Human Molecular Genetics

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Human Molecular Genetics, 2003, Vol. 12, No. 14 1713-1723
DOI: 10.1093/hmg/ddg179
© 2003 Oxford University Press

Myotubularin-related 2 protein phosphatase and neurofilament light chain protein, both mutated in CMT neuropathies, interact in peripheral nerve

Stefano C. Previtali¹, Barbara Zerega², Diane L. Sherman³, Peter J. Brophy³, Giorgia Dina¹, Rosalind H.M. King⁴, Mustafa M. Salih⁵, Laura Feltri⁶, Angelo Quattrini¹, Roberto Ravazzolo⁷, Lawrence Wrabetz⁶, Anthony P. Monaco⁸ and Alessandra Bolino^2,*

¹Neuropathology Unit, Department of Neurology, San Raffaele Scientific Institute, 20132 Milano, Italy, ²Laboratory of Molecular Genetics, Gaslini Institute and Dulbecco Telethon Institute, 16148 Genova, Italy, ³Department of Preclinical Veterinary Sciences, University of Edinburgh, Edinburgh EH9 1QH, UK, ⁴Department of Clinical Neurosciences, Royal Free and University College School of Medicine, Rowland Hill Street, London NW3 2PF, UK, ⁵Division of Pediatric Neurology, Department of Pediatrics, College of Medicine, King Saud University, Riyadh, Saudi Arabia, ⁶DIBIT, San Raffaele Scientific Institute, 20132 Milano, Italy, ⁷Laboratory of Molecular Genetics, Gaslini Institute and Department of Pediatrics and CEBR, University of Genova, Genova, Italy and ⁸Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK

Received March 17, 2003; Accepted May 9, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Charcot–Marie–Tooth disease type 4B1, CMT4B1, is a severe, autosomal-recessive, demyelinating peripheral neuropathy, due to mutations in the Myotubularin-related 2 gene, MTMR2. MTMR2 is widely expressed and encodes a phosphatase whose substrates include phosphoinositides. However, this does not explain how MTMR2 mutants specifically produce demyelination in the peripheral nerve. Therefore, we analysed the cellular and subcellular distribution of Mtmr2 in nerve. Mtmr2 was detected in all cytoplasmic compartments of myelin-forming Schwann cells, as well as in the cytoplasm of non-myelin-forming Schwann cells and both sensory and motorneurons. In contrast, Mtmr2 was detected in the nucleus of Schwann cells and motorneurons, but not in the nucleus of sensory neurons. As Mtmr2 is diffusely present also within the nerve, a specific function could derive instead from nerve-specific interacting proteins. Therefore, we performed two yeast two-hybrid screenings, using either fetal brain or peripheral nerve cDNA libraries. The neurofilament light chain protein, NF-L, was identified repeatedly in both screenings, and found to interact with MTMR2 in both Schwann cells and neurons. Interestingly, NF-L, encoding NF-L, is mutated in CMT2E. These data may provide a basis for the nerve-specific pathogenesis of CMT4B1, and further support for the notion that hereditary demyelinating and axonal neuropathies may represent different clinical manifestations of a common pathological mechanism.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Charcot–Marie–Tooth disease type 4B (CMT4B) is an autosomal-recessive peripheral neuropathy characterized by childhood onset; symmetrical distal and proximal muscular weakness starting at the lower extremities, sensory loss and severely decreased nerve conduction velocity. A hallmark of this disorder is the presence in peripheral nerves of redundant and irregular loops of myelin wrapping the axon, called myelin outfoldings (1,2). As the disease progresses, patients often become wheelchair-bound by the third decade of life and, in more severe cases, death follows, probably due to respiratory failure (3). Although CMT4B displays a homogeneous clinical picture, this disorder has been linked to at least two different loci, CMT4B1 located on chromosome 11q22 and CMT4B2 on chromosome 11p15 (4,5). We first demonstrated that CMT4B1 is caused by homozygous loss of function mutations in the Myotubularin-related 2 gene, MTMR2 (6–8). More recently, mutations in the MTMR13/SBF2 gene on chromosome 11p15 have been found to be responsible for pure CMT4B2 with myelin outfoldings as well as for CMT4B2 associated with infantile glaucoma (9,10). Both MTMR2 and MTMR13 belong to the Myotubularin-related (MTMR) family of dual specificity PTP-like protein phosphatases (11,12). It has recently been shown that MTMR2 possess enzymatic activity toward the phosphatidylinositol 3-phosphate, PI(3)P, both in vitro (13,14) and in vivo, using S. pombe (13). Subsequently, Berger et al. (15) reported that in vitro MTMR2 has a preferential phosphatase activity toward the phosphatidylinositol 3,5-biphosphate, PI(3,5)P2 and not PI(3)P. Despite these findings, a specific biological function for this phosphatase in the peripheral nerve still remains to be assessed and it is not suggested by its ubiquitous expression or by its enzymatic activity toward phospholipids. Even taking into account the characteristic morphological alterations observed in the peripheral nerve of CMT4B patients, it is not clear whether this demyelinating phenotype with myelin outfoldings arises from a primary neuronal defect, followed by secondary demyelination, or, conversely, from a primary Schwann cell perturbation leading to axonal degeneration.

