Urokinase Plasminogen Receptor and the Fibrinolytic Complex Play a Role in Nerve Repair after Nerve Crush in Mice, and in Human Neuropathies

Remodeling of extracellular matrix (ECM) is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR) is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA) promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies

Alteration of the late endocytic pathway in Charcot-Marie-Tooth type 2B disease

The small GTPase RAB7A regulates late stages of the endocytic pathway and plays specific roles in neurons, controlling neurotrophins trafficking and signaling, neurite outgrowth and neuronal migration. Mutations in the RAB7A gene cause the autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) disease, an axonal peripheral neuropathy. As several neurodegenerative diseases are caused by alterations of endocytosis, we investigated whether CMT2B-causing mutations correlate with changes in this process. To this purpose, we studied the endocytic pathway in skin fibroblasts from healthy and CMT2B individuals. We found higher expression of late endocytic proteins in CMT2B cells compared to control cells, as well as higher activity of cathepsins and higher receptor degradation activity. Consistently, we observed an increased number of lysosomes, accompanied by higher lysosomal degradative activity in CMT2B cells. Furthermore, we found increased migration and increased RAC1 and MMP-2 activation in CMT2B compared to control cells. To validate these data, we obtained sensory neurons from patient and control iPS cells, to confirm increased lysosomal protein expression and lysosomal activity in CMT2B-derived neurons. Altogether, these results demonstrate that in CMT2B patient-derived cells, the endocytic degradative pathway is altered, suggesting that higher lysosomal activity contributes to neurodegeneration occurring in CMT2B.Peer reviewe

Niacin‐mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination

Abstract Charcot–Marie–Tooth (CMT) neuropathies are highly heterogeneous disorders caused by mutations in more than 70 genes, with no available treatment. Thus, it is difficult to envisage a single suitable treatment for all pathogenetic mechanisms. Axonal Neuregulin 1 (Nrg1) type III drives Schwann cell myelination and determines myelin thickness by ErbB2/B3‐PI3K–Akt signaling pathway activation. Nrg1 type III is inhibited by the α‐secretase Tace, which negatively regulates PNS myelination. We hypothesized that modulation of Nrg1 levels and/or secretase activity may constitute a unifying treatment strategy for CMT neuropathies with focal hypermyelination as it could restore normal levels of myelination. Here we show that in vivo delivery of Niaspan, a FDA‐approved drug known to enhance TACE activity, efficiently rescues myelination in the Mtmr2−/− mouse, a model of CMT4B1 with myelin outfoldings, and in the Pmp22+/− mouse, which reproduces HNPP (hereditary neuropathy with liability to pressure palsies) with tomacula. Importantly, we also found that Niaspan reduces hypermyelination of Vim (vimentin)−/− mice, characterized by increased Nrg1 type III and Akt activation, thus corroborating the hypothesis that Niaspan treatment downregulates Nrg1 type III signaling

Fibrinolytic molecules in myelination.

<p>(A) Time course zymography of uPA and tPA activity in Wt DRG explant homogenate (as a pool of at least 8 coverslips) and their conditioned media without ascorbic acid treatment (-) or after 5, 10, 15, 20 days of ascorbic acid (+). Note both tPA and uPA activity are induced after ascorbic acid in both cell homogenate and media, although uPA activity increases in parallel with myelination. (B–E) DRG explants from Wt and uPAR null mice stained for neurofilament (green) and MBP (red) 7 days after ascorbic acid. The number of myelinated segments were similar between Wt and uPAR null explants. Bar = 50 µm.</p

Histological characteristics of uPAR null nerve.

