Tadalafil and CYR119 increase proteasome peptidase activity and cGMP in peripheral nerves

We previously showed that administration of the PDE5 inhibitor sildenafil via intraperitoneal injection to WT and S63del mice increased proteasome peptidase activities in the sciatic nerve lysates [20]. To determine whether the PDE5 inhibitor tadalafil has similar effects, S63del mice and WT littermates were treated with tadalafil for 7 days, beginning at P30, and then proteasomal peptidase activity was measured in the sciatic nerve lysates. Tadalafil was administered in the chow, to which the mice had ad libitum access. The tadalafil treatment increased proteasomal chymotrypsin-like activity by ~ 30% in WT and S63del mice (Fig. 1A). The proteasome’s caspase-like activity in the lysates was increased by a similar magnitude (Supplemental Fig. 1A). The coordinated increase in both peptidase activities strongly suggests that the proteasome activation involves more rapid entry of peptides into the 20S core, as seen previously with 26S proteasomes purified from human cells that had been treated with tadalafil [16], rather than stimulation of an individual 20S active site.

Fig. 1

Tadalafil and CYR119 increase proteasome peptidase activities and cGMP in sciatic nerves of WT and S63del mice. A.) Tadalafil per os for 7 days increased the chymotrypsin-like activity of proteasomes in sciatic nerve lysates from WT and S63del mice. Here and below, n = 3–5 mice per genotype, per condition, and one-way ANOVA with a Bonferroni post-hoc analysis comparing WT and WT treated, WT and S63del, and S63del and S63del treated. The experiment was repeated with similar results. B.) CYR119 per os for 7 days increased proteasome chymotrypsin-like activity in sciatic nerve lysates from WT and S63del mice. C.) In sciatic nerve lysates from S63del mice, protein levels of the 26S proteasome subunits Rpn6 and β5 were increased. Tadalafil treatment for 7 days did not alter the levels of these subunits in WT or S63del. D.) CYR119 treatment for 7 days did not alter the levels of the 26S proteasome subunits Rpn6 or β5 in WT or S63del. E.) Tadalafil or CYR119 for 7 days increased the levels of cGMP in the sciatic nerve lysates, as measured by a cGMP ELISA. T indicates tadalafil and C indicates CYR119. Sciatic nerves from S63del mice had ~ 50% less cGMP than WT littermates

A second cohort of mice was treated with CYR119, a CNS-penetrant stimulator of the soluble guanylyl cyclase (sGC) [24], which was administered in the chow at the same dose as tadalafil. Sciatic nerve lysates from WT and S63del mice treated for 7 days with CYR119 exhibited a ~ 50% increase in proteasomal chymotrypsin-like activity (Fig. 1B) and caspase-like activity (Supplemental Fig. 1B).

Tadalafil and CYR119 both increased proteasomal peptidase activity in the lysates without altering the levels of 26S proteasome subunits, analyzed by western blot for the 19S subunit Rpn6 and the 20S subunit β5 (Fig. 1C and D). Therefore, the observed proteasome activation is likely due to the phosphorylation of pre-existent 26S proteasomes by PKG, as seen previously in several cultured human cell lines when PKG was activated by PDE5 inhibitors or sGC stimulators [16].

The lysates of sciatic nerves from untreated S63del mice contained higher levels of proteasome subunits than those from WT littermates (Fig. 1C and D), as reported previously [7, 20]. This increase in proteasomes is likely the reason why total proteasomal peptidase activities in the S63del lysates are similar to WT, because the peptide hydrolysis is normalized to total protein, not the number of proteasomes. Previous studies on 26S proteasomes purified from sciatic nerve lysates of S63del mice showed a decrease in specific activity [7]. It seems that Schwann cells in the sciatic nerves of S63del mice attempt to compensate for the decreased functionality of individual proteasomes by increasing the expression of proteasome subunits, as seen in many other cell types when proteasomes are inhibited pharmacologically [27]. However, more proteasomes do not fully compensate for the decrease in specific activity in sciatic nerves of S63del mice because polyubiquitinated proteins still accumulate (see below).