Therefore, to elucidate the cell-autonomous role of MTMR2 in Schwann cells, neurons or both we decided to investigate in more detail the expression pattern of this protein in the peripheral nervous system. Its ubiquitous expression has only been demonstrated at the mRNA level in both human and mouse (7,15,16). We detected Mtmr2 protein in vivo in the cytoplasm of Schwann cells, DRG sensory neurons and motorneurons, as well as in their axonal processes. Mtmr2 was also observed in the nucleus of Schwann cells and motorneurons but not in the nucleus of sensory neurons. Localization of endogenous Mtmr2 was also demonstrated in vitro using purified Schwann cells and Schwann cell/sensory neuron co-cultures.

Since the expression pattern of MTMR2 did not suggest a specific function in peripheral nerve, we sought nerve-specific interactors. To identify these protein partners, we performed two independent yeast two-hybrid screenings using cDNA libraries from human fetal brain or rat sciatic nerve, highly enriched in Schwann cell mRNAs. The neurofilament light chain protein, NF-L, mutated in CMT2E and in severe forms of CMT often diagnosed as Dejerine-Sottas syndrome, was found to interact with MTMR2 in Schwann cells as well as in neurons. Since NF-L is specifically expressed in the nervous system, the interaction between MTMR2 and NF-L may explain why loss of a ubiquitously expressed phosphatase affects specifically the nerve.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mtmr2 is expressed in both Schwann cells and neurons in vivo
To dissect the expression pattern of MTMR2 in the peripheral nervous system we employed four different polyclonal antibodies. A first anti-rat MTMR2 antibody was raised against a 22 amino acid peptide (amino acids 156–177) of the rat Mtmr2 protein as already reported (17). The other three, named M42, M52 and M53, were raised against two different peptides from the N-terminus of the human MTMR2 protein and characterized by western blotting, using COS-7 cells transfected with Flag-tagged MTMR2 cDNA. A common band of 70 kDa, corresponding to the MTMR2 protein, was recognized by the M42, M52, and M53 antibodies as well as by the anti-Flag antibody (Fig. 1A). To confirm the specificity of MTMR2 antibodies, we performed immunohistochemistry in a muscle biopsy sample from a patient affected by CMT4B with loss of function mutation in MTMR2 as compared with a control muscle. We did not detect any MTMR2 staining in the muscle of the CMT4B patient, whereas cytoplasmic staining was observed in the control (data not shown and De Sandre-Giovannoli et al., manuscript in preparation).

View larger version (119K): [in this window] [in a new window]   Figure 1. Expression of Mtmr2 in the peripheral nervous system. (A) Characterization of myotubularin antibodies by western blotting in COS-7 cells transfected with Flag-tagged MTMR2 cDNA; M42, M52, M53 recognized a band of 70 kDa corresponding to the band identified by anti Flag antibody. The faint band below might represent a degradation product or the result of post-translational modification and it has also been revealed using anti-Flag antibody. (B, C) immunohistochemical localization of Mtmr2 in rat sciatic nerve by double staining using anti-MTMR2 and anti-NF antibodies; Mtmr2 is expressed in Schwann cells and in axons (merge in C). Mtmr2 is expressed in the cytoplasm of myelin forming Schwann cells, as shown by double staining with S100ß (D–F), as an external ring to myelin (G–I), and in teased nerve fibres, especially in paranodal area (arrows in M and N) and in Schmidt–Lanterman incisures (arrowhead in N). Expression of Mtmr2 in non-myelin forming Schwann cells is shown in double staining with GFAP (J–L). Expression of Mmtr2 is also shown in motorneuron (O), DRG sensory neurons and satellite cells (arrows) (P), motor root (Q) and sensory root (R). Bar=30 µm (B, C, O–R); 10 µm (D–L); 20 µm (M and N).

