The vitamin D activator CYP27B1 is upregulated in muscle fibers in denervating disease and can track progression in amyotrophic lateral sclerosis

Abstract

Extra-renal expression of Cytochrome P450 Family 27 Subfamily B Member 1 (CYP27B1) has been well recognized and reflects the importance of intracrine/paracrine vitamin D signaling in different tissues under physiological and pathological conditions. In a prior RNA sequencing project, we identified CYP27B1 mRNA as upregulated in muscle samples from patients with amyotrophic lateral sclerosis (ALS) compared to normal controls. Our aims here were: (1) to validate this finding in a larger sample set including disease controls, (2) to determine which cell type is expressing CYP27B1 protein in muscle tissue, (3) to correlate CYP27B1 mRNA expression with disease progression in the SOD1 G93A ALS mouse and in ALS patients. We assessed CYP27B1 expression by qPCR, western blot, and immunohistochemistry in a repository of muscle samples from ALS, disease controls (myopathy and non-ALS neuropathic disease), normal subjects, and muscle samples from the SOD1 G93A mouse. Eight ALS patients were studied prospectively over 6-12 months with serial muscle biopsies. We found that CYP27B1 mRNA and protein levels were significantly increased in ALS versus normal and myopathy muscle samples. Neuropathy samples had increased CYP27B1 mRNA and protein expression but at a lower level than the ALS group. Immunohistochemistry showed that CYP27B1 localized to myofibers, especially those with features of denervation. In the SOD1 G93A mouse, CYP27B1 mRNA and protein were detected in skeletal muscle in early pre-symptomatic stages and increased through end-stage. In the human study, increases in CYP27B1 mRNA in muscle biopsies correlated with disease progression rates over the same time period. In summary, we show for the first time that CYP27B1 mRNA and protein expression are elevated in muscle fibers in denervating disease, especially ALS, where mRNA levels can potentially serve as a surrogate marker for tracking

Wnt antagonist FRZB is a muscle biomarker of denervation atrophy in amyotrophic lateral sclerosis

“…In the SOD1 G93A mouse, however, FRZB mRNA did not decline, but remained significantly elevated beginning at 60 days (P < 0.05) compared to WT. This time frame is similar to what we previously observed with other muscle biomarkers in this model [8][9][10][11] . The fold-difference increased to ~ 2.5 through 125 days of age.…”

“…Its upregulation started between 40 and 60 days of age which is a very early pre-clinical stage in this model, suggesting that it may be a marker of disease onset. This time frame is in keeping with the pattern we observed for a number of muscle biomarkers in ALS including Smads 1, 2, 3, 5, 8, all three isoforms www.nature.com/scientificreports/ of TGF-β, CYP27B1 and select myomiRs [8][9][10][11] . Taken together, these findings suggest a synchronized molecular response in skeletal muscle at or near the onset of disease in the SOD1 G93A mouse and prior to clinical symptoms determined by rotarod testing and weight loss 9 .…”

“…The increase in FRZB with other denervating disease is not unexpected as there are likely many shared pathways that become activated. Some components of the molecular signature that we have characterized previously, such as CYP27B1 and certain miRNAs, are also not specific for ALS 8,10 . While FRZB is a secreted protein, we could not detect increases in plasma samples from ALS patients (not shown).…”

FGF23, A Novel Muscle Biomarker Detected in The Early Stages of ALS

“…This difference progressively increased to nearly 8-fold by end-stage (150 d). This temporal pattern is similar to what we previously observed with other muscle biomarkers in this model [10][11][12][13] .…”

“…Early involvement of skeletal muscle is underscored by our prior work which defines a molecular signature in ALS skeletal muscle beginning in the earliest pre-clinical phases of disease based on correlative studies with the SOD1 G93A mouse. Characterization of this signature emerged from RNA sequencing of human ALS muscle, and encompasses genes of diverse pathways including Smads, TGF-β, vitamin D (CYP27-B1), FRZB/Wnt signaling, and select microRNAs [9][10][11][12][13] . Some of the markers, such as Smad 1, 5, 8, and TGF-β, appear to be specific for ALS whereas FRZB and CYP27B1 were increased in non-ALS neurogenic processes.…”