss of water from thin and permeant skin, non-healing skin ulceration, neuropathy and pain. In conclusion, through genetic and proteomic approaches, we have identified Sprr1a as a novel treatable target for several consequences of SCD.