Initially, we studied PMP-22 expression in 3-month old mouse brain by Western blotting. As shown in , the PMP-22 protein could not be detected in total brain homogenate even when 100 μg of total protein was analyzed. In comparison, PMP-22 expression was found in the crude myelin fraction isolated from total brain, although its levels were considerably lower than those found in the sciatic nerve (). To substantiate the expression of PMP-22 in CNS white matter, purified myelin fractions were subsequently prepared from total brain and forebrain and analyzed using two different polyclonal antibodies (). As shown on the Western blot, PMP-22 protein expression was detected in myelin isolated from both regions.
Figure 1 Detection of PMP-22 protein in CNS myelin by Western blotting. (A) Crude myelin fraction was isolated from total brain of a 3-month old mouse by density gradient centrifugation and myelin proteins were analyzed by Western blot with a rabbit polyclonal (more ...)
We next determined whether PMP-22 could be detected in myelin from human brain. Myelin was isolated from frozen samples dissected from Brodmann area 9 obtained from three subjects. As shown in , in each of the three human brain myelin samples, a band that migrated slightly slower than mouse PMP-22 was detected. This observation is consistent with previous reports indicating that human PMP-22 has a slowed mobility as compared to the mouse protein, probably due to altered post-translational modifications [18
]. To further demonstrate that the PMP-22 core protein was present, myelin extracts were treated with PNGase F to remove the N-linked carbohydrate side chain ([19
]). Treatment with PNGase F resulted in an approximately 4 kDa shift in the mobility of PMP-22, with a complete disappearance of the 22-kDa band and the appearance of an approximately 18 kDa band (). This pattern was consistent among all four samples analyzed and confirms the expression of the PMP-22 protein in mouse and human white matter.
Figure 2 (A) Myelin was isolated from frozen human brain samples from three different subjects and analyzed for PMP-22 expression. Gels were loaded with 25–30 μg of protein from human or mouse brain (MuBr) myelin fractions or with 50 ng of a mouse (more ...)
Although the presence of the PMP-22 mRNA has been documented in both rodent and human brain, detection of the protein has been challenging probably due to its low level of expression. Here we show that PMP-22 protein can be detected by Western blotting in myelin isolated from both mouse and human brain. We have not been able to detect PMP-22 protein by immunohistochemical staining of either frozen sections or perfusion-fixed Vibratome sections from adult mouse brain (data not shown). We suspect that this is likely due to the low level of protein expression and possibly the masking of the antigen in the lipid-rich CNS myelin.
PMP-22 is thought to function as a modulator of cell-cell or cell-matrix interactions that are important for myelin formation and maintenance [10
]. Misexpression of the protein is associated with hereditary demyelinating peripheral neuropathies in humans. Deletion of PMP-22 is linked with hereditary neuropathy with liability to pressure palsies (HNPP), while duplication of the gene is associated with Charcot-Marie-Tooth disease type 1A [10
]. CNS white matter lesions have been detected in families with HNPP [21
], although the reasons for these findings were unclear. The present demonstration of PMP-22 expression in brain white matter suggests that this protein may play a role in the proper maintenance and function of CNS myelin and points to a need for further studies addressing its role in normal CNS function and disease. Our data also provide an explanation for why changes in PMP-22 expression may be relevant to the pathophysiology of schizophrenia [4