PERIPHERAL MYELIN PROTEIN-22 IS EXPRESSED IN CNS MYELIN

doi:10.2478/v10134-010-0038-3

Transl Neurosci. Author manuscript; available in PMC 2011 May 12.

Published in final edited form as:

Transl Neurosci. 2010 December 1; 1(4): 282–285.

doi: 10.2478/v10134-010-0038-3

PMCID: PMC3093192

NIHMSID: NIHMS284950

PERIPHERAL MYELIN PROTEIN-22 IS EXPRESSED IN CNS MYELIN

Rita De Gasperi,^1,³ Miguel A. Gama Sosa,^1,³ Zuzanna Naumowicz,^1,³ Patrick R. Hof,^4,⁵ Lucia Notterpek,⁸ Kenneth L. Davis,³ Joseph D. Buxbaum,^3,^4,⁶ and Gregory A. Elder^2,^3,⁷^*

¹ Research and Development, James J. Peters Department of Veterans Affairs Medical Center, Bronx, New York 10468, USA

² Neurology Services, James J. Peters Department of Veterans Affairs Medical Center, Bronx, New York 10468, USA

³ Department of Psychiatry, Mount Sinai School of Medicine, New York, New York 10029, USA

⁴ Department of Neuroscience, Mount Sinai School of Medicine, New York, New York 10029, USA

⁵ Department of Geriatrics and Palliative Care, Mount Sinai School of Medicine, New York, New York 10029, USA

⁶ Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York 10029, USA

⁷ Department of Neurology, Mount Sinai School of Medicine, New York, New York 10029, USA

⁸ Department of Neuroscience, College of Medicine, McKnight Brain Institute of the University of Florida, Gainesville, Florida 32610, USA

^* ; Email: gregory.elder/at/mssm.edu

Abstract

Myelin abnormalities exist in schizophrenia leading to the hypothesis that oligodendrocyte dysfunction plays a role in the pathophysiology of the disease. The expression of the mRNA for the peripheral myelin protein-22 (PMP-22) is decreased in schizophrenia and recent genetic evidence suggests a link between PMP-22 and schizophrenia. While PMP-22 mRNA is found in both rodent and human brain it has been generally thought that no protein expression occurs. Here we show that PMP-22 protein is present in myelin isolated from adult mouse and human brain. These results suggest that PMP-22 protein likely plays a role in the maintenance and function of central nervous system (CNS) myelin and provide an explanation for why altered PMP-22 expression may be pathophysiologically relevant in a CNS disorder such as schizophrenia.

Keywords: Myelin, Peripheral myelin protein-22, Schizophrenia

1. Introduction

Multiple lines of evidence including molecular studies, neuroimaging, as well as histopathological and ultrastructural analyses have demonstrated myelin abnormalities in schizophrenia, leading to the hypothesis that oligodendrocyte dysfunction plays a central role in its pathophysiology (reviewed in [1,2]). In particular, both microarray and quantitative polymerase chain reaction studies showed that expression of a subset of myelin-related genes is decreased in postmortem brain in schizophrenia [3–7]. Peripheral myelin protein-22 (PMP-22) is one of the myelin-related genes whose RNA is decreased in schizophrenia [4,6]. Both a copy number variation in PMP-22 as well as a deletion in chromosome 17p12 which contains the PMP-22 gene have been linked to schizophrenia [8].

PMP-22 is a transmembrane protein whose expression is most abundant in compact myelin of the peripheral nervous system (PNS) [9] where it is important for the establishment and maintenance of myelin [10]. The gene has two alternatively spliced forms containing either exon 1A or 1B, respectively, which are regulated by use of an alternative promoter with form 1A being myelin-specific, whereas form 1B is expressed more broadly [11]. As both exons 1A and B are non-coding, the two forms produce the same protein.

PMP-22 was initially identified as growth arrest specific gene 3 (gas 3) and subsequently as a protein whose expression is severely decreased post-peripheral nerve injury [12]. PMP-22 mRNA is detected in both rodent [9,13] and human brain [14] where relatively high levels of expression are found in the corpus callosum by Northern blotting. However, no protein expression has been detected in Western blots of total brain homogenates [9,13]. Recently it was suggested that the lack of PMP-22 protein expression in the CNS, despite the presence of RNA, results from the fact that in developing oligodendrocytes translation of PMP-22 RNA is negatively regulated by the microRNA mir-9 [15]. Therefore, although present in PNS myelin, PMP-22 is not considered a component of CNS myelin.