 
All the anti-MTMR2 antibodies showed similar results in both rat and mouse tissues. In the sciatic nerve, we clearly detected Mtmr2 staining in the cytoplasm of both myelin forming and non-myelin forming Schwann cells. S100ß, specifically expressed in the Schwann cell cytoplasm, co-localized with Mtmr2 in the cytoplasm of both myelin forming and non-myelin forming Schwann cells, which are identified by MBP and GFAP staining, respectively (Fig. 1C, D–F, G–I and J–L). Finally, Mtmr2 staining was also detected in axons, as shown by double staining with NF-M (Fig. 1B and C).

Single teased nerve fibres confirmed Mtmr2 expression in Schwann cell cytoplasm. Positive staining was detected in paranodes, Schmidt–Lanterman incisures, cytoplasmic layer external to myelin sheath, perinuclear region and in the nucleus (Fig. 1M and N).

We further investigated if Mtmr2 was expressed in the cell body of motorneurons and DRG sensory neurons that send axons into the peripheral nerve. As shown in Figure 1, motorneurons in the ventral horns of spinal cord stained for Mtmr2 in both cytoplasm and nucleus (Fig. 1O), whereas DRG sensory neurons showed a diffuse Mtmr2 staining in the cytoplasm but not in the nucleus (Fig. 1P). In addition, satellite cells, the glial cells in DRG, also stained for Mtmr2 (Fig. 1P, arrows). Finally, both axons and Schwann cells in motor and sensory roots were positively stained for Mtmr2 (Fig. 1Q and R).

Schwann cells and neurons synthesize Mtmr2 in vitro
To further confirm the expression of Mtmr2 in Schwann cells and neurons, we performed northern blot analysis and immunohistochemistry in purified primary cultures. Mtmr2 mRNA expression in neurons has already been reported (15,16). Purified Schwann cells expressed Mtmr2 mRNA both in the presence or absence of forskolin, an inducer of cAMP that mimics axonal signals in vitro (18) (Fig. 2A). Anti-MTMR2 antibodies stained the cytoplasm and nucleus of Schwann cells (Fig. 2B and C). To confirm the expression of Mtmr2 also in myelin-forming Schwann cells, we established Schwann cell–DRG neuron co-cultures since Schwann cells seeded on DRG neurons can myelinate axons in vitro. Even in vitro, myelin forming Schwann cells (identified by staining for MBP) showed diffuse cytoplasmic and nuclear staining (Fig. 2D). Similarly to in vivo studies, DRG sensory neurons showed Mtmr2 staining exclusively in the cytoplasm (Fig. 2E).

View larger version (98K): [in this window] [in a new window]   Figure 2. Expression of Mtmr2 in Schwann cells. Northern blot analysis (A) and immunohistochemical detection of MTMR2 in primary culture of Schwann cells (B, C) and Schwann cell-DRG neuron co-culture (D, E). (A) Using as a probe the full length of rat Mtmr2, the corresponding mRNA was detected in rat Schwann cells grown both in the presence or in the absence of forskolin. MTMR2 was not detectable in N20, an oligodendrocyte cell line, although the amount of total RNA was low, whereas sciatic nerve is shown as positive control. The presence of mRNA in all lanes was verified by hybridizing with a probe specific for GAPDH. (B, C) immunocytochemical detection of Mtmr2 in the cytoplasm and nucleus (identified by DAPI in C) of primary cultured Schwann cells. (D) Mtmr2 expression was equally observed in myelin-forming (identified by MBP, arrow) and non-myelin-forming Schwann cells (arrowheads) co-cultured with DRG sensory neurons. (E) Localization of Mtmr2 in the cytoplasm and nucleus of satellite cells (arrowhead), and in the cytoplasm but not nucleus of DRG sensory neurons in co-culture (arrow).

 
MTMR2/Mtmr2 interacts with NF-L
Since the expression pattern of Mtmr2 in nerve was not suggestive, we hypothesized that a specific role in the nerve might derive from nerve-specific interactors. To identify such proteins, we performed two independent yeast two-hybrid screenings using MTMR2/Mtmr2 as baits. In a first experiment, a 1972 bp ORF of human MTMR2 cDNA, carrying the aspartate at codon 320 mutated to alanine, was used as a bait to screen a cDNA library from human fetal brain. Codon 320 corresponds to the aspartate in position 278 of Myotubularin, MTM1, which is predicted to be the catalytic residue of protein phosphotyrosine phosphatases (PTPs) responsible for the release of the substrate (19). In MTM1 its replacement with alanine abolishes the enzymatic activity of the protein and causes a re-localization to the plasma membrane in transfected HeLa cells (20,21). Thus, this mutant might act as a substrate trap increasing the affinity of the enzyme for its substrate, as shown for other PTPs and dual specificity phosphatases (19). Among more than 6x10⁶ colonies, 44 colonies grew on selective plates, thus demonstrating activation of the expression of the HIS3 reporter gene belonging to the MaV203 yeast strain. After evaluating the expression of the remaining two reporter genes of this strain, LacZ and URA3 and reassessment of the interaction using individually isolated rescued plasmids and re-transformation in MaV203, four positive clones were confirmed. Two of them were found to contain a partial cDNA, encoding the neurofilament light protein, NF-L (amino acids 1–500; Fig. 3A). A similar degree of ß-gal activity was found using the wild-type MTMR2 cDNA as a bait.