To verify that both compounds increased cGMP in the tissue of interest, we measured the concentration of cGMP in the sciatic nerve lysates of WT and S63del mice by ELISA. These experiments produced the unexpected finding that S63del sciatic nerve lysates had approximately half the amount of cGMP as WT (Fig. 1E). This deficiency in cGMP may be a previously unidentified contributor to the nerve pathology seen in the S63del mouse model of CMT1B. Tadalafil (T) increased cGMP levels by ~ 30% in both WT (0.32 pmol/mL to 0.38 pmol/mL) and S63del mice (0.2 pmol/mL to 0.28 pmol/mL) (Fig. 1E). CYR119 (C) increased the relative concentration of cGMP in both WT and S63del sciatic nerves to ~ 0.45 pmol/mL (Fig. 1E). In WT, this correlated to an increase of ~ 50%, and in S63del, to an increase of ~ 150%. The causes of the reduced levels of cGMP in S63del are unknown, but because CYR119, and not tadalafil, increased cGMP levels to the same relative concentration in WT and S63del, the deficiency in S63del is likely due to a reduction in the synthesis of cGMP, and not an increase in its breakdown. Analysis by qRTPCR of the levels of transcripts encoding the α and β subunits of sGC showed a ~ 25% decrease in the sciatic nerves from S63del mice (Supplemental Fig. 2A and 2B), suggesting that less expression of sGC could be one reason for the lower levels of cGMP. Combined, these experiments demonstrate that a 7-day treatment with tadalafil or CYR119 increases cGMP levels and proteasomal peptidase activity in the sciatic nerves of WT and S63del mice.

Increasing cGMP in S63del mice for 21 days improves proteostasis

Because treatment for 7 days with CYR119 activated proteasome peptidase activities and increased the levels of cGMP in sciatic nerves of WT and S63del mice (Fig. 1), we extended the treatment to 21 days to evaluate its effects on proteostasis, myelination, and nerve conduction. Treatment began at P30, an age at which S63del mice already exhibit hypomyelination, slow electrical conduction, a UPR, and impaired protein degradation by the proteasomes in their sciatic nerves [6, 7]. We also included a tadalafil treatment group, enabling a comparison between tadalafil and CYR119 in these mice.

Treatment with CYR119 for 21 days increased proteasomal chymotrypsin-like activity by 2-3-fold in sciatic nerve lysates of WT and S63del mice (Fig. 2A). In WT mice, this treatment did not alter the protein levels of the 26S proteasome subunits Rpt1 and α1 in the sciatic nerve lysates (Fig. 2B). In untreated S63del mice, the levels of these subunits were higher than in WT, and the treatment with CYR119 reduced them to WT levels (Fig. 2B). Because the levels of 26S proteasome subunits decreased only in S63del, the treatment likely reduced the cause of the increased expression, which is, presumably, the accumulation of polyubiquitylated proteins.

Fig. 2

Treating S63del mice for 21 days with tadalafil or CYR119 improves proteostasis in the sciatic nerves A.) Raising cGMP with tadalafil (T) or CYR119 (C) increased proteasomal chymotrypsin-like activity in the sciatic nerve lysates of S63del mice. Here and below, n = 4–7 mice per genotype, per condition, and one-way ANOVA with a Bonferroni post-hoc analysis comparing WT and WT CYR, WT and S63del, S63del and S63del Tad, and S63del and S63del CYR. The experiment was repeated with similar results. B.) The levels of 26S proteasome subunits are higher in the sciatic nerves of S63del mice than in WT littermates. Tadalafil and CYR119 decreased the levels of the 20S subunit α1 and the 19S subunit Rpt1 in S63del mice, as determined by western blot. C.) S63del sciatic nerve lysates exhibit a 2-fold increase in polyubiquitinated proteins over WT littermates. Treatment with tadalafil or CYR119 reduced the levels of polyubiquitinated proteins. D.) Two indicators of proteotoxic stress, BiP and p-eif2α, are increased in the sciatic nerve lysates of S63del mice. Tadalafil and CYR119 reduced the levels of p-eiF2α. Only CYR119 reduced the protein levels of BiP

Proteasomal protein degradation is reduced in the sciatic nerves of S63del mice [7], causing a 2-fold increase in polyubiquitinated proteins, including those linked through K48 which target proteins for degradation by 26S proteasomes (Fig. 2C). CYR119 treatment for 21 days reduced these ubiquitin conjugates to WT levels (Fig. 2C), presumably by increasing their degradation. The treatment did not alter the levels of polyubiquitinated proteins in WT mice (Fig. 2C).

