Macrophages contribute to peripheral nerve regeneration and produce collagen VI, an extracellular matrix protein involved in nerve function. Here, we show that collagen VI is critical for macrophage migration and polarization during peripheral nerve regeneration. Nerve injury induces a robust upregulation of collagen VI, whereas lack of collagen VI in Col6a1(-/-) mice delays peripheral nerve regeneration. In vitro studies demonstrated that collagen VI promotes macrophage migration and polarization via AKT and PKA pathways. Col6a1(-/-) macrophages exhibit impaired migration abilities and reduced antiinflammatory (M2) phenotype polarization, but are prone to skewing toward the proinflammatory (M1) phenotype. In vivo, macrophage recruitment and M2 polarization are impaired in Col6a1(-/-) mice after nerve injury. The delayed nerve regeneration of Col6a1(-/-) mice is induced by macrophage deficits and rejuvenated by transplantation of wild-type bone marrow cells. These results identify collagen VI as a novel regulator for peripheral nerve regeneration by modulating macrophage function.

Charcot-Marie-Tooth disease type 1 (CMT1) 3 is a progressive hereditary motor and sensory neuropathy, characterized by distal muscle wasting and weakness, foot deformities, and severe slowing of nerve conduction, because of progressive demyelination (1). With a prevalence of 1 case in 2500, CMT1 is the most common hereditary neurologic disorder, and in the majority of cases (CMT1A) the disease is associated with a duplication on chromosome 17p11.2 of the gene for PMP22 (peripheral myelin protein 22) (2). PMP22 is a 22-kDa glycoprotein mainly expressed by myelinating Schwann cells (SC) and localized in compact myelin (3). The transgenic rat model of CMT1A, obtained by overexpression of PMP22 (4), confirms a role of PMP22 in the pathogenesis of CMT1A. Both PMP22 overexpression because of gene duplication and point mutations of PMP22 are associated with a CMT1A phenotype.The biochemical mechanisms correlating PMP22 dysfunction with demyelination are still unclear. Some reports indicate that a perturbed homeostasis of the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) might be causally involved in the demyelination process. Conditions inducing an increased [Ca 2ϩ ] i in SC impair cell differentiation and myelination (5, 6), similarly to what occurs in CMT1A. Incubation of intact rat nerves with Ca 2ϩ and ionophores causes a progressive demyelination, spreading from the paranodes and invading regions of formerly compact myelin, which is dependent upon a rise in the [Ca 2ϩ ] i of SC (5). Additional evidence for the detrimental effect of a [Ca 2ϩ ] i elevation on myelin production by SC comes from application of ATP to murine SC monocultures, inducing an immediate and large increase in the [Ca 2ϩ ] i . As a result of ATP treatment, maturation and differentiation of SC, as well as expression of the myelin basic protein and production of compact myelin, are completely prevented (6). Taken together, the above observations indicate that abnormally elevated Ca 2ϩ levels are causally related to impairment of myelin production by SC. * This work was supported in part by Telethon Grants GGP06178 and GUP05007 (to A. S.)

Mutations in the gene MPZ, encoding myelin protein zero (MPZ), cause inherited neuropathies collectively called Charcot-Marie-Tooth type 1B (CMT1B). Based on the age of onset, clinical and pathological features, most MPZ mutations are separable into two groups: one causing a severe, early-onset, demyelinating neuropathy and a second, causing a late-onset neuropathy with prominent axonal loss. To investigate potential pathomechanisms underlying the two phenotypes, we transiently transfected HeLa cells with two late-onset (T95M, H10P) and two early-onset (H52R, S22_W28 deletion) mutations and analyzed their effects on intracellular protein trafficking, glycosylation, cell viability and intercellular adhesion. We found that the two late-onset mutations were both transported to the cell membrane and moderately reduced MPZ-mediated intercellular adhesion. The two early-onset mutations caused two distinct abnormalities. H52R was correctly glycosylated and trafficked to the plasma membrane, but strongly affected intercellular adhesion. When co-expressed with wild-type MPZ (wtMPZ), a functional dominant negative effect was observed. Alternatively, S22_W28 deletion was retained within the cytoplasm and reduced both adhesion caused by wtMPZ and cellular viability. Since the same trafficking patterns were observed in transfected murine Schwann cells, they are not an artifact of heterologous cell expression. Our results suggest that at least some late-onset mutations cause a partial loss of function in the transfected cells, whereas multiple abnormal gain of function pathways can result in early-onset neuropathy. Further characterization of these pathways will lead to a better understanding of the pathogenesis of CMT1B and a rational basis for treating these debilitating inherited neuropathies.

Growth arrest specific 3 (Gas3)/peripheral myelin protein 22 (PMP22) is a component of the compact peripheral nerve myelin, and mutations affecting gas3/PMP22 gene are responsible for a group of peripheral neuropathies in humans. We have performed in vivo imaging in order to investigate in detail the phenotype induced by Gas3/PMP22 overexpression in cultured cells. Here we show that Gas3/PMP22 triggers the accumulation of vacuoles, before the induction of cell death or of changes in cell spreading. Overexpressed Gas3/PMP22 accumulates into two distinct types of intracellular membrane compartments. Gas3/PMP2 accumulates within late endosomes close to the juxtanuclear region, whereas in the proximity of the cell periphery, it induces the formation of actin/phosphatidylinositol (4,5)-bisphosphate(PIP2)-positive large vacuoles. Gas3/PMP22-induced vacuoles do not contain transferrin receptor, but instead they trap membrane proteins that normally traffic through the ADP-ribosylation factor 6 (Arf6) endosomal compartment. Arf6 and Arf6-Q67L co-localize with Gas3/PMP22 in these vacuoles,and the dominant negative mutant of Arf6, T27N, blocks the appearance of vacuoles in response to Gas3/PMP22, but not its accumulation in the late endosomes. Finally a point mutant of Gas3/PMP22 responsible for the Charcot-Marie-Tooth 1A disease is unable to trigger the accumulation of PIP2-positive vacuoles. Altogether these results suggest that increased Gas3/PMP22 levels can alter membrane traffic of the Arf6 plasma-membrane–endosomal recycling pathway and show that, similarly to other tetraspan proteins, Gas3/PMP22 can accumulate in the late endosomes.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with increased gene dosage for PMP22. Therapeutic approaches are currently aiming at correcting PMP22 over-expression. It is unknown whether PMP22 can be used as a biological marker of disease progression and therapy efficacy. We performed quantitative real-time polymerase chain reaction on skin biopsies of 45 patients with CMT1A, obtained at study entry and after 24-months of treatment either with ascorbic acid or placebo. Data of a subgroup of patients were also compared with matched healthy subjects. Finally, we analysed PMP22 messenger RNA levels in sural nerve biopsies. We did not find significant differences in the levels of any known PMP22 transcripts in treated or untreated patients with CMT1A, thus confirming that ascorbic acid does not impact on the molecular features of CMT1A. Most importantly, we did not observe any correlation between PMP22 messenger RNA levels and the different clinical and electrophysiological outcome measures, underscoring the weakness of PMP22 to mirror the phenotypic variability of patients with CMT1A. We did not find increased PMP22 messenger RNA levels in skin and sural nerve biopsies of patients with CMT1A compared with relative controls. In conclusion, this study shows that ascorbic acid does not impact on PMP22 transcriptional regulation and PMP22 is not a suitable biomarker for CMT1A.