View larger version (23K): [in this window] [in a new window]   Figure 3. Interaction of NF-L and MTMR2. (A) Structure of the NF-L protein of 543 amino acids in length and identification of the NF-L putative binding domain for MTMR2. Deletion constructs of NF-L were made as PCR amplification products of the full-length NF-L cDNA and cloned into SalI and NotI sites of the pPC86 vector, in frame with the GAL4 activating domain. Interaction between these constructs and MTMR2 cDNA cloned into the pDBLeu vector was assessed by monitoring the ß-gal activity in a yeast two-hybrid assay, using the MaV203 yeast strain. Numbers flanking the constructs refer to their amino acid length. (+++) is the blue colour visualized after less than 3 h incubation of colonies (ProQuest Yeast Two-Hybrid system, Life Technologies). (++) is the colour visualized after 8 h and (-) is indicating that no colour was observed after 24 h. The region of the NF-L protein from amino acids 255–400 still interacting with MTMR2 was determined by sequencing the 63 NF-L cDNA fragments cloned into the pACT-II vector. These 63 cDNA clones were found to be positive in a second yeast two-hybrid screening performed using a rat sciatic nerve cDNA library and a Mtmr2 rat cDNA as a bait. (B) Structure of the MTMR2 protein of 643 amino acids in length and identification of the MTMR2 putative binding domain for NF-L: PH-G, pleckstrin homology-GRAM domain; RID, Rac1-induced localization domain; PTP, phosphotyrosine phosphatase protein-like domain; SID, SET interacting domain; PDZ-BD, PDZ-binding domain. Deletion constructs of MTMR2 cloned into SalI and NotI sites pDBLeu vector in frame with the GAL4 binding domain were tested in a yeast two-hybrid assay with NF-L cloned into pPC86 vector. Interaction was determined as above, by monitoring the ß-gal activity.

 
To define a putative domain of interaction with MTMR2, deletion constructs of NF-L were generated and interaction with MTMR2 was assessed by monitoring ß-gal activity. In this way, a putative domain of interaction was found to correspond to amino acids 250–430 of NF-L, encoding the third -helical structure of the rod region of the protein. Similarly, a putative binding site for NF-L was demonstrated in MTMR2 (amino acids 160–300, Fig. 3B), where a Rac1-induced localization domain (RID) has been mapped (21). In order to identify specific interactors of MTMR2 in Schwann cells, we also performed a second yeast two-hybrid screening using a rat sciatic nerve cDNA library (22). As a bait, a rat 1710 bp Mtmr2 cDNA was used. On a total of 1x10⁶ yeast transformants, 430 clones were growing on selective plates. Among these clones, 339 were found to activate expression of the LacZ reporter gene of the Y190 yeast strain. After reassessment of interaction, 118 clones were confirmed, 63 of which represented partial rat Nf-l cDNAs. On the basis of the length of the cDNA inserts determined by sequencing these 63 clones, a smallest region (amino acids 255–400) still able to interact and thereby activate expression of the reporter genes was found. This region corresponds to the putative protein-protein interaction domain of human NF-L, as assessed by creating deletion constructs of NF-L and monitoring the ß-gal activity (Fig. 3A).

Further evidence that MTMR2 interacts with NF-L
To provide further evidence that MTMR2 interacts with NF-L, we performed co-immunoprecipitation experiments using transfected cells. COS-7 cells were first transiently co-transfected with Flag-tagged MTMR2 and NF-L cloned into pcDNA 3.1 vector. When lysates were immunoprecipitated with anti-Flag antibody and immunoblotted using anti-NF-L antibody, the NF-L protein was revealed. NF-L was detected in immunoprecipitates of MTMR2-Flag co-transfected cells but not in cells transiently transfected with the Flag-tagged empty vector or in cells not transfected (Fig. 4A). COS-7 cells were then transiently co-transfected with NF-L cloned into pcDNA-Myc vector and MTMR2 Flag-tagged and the lysates were immunoprecipitated using an anti-Myc antibody. After western blot analysis of the immunoprecipitates, using anti-Flag (not shown) or anti-MTMR2 antibodies, we were also able to detect co-immunoprecipitation of MTMR2 (Fig. 4B).