In the sciatic nerves of S63del mice, MPZS63del accumulates in the ER of Schwann cells and induces a canonical UPR [5, 6]. Consequently, the levels of p-eif2α and the ER chaperone BiP are ~ 2-fold higher in the sciatic nerves of S63del mice than in WT littermates (Fig. 2D). The treatment with CYR119 reduced the levels of p-eif2α in S63del mice to levels below those seen in WT controls (Fig. 2D). The protein levels of BiP were also reduced (Fig. 2D). These decreases in markers of the UPR and in polyubiquitinated proteins strongly suggest that the CYR119 treatment ameliorated the proteotoxic stress observed in the sciatic nerves of S63del mice.

Tadalafil treatment of S63del mice for 21 days also increased proteasomal chymotrypsin-like activity (Fig. 2A) and reduced the levels of proteasome subunits (Fig. 2B), polyubiquitinated proteins (Fig. 2C), and p-eIF2α (Fig. 2D). In each case the magnitude of change with tadalafil was less than that observed with CYR119 - likely because tadalafil increased cGMP in sciatic nerves to a lesser extent than CYR119 (Fig. 1E). Tadalafil, like our previous treatments of S63del mice with sildenafil [20], did not reduce the levels of BiP (Fig. 2D), further demonstrating a greater reduction in proteotoxic stress with CYR119-mediated stimulation of sGC than that achieved with inhibition of PDE5.

Treating S63del mice for 21 days with tadalafil or CYR119 restores myelin thickness in sciatic nerves

The pathological hallmarks of human patients with CMT1B [28], and S63del mice [25], are the presence of unmyelinated axons in peripheral nerves, and demyelination. Unmyelinated axons are those that meet the requirements for myelination – a diameter greater than 1 μm and are present in a one-to-one relationship with a Schwann cell – but are not myelinated. Such fibers are rare in the sciatic nerves of WT mice (< 1% of all axons) (Fig. 3A and B, Supplemental Fig. 3), but are more prevalent in S63del, where they constitute ~ 10% of all axons (Fig. 3A and B, Supplemental Fig. 3). Treatment of S63del mice with CYR119 for 3 weeks reduced the incidence of unmyelinated axons to ~ 1% (Fig. 3A and B, Supplemental Fig. 3), a frequency indistinguishable from WT.

Fig. 3

Treating S63del mice for 21 days with tadalafil or CYR119 restores myelin thickness and nerve conduction in sciatic nerves. A.) Representative electron microscopic images of ultrathin sections of sciatic nerves. The yellow asterisk indicates an unmyelinated fiber. B.) The % of unmyelinated fibers in sciatic nerves is higher in S63del mice than in WT littermates. The treatment with tadalafil (T) or CYR119 (C) reduced the number of unmyelinated fibers in the sciatic nerves of S63del mice. Here and below, n = 5–7 mice per genotype, per condition and one-way ANOVA with a Bonferroni post-hoc analysis comparing WT and WT CYR, WT and S63del, S63del and S63del Tad, and S63del and S63del CYR. C.) Average myelin thickness was lower in S63del sciatic nerves than in WT and was increased by tadalafil or CYR119. G-ratio = axon diameter / fiber diameter. Therefore, the higher the g-ratio, the thinner the myelin sheath. D.) A scatterplot of the g-ratio distribution across the measured axon diameters. S63del sciatic nerves have thinner myelin across all axon diameters, as indicated by the red line (S63del) being always above the black line (WT). Tadalafil and CYR119 treatments of S63del mice increased myelin thickness across all axon diameters, as indicated by the purple (tadalafil) and gold (CYR119) lines always being under the red line (S63del). E.) Graphs of the g-ratio distribution by axon diameter shown in D. S63del has thinner myelin (higher g-ratio) than WT across all axon diameters. Tadalafil or CYR119 treatments of S63del mice increased myelin thickness (lowered g-ratio) across all axon diameters. F.) The three-week treatment with tadalafil or CYR119 did not cause statistically significant changes in the percentage of myelinated axons of any diameter. G.) Treatment with tadalafil or CYR119 increased conduction velocity in the peripheral nerves of S63del mice. Here and below, n = 5–7 mice per genotype, per condition, corresponding to 10–14 measurements, one per sciatic nerve, and one-way ANOVA with a Bonferroni post-hoc analysis comparing WT and WT CYR, WT and S63del, S63del and S63del Tad, and S63del and S63del CYR. H.) Tadalafil or CYR119 treatment reduced the longer distal latencies in the peripheral nerves of S63del mice. I.) Treatment with tadalafil or CYR119 reduced the longer F-wave latencies in S63del mice

