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Previous clinical observations and data from mouse models with defects in lipid metabolism suggested that epineurial adipocytes may play a role in peripheral nervous system myelination. We have used adipocyte-specific Lpin1 knockout mice to characterize the consequences of the presence of impaired epineurial adipocytes on the myelinating peripheral nerve. Our data revealed that the capacity of Schwann cells to establish myelin, and the functional properties of peripheral nerves, were not affected by compromised epineurial adipocytes in adipocyte-specific Lpin1 knockout mice. To evaluate the possibility that Lpin1-negative adipocytes are still able to support endoneurial Schwann cells, we also characterized sciatic nerves from mice carrying epiblast-specific deletion of peroxisome proliferator-activated receptor gamma, which develop general lipoatrophy. Interestingly, even the complete loss of adipocytes in the epineurium of peroxisome proliferator-activated receptor gamma knockout mice did not lead to detectable defects in Schwann cell myelination. However, probably as a consequence of their hyperglycemia, these mice have reduced nerve conduction velocity, thus mimicking the phenotype observed under diabetic condition. Together, our data indicate that while adipocytes, as regulators of lipid and glucose homeostasis, play a role in nerve function, their presence in epineurium is not essential for establishment or maintenance of proper myelin.
Peripheral nerves are composed of three distinct tissue compartments: the epineurium, perineurium, and endoneurium. A substantial amount of studies have focused on the endoneurial compartment of the peripheral nerve, harboring Schwann cells surrounding axons, which communicate information between the central neural system and the periphery. The role of both perineurium and epineurium remains less clear. The perineurium, which is mostly composed of fibroblasts, is thought to play a role as a diffusion barrier between the endoneurium and the epineurium (Parmantier et al. 1999; Pina-Oviedo and Ortiz-Hidalgo 2008). The adipocyte-rich epineurium was previously suggested to play a role in the mechanical protection of the nerve from compression damage or to serve as a source of fatty acids for the endoneurium (Barkmeier and Luschei 2000; Verheijen et al. 2003; Moayeri and Groen 2009). Epineurial adipose tissue may also function as an endocrine organ (Deng and Scherer 2010). Inactivation of either of the above mentioned epineurial adipocyte functions may potentially lead to defects in PNS function.
Similar to the situation in adipocytes, strictly controlled lipid metabolism plays a crucial role in glial cell ability to produce and maintain myelin membrane (Chrast et al. 2011). Interestingly, a previously described mutation in the Lpin1 gene present in ‘fatty liver dystrophy’ (Lpin1fld/fld) mice affects both glial cells and adipocytes, leading to a demyelinating peripheral nerve neuropathy and epineurial lipoatrophy (Langner et al. 1991; Peterfy et al. 2001; Verheijen et al. 2003). We previously demonstrated that Schwann cell-specific Lpin1 inactivation contributes to the development of pronounced peripheral neuropathy (Nadra et al. 2008). Here, we use aP2Cre/+/LpfEx2-3/fEx2-3 mice, in which Lpin1 is deleted in adipocytes, to address whether the compromised function of epineurial adipocytes also contributes to the neuropathy observed in Lpin1fld/fld mice. In addition, we have used recently generated Sox2Cre/+/PPARγemL–/L– mice, which completely lack adipocytes as a consequence of epiblastic deletion of peroxisome proliferator-activated receptor gamma-γ (Pparg) (Nadra et al. 2010), to evaluate the consequence of lipoatrophy present in this model on myelinated Schwann cells. Our characterization of these two models shows that proper function or presence of epineurial adipocytes is not critical for establishment or maintenance of Schwann cell myelin.
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Our data, generated through analysis of the aP2Cre/+/LpfEx2-3/fEx2-3 and Sox2Cre/+/PPARγemL–/L– knockout mice, present the first insight into the consequences of either disturbed epineurial adipocyte function or of complete adipocyte absence on peripheral nerve myelination.
Our analysis of PNS morphology and function in aP2Cre/+/LpfEx2-3/fEx2-3 knockout mice indicated that the functional integrity of epineurial adipocytes does not play a major role in myelination. This conclusion is further strengthened by the data from the analysis of Sox2Cre/+/PPARγemL–/L– mice. In accordance with the known requirement of PPARγ for adipocyte differentiation (Rosen et al. 2000), the PPARγ null mice have no BAT or WAT, thus providing a unique model to study the physiological consequences of a complete congenital lipodystrophy on peripheral nerve myelination. Contrary to previously described PNS deficits in Lpin1 complete knockout mice (Lpin1fld/fld), which also develops severe lipodystrophy (Langner et al. 1991; Verheijen et al. 2003), the Sox2Cre/+/PPARγemL–/L– mice show no detectable structural myelin abnormalities despite complete absence of epineurial fat.
It is possible that lipids originating from non-glial endoneurial cells and/or from the circulation compensate through horizontal lipid transfer for lipid-compromised epineurial adipocytes, as previously suggested in the situation of glia-specific lipid mutants (Saher et al. 2005; Verheijen et al. 2009). Alternatively, Schwann cells may increase their lipid synthesis to respond to reduced and/or abolished epineurial lipid input. Both of these possibilities will require further exploration.
The observed slight increase (~50%) in the level of expression of Mpz and Pmp22 in aP2Cre/+/LpfEx2-3/fEx2-3 may potentially have pathological consequences as, in humans, gene dosage variation affecting genes encoding myelin proteins leads to peripheral neuropathy (Lupski et al. 1991; Maeda et al. 2012). However, previous data from mice suggest that for Pmp22, approximately 100% over-expression and for MPZ approximately 80% over-expression is necessary to reproduce this phenotype (Magyar et al. 1996; Huxley et al. 1998; Wrabetz et al. 2000). The absence of the PNS phenotype in aP2Cre/+/LpfEx2-3/fEx2-3 mice, where the level of Pmp22 and Mpz mRNA over-expression is 50–60%, therefore, indicates that the level of Pmp22 and Mpz over-expression is not reaching the threshold necessary to induce a detectable PNS phenotype and/or that this over-expression is absent during the process of myelination which, in mice, is mostly completed during the first post-natal month. In addition, the fact that we observed increased Pmp22 and Mpz mRNA expression level in the absence of increased Krox20 levels provides supplementary evidence for the previously suggested role of Krox20-independent myelin gene activation (Parkinson et al. 2003).
Interestingly, we have detected a reduced MNCV in Sox2Cre/+/PPARγemL–/L– animals. A reduced nerve conduction velocity was previously observed in both type I and type II diabetic situations even before the presence of PNS structural changes (de Preux Charles et al. 2010; Zenker et al. 2012). It is therefore possible that the reduced MNCV, which is present in Sox2Cre/+/PPARγemL–/L– mice and not accompanied by any detectable nerve structural changes, is a consequence of their substantial hyperglycemia.
Although we cannot formally exclude that defective or missing epineurial adipocytes in aP2Cre/+/LpfEx2-3/fEx2-3, and Sox2Cre/+/PPARγemL–/L– mice, respectively, do not lead to subtle changes in lipid composition of peripheral nerve myelin, the comparison between the data presented in this study and previously observed strong myelin defects in peripheral nerves of animals with Schwann cell-specific lipid defects (Nadra et al. 2008; Verheijen et al. 2009) further strengthen the concept that myelinating glial cells in the PNS predominantly rely on local, cell autonomous, lipid metabolism.