View larger version (41K): [in this window] [in a new window]   Figure 4. MTMR2 interacts with NF-L in transfected COS7 cells. (A) COS-7 cells were transiently transfected with Flag empty vector alone or with both NF-L and MTMR2-Flag. Harvested cells were lysed in NP40 lysis buffer, immunoprecipitated using anti-Flag antibody and immunoblotted using anti-NF-L antibody. NF-L and MTMR2 input are the immunoblotted unbound fraction of the immunoprecipitation where both proteins are expressed. MTMR2 input was revealed in a separate blot using anti-Flag antibody. NF-L co-immunoprecipitation was not detected when immunoprecipitation was performed using lysates of non-transfected COS-7 cells (first lane) or lysates of COS-7 cells transfected with the Flag empty vector and immunoblotted using anti NF-L antibody (second lane). (B) COS-7 cells transiently transfected with empty vectors or with both NF-L-Myc and MTMR2-Flag. Lysates were immunoprecipitated using anti-Myc antibody and immunoblotted using anti-MTMR2 antibody. MTMR2-Flag and NF-L-Myc input are the immunoblotted un-bound fraction of the immunoprecipitation where both proteins are expressed. NF-L-Myc was revealed in a separate blot using anti-Myc antibody. As above, MTMR2-Flag co-immunoprecipitation was not detected when immunoprecipitation was performed using lysates of non-transfected cells (first lane) or lysates containing empty vectors and immunoblotted using anti-MTMR2 antibody (second lane).

 
Having established that MTMR2 and NF-L are potential interactors, we next asked whether they co-localize. First, we performed double immunocytochemistry in COS-7 and primary Schwann cells transiently co-transfected with Flag-tagged MTMR2 and Myc-tagged NF-L. Despite diffused expression of both MTMR2 and NF-L, we observed co-localization in some, but not all cytoplasmic locations in transfected COS-7, as visualized by confocal microscopy (Fig. 5A–C). Then in transfected Schwann cells, that somehow mimic the increased NF-L expression of denervated Schwann cells in nerve injury (23), there is also co-localization, but with some more intense co-localization in cell processes (Fig. 5D–F). Then, we confirmed in vivo the pattern of co-localization between MTMR2 and NF-L in motorneurons, DRG sensory neurons and axons from motor root, sensory root and peripheral nerve (Fig. 5G–N, these are the same sections shown in Fig. 1O–R; and data not shown). In neurons in vivo, which in adult nerve highly express both NF-L and MTMR2, the co-localization is brightest in axons of the motor roots.

View larger version (124K): [in this window] [in a new window]   Figure 5. Co-localization of NF-L and MTMR2. Immunohistochemical detection of NF-L/MTMR2 in COS-7 (A–C), and primary Schwann cells in vitro (D–F), motorneurons (G, H), and DRG sensory neurons in vivo (I, J), motor root (K, L) and sensory root (M, N). COS-7 and Schwann cells were co-transfected with Myc-tagged NF-L and Flag-tagged MTMR2 cDNAs and proteins revealed by anti-Myc (A, D) and anti-Flag (B, E) antibodies showing co-localization in the cell cytoplasm (C, F). Sections stained with anti NF-L (G, I, K, M) are the same stained with anti-MTMR2 in Figure 1 (O–R). Co-localization is shown in H, J, L, N. Bar=30 µm (G–N).

 
Influence of CMT4B mutations on the expression of MTMR2 and NF-L
To better understand the pathogenesis of CMT4B, we studied the effects of disease-associated mutations on the expression of MTMR2 and NF-L in human sural nerves. To this aim, we collected paraffin-embedded sural nerve biopsy samples from four unrelated CMT4B patients with known MTMR2 mutations. The first patient (KAY.690 family) has a Tyr579fs homozygous mutation in MTMR2 leading to a premature truncation of the protein at codon 599, whilst the second patient (GEN-ALS family) has two different homozygous mutations, consisting of a Glu276stop and an in-frame exon skipping, Phe494-Glu531del (7). The third patient (case 9 of family C) presents a Gly103Glu homozygous missense mutation and, finally, the fourth patient (case 8 of family V) has a Thr108fs homozygous mutation leading to a premature truncation codon in position 124 (24). As controls, we stained sample biopsies from two normal individuals and three unrelated patients negative for mutations in MTMR2 and affected by chronic demyelinating and axonal polyneuropathy of undetermined aetiology (Fig. 6). The staining was performed using both the anti-hMTMR2 (M42) antibody, that recognizes the N-terminus of the protein, and the anti-rat MTMR2, that recognizes a peptide from amino acids 156–177 of the rat protein corresponding to 230–251 of human MTMR2.