S63del mice have thinner myelin sheaths around the axons in their peripheral nerves than WT littermates due to uniform demyelination [25]. To determine whether raising cGMP and reducing proteotoxic stress restored myelination, we quantified myelin thickness per axon by electron microscopic analysis of ultrathin sections of sciatic nerves (Fig. 3A). Myelin thickness is quantified as a g-ratio, which is defined as axon diameter divided by fiber diameter. Therefore, the higher the g-ratio, the thinner the myelin sheath surrounding the axon. In S63del mice, the 21-day treatment with CYR119 reduced the average g-ratio of all axons to the levels of untreated WT mice (Fig. 3C). This increase in myelin thickness in S63del was seen across all axon diameters (Fig. 3D and E). CYR119 treatment did not alter the percentage of myelinated axons in S63del or WT mice (Fig. 3F).

Treating S63del mice with tadalafil also reduced the incidence of unmyelinated fibers and increased myelin thickness across all axon diameters without altering the percentage of myelinated axons (Fig. 3B and F). The magnitude of these improvements was less than those elicited by CYR119.

Treating S63del mice with tadalafil or CYR119 improves nerve conduction

We performed electromyography (EMG) to investigate whether the observed increases in myelin thickness improved nerve conduction. S63del mice have slow nerve conduction velocities, and prolonged distal and F-wave latencies due to uniform hypomyelination (Fig. 3G, 3, and I). Distal latency is the time required for a stimulus to travel from the dorsum of the vertebral column to muscles in the hind paw. F-wave latency is the time it takes a supramaximal electrical stimulus to travel antidromically from the ankle to the spinal cord and then orthodromically along the sciatic nerve to muscles in the hind paw. In S63del mice, the CYR119 treatment restored nerve conduction velocity (Fig. 3G), distal latency (Fig. 3H), and F-wave latency (Fig. 3I) to WT levels. The tadalafil treatment also improved these measures of nerve conduction, but to a lesser extent than the CYR119 treatment.

Taken together, these data demonstrate that pharmacologically raising cGMP in the S63del mouse model of CMT1B activates proteasomes and restores proteostasis, myelin thickness and nerve conduction.

The C3 mouse model of CMT1A exhibits similar pathomechanisms to the S63del mouse model of CMT1B

Our promising results in the S63del mouse model of CMT1B prompted us to investigate whether compounds that increase cGMP could also treat the hereditary peripheral neuropathy CMT1A. CMT1A has a similar clinical presentation and pathological progression as CMT1B – demyelination of peripheral nerves that causes progressive distal weakness and muscle atrophy [2]. A clinically relevant model of CMT1A is C3 mice, which overexpress human PMP22 [29]. To determine whether CMT1A also has similar pathomechansims as CMT1B, and might therefore be treated by raising cGMP, we first performed western blot analysis for polyubiquitinated proteins. Sciatic nerve lysates from C3 mice contained approximately 2-fold more ubiquitin conjugates than WT littermates (Fig. 4A), suggesting a defect in their degradation by 26S proteasomes, as seen in the S63del mouse model of CMT1B (Fig. 2C).