Given the apparent lack of PMP-22 protein expression in the CNS, it was surprising that genetic studies identified a link between PMP-22 and schizophrenia [8]. This led us to reexamine whether PMP-22 protein is expressed in the CNS, particularly focusing on white matter. Here we show that PMP-22 protein is present in myelin isolated from adult mouse and human brain.

2. Experimental Procedures

2.1 Isolation of myelin

Myelin was prepared from 3-month old adult mouse brain by density gradient centrifugation [16]. Whole brain or forebrain with brainstem and cerebellum removed were homogenized in 20 mM Tris HCl, pH 7.45, 2 mM EDTA, 1 mM DTT (Tris-HCl buffer), supplemented with Halt protease and phosphatase inhibitor cocktail (Pierce Biotechnology, Rockford Il, USA) and containing 0.3 M sucrose. The homogenate was layered on 0.83 M sucrose in the above buffer and centrifuged at 35,000 g for 35 minutes. The myelin membranes were collected at the interphase of the sucrose solutions and further purified by hypo-osmotic shock in Tris HCl buffer yielding a crude myelin extract. To obtain purified myelin, the sucrose gradient centrifugation and hypo-osmotic shock were repeated once more. For Western blot analysis the myelin fraction was solubilized in 62.5 mM Tris HCl pH 6.8, 10% glycerol, 3% sodium dodecyl sulfate (SDS). As a positive control, mouse sciatic nerves were dissected and extracts prepared as described in Amici et al. [17].

For isolation of myelin from human brain, frozen samples dissected from Brodmann area 9 (~1 g each) were obtained from 3 normal cases (ages: 52, 81, 86, years; postmortem interval: average 11.9 hours, range 2.4–21.7 hours) from the Brain Bank of the Department of Psychiatry of the Mount Sinai School of Medicine/James J. Peters VA Medical Center. Myelin was isolated as described above.

2.2 Western blotting

Protein concentrations were determined by the BCA reagent (Pierce) and proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). The membranes were probed overnight at 4°C with either an affinity polyclonal antibody, (Abcam, Cambridge, UK, Ab 61220 diluted 1:750) or a polyclonal anti-PMP-22 antibody raised in chicken (L. Notterpek et al. unpublished, diluted 1:1,000). Both antibodies were diluted in TBST (50 mM Tris HCl pH 7.4, 1% Tween) containing 5% low-fat dry milk. After incubation with anti-rabbit horseradish peroxidase (HRP; GE Healthcare Bio-Sciences Corporation, Piscataway, NJ, USA; 1: 5,000 in blocking solution) or anti-chicken IgY-HRP (Jackson Immunolabs, West Grove, PA; 1:100,000) binding of secondary antibodies was visualized with the ECL+ reagent (GE Healthcare Bio-Sciences Corporation). In some experiments myelin extracts were treated for 2 hours at 37°C with PNGase F (New England Biolabs, Ipswich, MA USA) according to the manufacturer’s instructions and analyzed by Western blotting.

3. Results and Discussion

Initially, we studied PMP-22 expression in 3-month old mouse brain by Western blotting. As shown in Figure 1A, 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 (Figure 1A). 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 (Figure 1B). 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 Figure 2A, 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 (Figure 2B). 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 [10,17,20]. 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 [10,12]. 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 [4,6,8].

Acknowledgments

This research was supported by the Mount Sinai Silvio Conte Neuroscience Center (NIH grant P50 MH36692). We thank Dr. Vahram Haroutunian for providing frozen samples of postmortem human brain.

References

1. Davis KL, Stewart DG, Friedman JI, Buchsbaum M, Harvey PD, Hof PR, et al. White matter changes in schizophrenia: evidence for myelin-related dysfunction. Arch Gen Psychiat. 2003;60:443–456. [PubMed]

2. Hoistad M, Segal D, Takahashi N, Sakurai T, Buxbaum JD, Hof PR. Linking white and grey matter in schizophrenia: oligodendrocyte and neuron pathology in the prefrontal cortex. Front Neuroanat. 2009;3:9. [PMC free article] [PubMed]

3. Aston C, Jiang L, Sokolov BP. Microarray analysis of postmortem temporal cortex from patients with schizophrenia. J Neurosci Res. 2004;77:858–866. [PubMed]