View larger version (62K): [in this window] [in a new window]   Figure 6. MTMR2 expression in CMT4B patients. Immunohistochemical detection of MTMR2 by anti-hMTMR2 and anti-rat MTMR2, that recognize the N-terminus and the portion of MTMR2 protein encoded by exon 8 respectively, in control patient (A, B), Tyr579fs patient (C, D), Glu276stop and Phe494-Glu531del patient (E, F), Gly103Glu patient (G, H) and in the Thr108fs patient (I, J). A scheme with putative length of truncated protein produced in the four patients and the peptide recognized by the antibodies is shown at the bottom. In controls both antibodies detected MTMR2 in Schwann cell cytoplasm and axons (A, B). In the Tyr 579fs patient both antibodies detected MTMR2 only in Schwann cell cytoplasm (C, D). In the Glu276stop and Phe494-Glu531del patient, anti-hMTMR2 antibody detected the protein only in Schwann cell cytoplasm (E), the staining of anti-rat MTMR2 antibody was very faint but still limited to the Schwann cell cytoplasm (F) In the Gly103Glu patient both antibodies could not detect any staining (G, H). In the Thr108fs patient, a faint staining in Schwann cells was observed with anti-hMTMR2 (I), whereas no staining with anti-rat MTMR2 (J). Bar=7 µm.

 
In all control nerves, both antibodies confirmed the expression of MTMR2 in Schwann cell cytoplasm and in the axons (Fig. 6A and B). In the patient with the Tyr579fs mutation we observed staining with both antibodies exclusively in Schwann cell cytoplasm but not in the axon (Fig. 6C and D). Similar results were observed in the patient carrying both Glu276stop and Phe494–Glu531del mutations, although the staining with anti-rat MTMR2 antibody was very faint (Fig. 6E and F). In the third patient having the Gly103Glu missense mutation, we could not detect any staining using both antibodies either in Schwann cells or axons, consistently with the severity of the nerve pathology (Fig. 6G and H).

Finally, in the patient with the Thr108fs mutation, we could detect a very faint staining only in the Schwann cell cytoplasm using the anti-hMTMR2 antibody. The anti-rat MTMR2 did not produce any staining in this patient, as expected, since this antibody recognizes a portion of the protein predicted by virtual translation to be absent.

We further investigated if the lack of functional MTMR2 in CMT4B patients interferes with the expression of NF-L protein in peripheral nerve. To this purpose, we stained serial sural nerve sections from the four available patients and controls with anti-NF-L antibody and with anti-NF-H antibody to identify axons. We could not discern any significant difference in staining between NF-L and NF-H in both patients and controls (Fig. 7).

View larger version (155K): [in this window] [in a new window]   Figure 7. Detection of NF-L in CMT4B patients. Immunohistochemical detection of NF-H (A, C) and NF-L (B, D) in the patients with Tyr579fs and Gly103Glu mutations, respectively. Similar expression of NF-H (to identify axons) and NF-L was observed in the remaining two available CMT4B patients. Bar=25 µm.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
To elucidate the biological role of MTMR2 in the nerve, we followed two different strategies: determine its expression pattern in the tissue specifically affected and search for nerve-specific protein interactors. We observed Mtmr2 expression in vivo in Schwann cells and axons from peripheral nerves and roots, in DRG sensory neurons, and in motorneurons. In all these cell types, Mtmr2 was mainly cytoplasmatic and enriched in perinuclear area. We confirmed Mtmr2 expression in vitro, in Schwann cells and dissociated DRG sensory neurons. We also observed Mtmr2 staining in nuclei of Schwann cells and motorneurons, similarly to that previously reported in MTMR2 transfected COS and HeLa cells (13,14). Finally, we showed absent or limited MTMR2 expression in sural nerves from CMT4B patients. Whenever MTMR2 protein was detectable, it was restricted to Schwann cell cytoplasm where it might represent a non-functional protein that has to be degraded. Consistent with this hypothesis, we did not detect any staining in the axon since mutated proteins that undergo degradation would not be transported along the axons. Our results in patient biopsies demonstrate for the first time the consequence of CMT4B mutations on MTMR2 expression. Overall, our data in part confirm previously reports (15), but also clearly show that MTMR2 is expressed also in the glial component of the peripheral nerve, suggesting that CMT4B pathology might arise from a specific defect in neurons and/or Schwann cells.