Fig. 4

Polyubiquitinated proteins and PMP22 accumulate in the sciatic nerves of C3 mice. A.) The levels of polyubiquitinated proteins were higher in sciatic nerve lysates from C3 mice than from WT littermates. n = 3. Student’s t-test. B.) PMP22 accumulated in its glycosylated (20 kDa) and unglycosylated (16 kDa) forms in C3 sciatic nerves. Human PMP22 protein was detected only in lysates from C3 mice, as expected because C3 mice contain 3–4 copies of a human PMP22 transgene. Total PMP22 protein was detected by probing the blots simultaneously with one antibody specific for human PMP22 and a second antibody specific for mouse PMP22. Representative western blot of 3 experiments performed with lysates from 3 distinct mice per genotype. C.) EndoH-sensitive PMP22 is increased in the sciatic nerve lysates of C3 mice. The removal of all glycans in the lysates by PNGase showed that PMP22 is increased in the sciatic nerve lysates of C3 mice. n = 3 mice per genotype and one-way ANOVA with a Bonferroni post-hoc analysis comparing WT and C3. D.) PMP22 is localized in the ER of Schwann cells in sciatic nerves of C3 mice. Sciatic nerves from WT and C3 mice were teased into individual nerve fibers and immunohistochemical analysis was performed for KDEL, a marker of the ER, and human and mouse PMP22. The scale bar represents 5 μm. E.) The levels of cGMP were 50% lower in sciatic nerve lysates from C3 mice than from WT, as measured by cGMP ELISA. n = 5. One-way ANOVA with a Bonferroni post-hoc analysis comparing WT and C3, and WT and S63del. Experiment was repeated with similar results

To determine whether the disease-causing protein accumulates in the sciatic nerve lysates of C3 mice, a hallmark of proteotoxic diseases, we performed western blot analysis with two antibodies that detect PMP22 [30]. One antibody was specific for mouse PMP22 and detected a band at ~ 20 kDa, the observed molecular weight of PMP22, in both WT and C3 mice (Fig. 4B). C3 mice, in addition to their two copies of endogenous PMP22, harbor 3–4 copies of transgenic human PMP22 [29]. The second antibody we used was specific for human PMP22 and detected no PMP22 protein in lysates from WT mice (Fig. 4B), as expected. In sciatic nerve lysates from C3 mice, this antibody detected two bands, one at ~ 20 kDa and another at ~ 16 kDa (Fig. 4B), approximately the predicted molecular weight of PMP22. Enzymatic deglycosylation of the lysates with PNGase converted the 20 kDa band to the 16 kDa band (Fig. 4B), demonstrating that the 20 kDa band is glycosylated PMP22. The 16 kDa unglycosylated PMP22 was detected only in C3 mice in this study (Fig. 4B) and has been detected previously in the sciatic nerve lysates of C22 mice, a model which harbors 7 copies of human PMP22 and presents with a phenotype much more severe than that observed in patients with CMT1A [31], and in cultured rat Schwann cells in which glycosylation was inhibited by tunicamycin [32]. Probing the blots with both antibodies to simultaneously detect the endogenous and transgenic PMP22 revealed an increase in the levels of PMP22 protein in C3 sciatic nerves (Fig. 4B), shown even more clearly by enzymatic deglycosylation of the lysates with PNGase to convert PMP22 to one band (Fig. 4B and C). Thus, the level of PMP22 protein, especially pathological unglycosylated PMP22, is increased in the sciatic nerve lysates of C3 mice.

We also enzymatically deglycosylated the sciatic nerve lysates of WT and C3 mice with endoglycosidase (EndoH), which cleaves the high mannose N-linked glycans found on glycoproteins that have not fully trafficked through the Golgi. Sciatic nerve lysates from C3 mice contained approximately 3-times more EndoH-sensitive PMP22 than those from WT littermates (Fig. 4C). When PMP22 is incorporated into the myelin sheath, it contains mature glycans that are insensitive to hydrolysis by EndoH [33]. Therefore, in C3 mice, the levels of PMP22 are increased in the pre-Golgi space in the Schwann cells. PMP22 is a transmembrane protein. The pre-Golgi space in which it would most likely accumulate is the ER. We performed immunohistochemical analysis of teased sciatic nerve fibers from WT and C3 mice for KDEL, a marker of the ER, and PMP22. More PMP22 signal was detected in C3 mice than WT (Fig. 4D), as seen above by western blot analysis (Fig. 4B). In the WT nerves, there was little to no co-localization of KDEL and PMP22 (Fig. 4D), as expected because there was little EndoH-sensitive PMP22 in these nerves (Fig. 4C). In the nerves of C3 mice, KDEL and PMP22 co-localized, indicating that there is accumulation of PMP22 in the ER of Schwann cells in C3 mice (Fig. 4D).

Next, we measured the levels of cGMP in the sciatic nerve lysates of C3 mice and found a decrease of ~ 50% (Fig. 4E), similar to the magnitude of decrease seen in S63del mice (Figs. 1E and 4E). qRTPCR analysis found a 25–30% reduction in the levels of transcripts of the α and β sGC subunits in the sciatic nerves of C3 mice (Supplemental Fig. 4A and 4B), similar to the decreases seen in S63del (Supplemental Fig. 2A and 2B).