<p>(A–B) semithin sections from sciatic nerve of Wt (A) and uPAR null mouse (B) showing normal fiber appearance. (C) Similar fiber type distribution in Wt and uPAR null sciatic nerve. (D) Neurophysiology analysis showing similar values of cMAP and NCV between Wt and uPAR−/− mice (n. 10 mice per group). (E) g-ratio did not show differences in myelin thickness between Wt and uPAR−/− nerves (n. 20000). (F–K) sciatic nerve cryosections of Wt and uPAR null mouse stained for fibrin fibronectin and vitronectin. Fibrin and vitronectin staining was mildly increased in uPAR null endoneurium (G and K) as compared to Wt (F and J), whereas fibronectin was similarly expressed (H–I). (L) Western blot analysis of fibrin and fibronectin in Wt and uPAR null sciatic nerve homogenate. Calnexin was used to normalize loading (fibronectin and vitronectin were loaded on the same gel, hence they have the same calnexin bands). r = densitometric ratio between the band of interest and calnexin; Wt was always assigned as r = 1. Fibrin and vitronectin levels were increased in uPAR null nerves as compared to Wt, whereas levels of fibronectin were similar. Fg = fibrin; Fn = fibronectin; Vn = vitronectin; Nf = neurofilaments. Bar = 15 µm in A and B; 50 µm in F–K.</p

Different expression of ECM in uPAR−/− nerves after damage.

<p>Sciatic nerve cryosections from Wt (A, C, E, G, I, K) and uPAR−/− (B, D, F, H, J, L) mice stained for fibrin (Fg), fibronectin (Fn) or vitronectin (Vn), and neurofilaments (Nf) at 15 and 45 dpc. Both at 15 and 45 dpc Fibrin expressions was higher in uPAR−/− as compared to Wt endoneurium (B versus A, and H versus G). Fibronectin expression was similarly in Wt and uPAR−/− mice at both time points (D versus C, and J versus I). Vitronectin expression was higher in uPAR−/− mice as compared to wt at 15dpc (F versus E), whereas it was similar at 45 dpc (L and K). Bar = 50 µm.</p

Sciatic nerve regeneration after injury.

<p>(A–B, D–E) semithin section and (C, F) fiber type distribution from sciatic nerve of Wt and uPAR null mice at 15 and 45 dpc. At both 15 and 45 dpc we observed reduced number of regenerating fibers. (G) g-ratio was significantly increased in uPAR−/− regenerating fibers at 45 dpc (n. 20000; p = 0.01). (H) Neurophysiological analysis showing similar values of cMAP between Wt and uPAR−/− mice, whereas NCV was significantly reduced in uPAR−/− mice (n. 8; p = 0.001). (I–J) Staining for Mac-1/CD11b (Mac1) in Wt (I) and uPAR null (J) sciatic nerve 45dpc. (K) Quantification of number of macrophages observed in sciatic nerve 15, 21 and 45dpc; differences were significant at 21 and 45 dpc (*p = 0.04; **p = 0.008). Bar = 10 µm in A, B, D, E, I and J.</p

Impaired uPA activity and fibrin clearance in uPAR−/− nerves after damage.

<p>(A) western blot analysis of fibrin and fibronectin in Wt and uPAR null sciatic nerve homogenate at 15, 21 and 45 dpc. Calnexin was used to normalize loading. Quantification of western blot is reported as an average of 3 independent experiments, and represented as ratio fibrin/calnexin, vitronectin/calnexin and fibronectin/calnexin, assigning Wt 0dpc as 1±SEM). At each time point uPAR null homogenate showed increased levels of fibrin and vitronectin as compared to Wt, whereas fibronectin levels were higher at 15 and 21 dpc, but lower at 45 dpc. (B) Zymography of sciatic nerve homogenate from Wt and uPAR null mice measuring tPA and uPA activity 0, 7 and 15dpc. Bands were stained with Coomassie blue as loading control. Quantification of zymography is reported as an average of 3 independent experiments, and represented as ratio uPA/coomassie blue and tPA/coomassie blue, assigning Wt 0dpc as 1±SEM. Note the reduced increase of uPA in uPAR null homogenate as compared to Wt 7 and 15dpc, whereas there are no differences in the tPA activity between mutant and Wt mice. Fg = fibrin; Fn = fibronectin; Vn = vitronectin; Cln = calnexin.</p