Since Mtmr2 does not reveal a specific pattern of expression in the peripheral nerve, we performed two independent yeast two-hybrid screenings, aimed at identifying proteins specifically interacting with MTMR2/Mtmr2 in the nerve. Two different libraries from human fetal brain and rat sciatic nerve were screened against human MTMR2 and rat Mtmr2 bait proteins, respectively. In both Schwann cells and neurons, interaction between MTMR2 and NF-L, was found. Our finding of NF-L mRNA in Schwann cells is not surprising as NF-L expression has been demonstrated in both normal and severed sciatic nerve (23,25,26). Both NF-L and NF-M mRNAs as well as the corresponding proteins are detectable in dedifferentiating Schwann cells after injury (23).

We provided further evidence for this interaction using co-immunoprecipitation in co-transfected COS cells and co-localization by immunohistochemistry and confocal microscopy. In particular, the co-localization observed in neurons where both proteins are highly expressed supports interaction of these proteins in vivo at physiological concentrations.

The interaction we found between MTMR2 and NF-L, which is specifically expressed in the nervous system, might explain why the CMT4B pathology is restricted to the nervous system despite the ubiquitous expression of MTMR2. In the central nervous system, which is not affected in CMT4B, a different molecular mechanism could be involved or other members of the myotubularin-related protein family could compensate loss of the phosphatase activity.

However, the nature of the interaction between MTMR2 and NF-L, as well as its relevance in the CMT4B pathogenesis, remains to be assessed. NF-L could represent an interactor of MTMR2 that places the phospholipid phosphatase near to events it must regulate such as endosomal vesicle trafficking, which is very important for the plasma membrane remodelling of myelin forming Schwann cell. Loss of MTMR2 could thus cause an aberrant overgrowth of myelin, as observed in the CMT4B phenotype. Alternatively in neurons, the interaction between MTMR2 and NF-L might be important to transport, or localize, the phosphatase along the axon to its site of action. Another scenario might be represented by NF-L as substrate of the MTMR2 phosphatase. Phosphorylation is the major post-translational modification of neurofilaments. Extensive phosphorylation occurs when NFs enter the axon and phosphate groups incorporated in the cell body are lost and replaced during axonal transport (27,28). As the prevalent demyelinating phenotype in CMT4B would suggest, loss of dephosphorylation by MTMR2 on a particular residue of NF-L might affect local interactions between Schwann cells and axons (29). The generation of a mouse model for CMT4B disease in which the Mtmr2 gene is conditionally inactivated in either neurons or Schwann cells is in progress, which will help to prove the nature of the MTMR2 and NF-L interaction as well as to assess the cell-autonomous role of the protein phosphatase.

Interestingly, mutations in NF-L cause CMT2E, characterized by axonal loss and usually normal values of nerve conduction velocity, NCV (30–33). Neurofilaments, formed by the assembly of three subunit proteins NF-L, NF-M and NF-H, are the major constituents of the neuronal cytoskeleton (27,34). The mutant NF-L proteins are thought to disrupt the assembly of NF subunits in neurons, thus causing a decrease in the number of neurofilaments in the axon, producing a reduced axonal diameter and slowing of axonal transport, leading to distal nerve degeneration (35,36). More recently, mutations in NF-L have been also found in patients with more variable clinical and electrophysiological features, including severe cases initially diagnosed as Dejerine–Sottas syndrome, mainly characterized by severely slowed NCV (33). Interestingly, the sural nerve biopsy of one of these patients with NF-L mutations clearly showed signs of primary myelin damage including irregular foldings of the myelin sheaths (33). These findings support the increasing evidence of overlapping CMT forms in which both demyelination and axonopathy coexist, despite the standard classification into demyelinating (CMT1) or axonal (CMT2) neuropathies (37). In this view, the interaction here reported between the MTMR2 protein phosphatase and NF-L, not only may provide a basis for the nerve-specific pathogenesis of CMT4B1 but also further supports the notion that demyelinating and axonal neuropathies may represent different clinical manifestations of a common pathological mechanism.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Patient material
Sural nerve biopsies from patients carrying the Tyr579fs and [Glu276stop+Phe494-Glu531del] mutations, respectively, have been characterized by Salih et al. (38). The clinical features of the patients having the Gly103Glu missense mutation and the Thr108fs mutation have been already reported (39).