A 7-day treatment of C3 mice with CYR119 increased proteasome activity and decreased polyubiquitinated proteins

Because C3 mice, like S63del mice, accumulate polyubiquitinated proteins (Fig. 4A) and the disease-causing protein in their sciatic nerves (Fig. 4B and C), and have decreased levels of cGMP (Fig. 4E), we sought to determine whether pharmacological agents that increase cGMP could also have therapeutic effects. First, we treated C3 and WT mice for 7 days with tadalafil or CYR119 and assessed proteasomal chymotrypsin-like activity in the sciatic nerve lysates. Both compounds increased this activity in WT mice (Fig. 5A) without changing the levels of proteasome subunits (Fig. 5B, Supplemental Fig. 5A and 5B), as seen above (Fig. 1A, B and C, and 1D). C3 sciatic nerve lysates exhibited proteasomal chymotrypsin-like activity that was ~ 40% higher than WT (Fig. 5A). This result was unexpected because polyubiquitinated proteins accumulated in the sciatic nerves of C3 mice (Fig. 4A), which often indicates a decrease in proteasomal degradation. To determine whether the increased proteasomal peptidase activity in C3 sciatic nerve lysates was caused by an increase in proteasomes, we performed western blot analysis for subunits of the 26S proteasome. The protein levels of the 19S subunit Rpt1 and the 20S subunit α1 in C3 sciatic nerve lysates were similar to that of WT (Fig. 5B, Supplemental Fig. 5A and 5B), showing that the higher peptide hydrolysis was not due to an increased level of proteasomes. Interestingly, C3 mice treated with tadalafil or CYR119 exhibited increased proteasomal peptidase activity in their sciatic nerves above the levels observed in untreated C3 mice (Fig. 5A).

Fig. 5

Treatment of C3 mice with CYR119 for 7 days increases proteasome peptidase activity in sciatic nerve lysates and reduces ubiquitin conjugates. A.) Treatment with tadalafil (T) or CYR119 (C) for 7 days increased proteasome chymotrypsin-like activity in sciatic nerve lysates of WT and C3 mice. Baseline proteasome peptidase activity was higher in C3 than in WT mice. Here and below, n = 3 mice per genotype, per condition and one-way ANOVA with a Bonferroni post-hoc analysis comparing WT and WT treated, WT and C3, and C3 and C3 treated. Experiment was repeated with similar results. B.) Levels of 26S proteasome subunits were similar in C3 mice and WT littermates. Neither treatment altered the protein levels of the 19S subunit Rpt1 or the 20S subunit α1. C.) CYR119 for 7 days reduced the levels of polyubiquitinated proteins in WT and C3 sciatic nerve lysates

The unexpected high levels of proteasomal peptidase activity in the sciatic nerve lysates of C3 mice prompted us to investigate whether further increasing this activity by pharmacologically raising cGMP could reduce the levels of polyubiquitinated proteins. Western blot analysis showed that the 7-day treatment of C3 mice with CYR119, but not tadalafil, reduced the levels of polyubiquitinated proteins towards levels seen in WT (Fig. 5C). Whatever the cause of the high proteasomal peptidase activity in C3 mice, this result suggests that the accumulation of polyubiquitinated proteins in C3 sciatic nerves is caused by a lack of degradation by 26S proteasomes, and that the treatment with CYR119 reduced the levels of polyubiquitinated proteins by increasing their degradation. We therefore chose to extend the treatment with CYR119 to 21-days to assess whether it could improve proteostasis, myelination, and nerve function in C3 mice.