4. Dracheva S, Davis KL, Chin B, Woo DA, Schmeidler J, Haroutunian V. Myelin-associated mRNA and protein expression deficits in the anterior cingulate cortex and hippocampus in elderly schizophrenia patients. Neurobiol Dis. 2006;21:531–540. [PubMed]

5. Hakak Y, Walker JR, Li C, Wong WH, Davis KL, Buxbaum JD, et al. Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc Natl Acad Sci USA. 2001;98:4746–4751. [PubMed]

6. Katsel P, Davis KL, Haroutunian V. Variations in myelin and oligodendrocyte-related gene expression across multiple brain regions in schizophrenia: a gene ontology study. Schizophren Res. 2005;79:157–173. [PubMed]

7. Tkachev D, Mimmack ML, Ryan MM, Wayland M, Freeman T, Jones PB, et al. Oligodendrocyte dysfunction in schizophrenia and bipolar disorder. Lancet. 2003;362:798–805. [PubMed]

8. Kirov G, Grozeva D, Norton N, Ivanov D, Mantripragada KK, Holmans P, et al. Support for the involvement of large copy number variants in the pathogenesis of schizophrenia. Hum Mol Genet. 2009;18:1497–1503. [PMC free article] [PubMed]

9. Snipes GJ, Suter U, Welcher AA, Shooter EM. Characterization of a novel peripheral nervous system myelin protein (PMP-22/SR13) J Cell Biol. 1992;117:225–238. [PMC free article] [PubMed]

10. Suter U, Scherer SS. Disease mechanisms in inherited neuropathies. Nat Rev Neurosci. 2003;4:714–726. [PubMed]

11. Suter U, Snipes GJ, Schoener-Scott R, Welcher AA, Pareek S, Lupski JR, et al. Regulation of tissue-specific expression of alternative peripheral myelin protein-22 (PMP22) gene transcripts by two promoters. J Biol Chem. 1994;269:25795–25808. [PubMed]

12. Naef R, Suter U. Many facets of the peripheral myelin protein PMP22 in myelination and disease. Microsc Res Tech. 1998;41:359–371. [PubMed]

13. Parmantier E, Cabon F, Braun C, D’Urso D, Muller HW, Zalc B. Peripheral myelin protein-22 is expressed in rat and mouse brain and spinal cord motoneurons. Eur J Neurosci. 1995;7:1080–1088. [PubMed]

14. Ohsawa Y, Murakami T, Miyazaki Y, Shirabe T, Sunada Y. Peripheral myelin 22 is expressed in human central nervous system. J Neurol Sci. 2006;247:11–15. [PubMed]

15. Lau P, Verrier JD, Nielsen JA, Johnson KR, Notterpek L, Hudson LD. Identification of dynamically regulated microRNA and mRNA networks in developing oligodendrocytes. J Neurosci. 2008;28:11720–11730. [PMC free article] [PubMed]

16. Norton WT, Poduslo SE. Myelination in rat brain: method of myelin isolation. J Neurochem. 1973;21:749–757. [PubMed]

17. Amici SA, Dunn WA, Jr, Murphy AJ, Adams NC, Gale NW, Valenzuela DM, et al. Peripheral myelin protein 22 is in complex with alpha6beta4 integrin, and its absence alters the Schwann cell basal lamina. J Neurosci. 2006;26:1179–1189. [PubMed]

18. Miyazaki T, Takeda Y, Murakami Y, Kawano H, Shimazu T, Toya S, et al. Distribution of PASII/PMP22 and connexin 32 proteins in the peripheral nervous system. Neurochem Int. 1995;27:377–383. [PubMed]

19. Pareek S, Suter U, Snipes GJ, Welcher AA, Shooter EM, Murphy RA. Detection and processing of peripheral myelin protein PMP22 in cultured Schwann cells. J Biol Chem. 1993;268:10372–10379. [PubMed]

20. Jetten AM, Suter U. The peripheral myelin protein 22 and epithelial membrane protein family. Prog Nucleic Acid Res Mol Biol. 2000;64:97–129. [PubMed]

21. Sanahuja J, Franco E, Rojas-Garcia R, Gallardo E, Combarros O, Begue R, et al. Central nervous system involvement in hereditary neuropathy with liability to pressure palsies: description of a large family with this association. Arch Neurol. 2005;62:1911–1914. [PubMed]