Treating C3 mice with CYR119 for 21 days improves proteostasis, myelination, and nerve conduction

We treated a cohort of C3 and WT mice with CYR119 for 21 days and measured proteasomal chymotrypsin-like activity in the sciatic nerve lysates. C3 mice exhibited ~ 40% more of this activity than WT (Fig. 6A), as seen above (Fig. 5A). The treatment increased proteasomal chymotrypsin-like activity in both WT and C3 mice (Fig. 6A) without altering the levels of 26S proteasome subunits (Fig. 6B, Supplemental Fig. 6A and 6B). Western blot analysis for ubiquitin in the sciatic nerve lysates showed an approximately 2-fold higher level of polyubiquitinated proteins in C3 mice than in WT (Fig. 6C). The CYR119 treatment reduced the levels of polyubiquitinated proteins in C3 mice to those seen in WT (Fig. 6C). The sciatic nerve lysates were also analyzed for PMP22 by western blot with two antibodies to detect both the endogenous and transgenic PMP22, as performed above (Fig. 4B and C). In C3 mice, the CYR119 treatment reduced the ~ 20 kDa PMP22 to levels seen in WT and reduced the levels of the pathological ~ 16 kDa PMP22 by ~ 50% (Fig. 6D-F), presumably by increasing their degradation, as seen previously with MPZS63del [20].

Fig. 6

Treating C3 mice with CYR119 for 21 days increases proteasome peptidase activity and decreases the levels of PMP22. A.) CYR119 (C) for 21 days increased proteasome chymotrypsin-like activity in the sciatic nerve lysates of WT and C3 mice. Here and below, n = 4–5 mice per genotype, per condition and one-way ANOVA with a Bonferroni post-hoc analysis comparing WT and WT treated, WT and C3, and C3 and C3 treated. Experiment was repeated with similar results. B.) The levels of the 19S subunit Rpt1 and the 20S subunit α1 were similar in sciatic nerve lysates of WT and C3 mice and were unaltered by the treatment with CYR119. C.) Treating C3 mice for 3 weeks with CYR119 reduced the levels of polyubiquitinated proteins in the sciatic nerve lysates. D.) Glycosylated (20 kDa) and unglycosylated (16 kDa) PMP22 accumulated in the sciatic nerve lysates of C3 mice and their levels were reduced by the 3-week treatment with CYR119. E.) Treatment of C3 mice with CYR119 reduced the levels of glycosylated PMP22 (20 kDa). Here and below, n = 3 mice per genotype, per condition and one-way ANOVA with a Bonferroni post-hoc analysis comparing WT and WT treated, WT and C3, and C3 and C3 treated. F.) Unglycosylated PMP22 (16 kDa) was present only in C3 sciatic nerves and its levels were reduced by the treatment with CYR119. Student’s t-test

To assess whether raising cGMP was also improving the morphological and functional deficits associated with CMT1A neuropathy, we first analyzed images of semithin sections of sciatic nerves from WT and C3 mice for the presence of unmyelinated axons. In the sciatic nerves of C3 mice, unmyelinated axons constituted ~ 6% of all axons (Fig. 7B and Supplemental Fig. 7). The treatment with CYR119 reduced their incidence to ~ 4% (Fig. 7B and Supplemental Fig. 7).

Fig. 7

CYR119 treatment for 21 days improves myelination in the sciatic nerves of C3 mice. A.) Representative electron microscopic images of ultrathin sections of sciatic nerves. The yellow asterisk indicates an unmyelinated fiber. B.) C3 mice had more unmyelinated fibers in their sciatic nerves than WT littermates. The treatment with CYR119 (C) reduced the percentage of unmyelinated fibers. Here and below, n = 5–6 mice per genotype, per condition and one-way ANOVA with a Bonferroni post-hoc analysis comparing WT and WT treated, WT and C3, and C3 and C3 treated. C.) Average myelin thickness was lower (higher g-ratio) in C3 sciatic nerves and was increased (lower g-ratio) by the treatment with CYR119. D.) Scatterplot of the g-ratio distribution across the measured axon diameters. C3 sciatic nerves had thinner myelin (higher g-ratio) than WT around axons with diameters > 2 μm and thicker myelin (lower g ratio) around axons with diameters < 2 μm. E.) Bar graphs of the g-ratio distribution by axon diameter shown in D. CYR119 treatment of C3 mice increased myelin thickness across all axon diameters < 5 μm. F.) The three-week treatment with CYR119 did not cause statistically significant changes in the percentage of myelinated axons of any diameter in C3 mice

C3 mice have a higher average g-ratio (i.e., thinner myelin sheaths) in their sciatic nerves than WT littermates (Fig. 7C) due to demyelination and dysmyelination [29]. However, in C3 mice, not all axons in the peripheral nerves are hypomyelinated, as they are in S63del mice (Fig.