Tissues and cell culture
All the experiments involving animals were performed in accordance with Italian national regulations and covered by protocols reviewed by local Institutional Animal Care and Use Committees. C57BL/6J mice and Sprague–Dawley rats were purchased from Charles River Italia.

For immunohistochemistry, sciatic nerves, spinal cord, dorsal root ganglia (DRG), and spinal roots were removed and rapidly snap-frozen in liquid nitrogen or previously fixed in 4% paraformaldehyde.

Neuronal cultures were prepared by dissociating purified DRG neurons form embryonic day 15.5 (E15.5) rats as described (40). Briefly, DRG were trypsinized (0.25%; GIBCO), mechanically dissociated, and cell suspension plated as a drop onto 12 mm glass coverslips (Greiner) coated with rat collagen (0.2 mg/ml; Biomedical Tech. Inc.) in CB10 media. CB10 consisted of Eagle's minimal essential medium (EMEM; GIBCO) supplemented with 10% fetal calf serum (FCS; Biological Industries Kibbutz), 5 mg/ml glucose (Sigma), 50 µg/ml crude nerve growth factor (NGF; Calbiochem). Non-neuronal cells were removed by alternating CB10 media with E2F media every 2 days for 12 days. EF2 consisted of EMEM supplemented with 4 mg/ml glucose, 5 µg/ml insulin (Sigma); 10 µg/ml transferrin (Jackson Collaborative Imm.), 100 µM putrescine (Sigma), 20 nM progesterone (Sigma), 30 nM sodium selenite (Sigma), 10 µM 5-fluorodeoxyuridine (Sigma), 10 µM uridine (Sigma), 50 µg/ml NGF. Schwann cells were prepared from sciatic nerves of P3 rats by the method of Brockes et al. (41) and expanded as described (42). Neuron-Schwann cell co-cultures were prepared by adding Schwann cells to coverslips 2.5 weeks after neuronal seeding. Co-cultures were maintained in medium consisting of DMEM/F12, 5 mg/ml glucose, 5 µg/ml insulin, 10 µg/ml transferrin, 100 µM putrescine, 20 nM progesterone, 30 nM sodium selenite and 50 mg/ml NGF. To initiate myelination, co-cultures were treated with DMEM, 15% FCS, 5 mg/ml glucose and 50 µg/ml ascorbic acid (Sigma).

COS-7 cells were grown in 100 mm petri dishes using Dulbecco's modified Eagle's medium containing 10% FCS, 50 units/ml penicillin, and 50 µg/ml streptomycin. COS-7 cells were transiently transfected using 20 µg of each construct DNA and Superfect transfection reagent (QIAGEN) according to the manufacturer's protocol.

Antibodies and immunohistochemistry
The following antibodies were used in immunohistochemistry: a rabbit polyclonal antibody against a 22 amino acid peptide corresponding to residues 156–177 of rat Mtmr2 (anti-rat MTMR2; kindly provided by Dr Y. Cheng) (17); three additional polyclonal antibodies, M42, M52 and M53, raised in rabbit against peptides SENSVHTKSAC and SADNFSSPDLRVLRE, corresponding to residues 33–42 and 53–67 of human MTMR2, respectively, coupled to Keyhole Limpet Hemocyanin (Gramsch Laboratories) and affinity purified; a rabbit polyclonal anti-NF-L (Chemicon) and a mouse anti-NF-L (Sigma); mouse anti-myelin basic protein (MBP, Chemicon), anti-Glial fibrillary acidic protein (GFAP, Chemicon), and anti-S100ß (Chemicon). To detect epitope tagged proteins, a mouse M2 monoclonal anti-Flag (Stratagene) and a mouse 9E10 monoclonal anti-Myc antibody (Roche) were used.

Secondary antibodies included fluorescein (FITC) or rhodamine (TRITC) conjugated-goat anti-mouse or rabbit IgG (Southern Biot. Ass.), or peroxidase conjugated-goat anti-mouse or rabbit IgG (Sigma).

Immunofluorescence on cryosections and cell cultures was performed as described (43), and examined with confocal (Biorad MRC 1024) or fluorescent microscopy (Olympus BX). For immunohistochemistry, paraffin-embedded sections were rehydrated, microwave treated in 0.01 M citrate buffer (only NF-L and NF-H) and then treated with H₂O₂ to block endogenous peroxidase. After incubation with normal goat serum (DAKO, 1 : 10 in PBS+BSA 1%), sections were incubated with primary antibody for one hour at room temperature, washed and then incubated for 30 min with secondary antibody-peroxidase conjugated. Staining was revealed with 3,3'-diaminobenzidine (DAKO) as peroxidase substrate, washed, mounted in Eukitt, and viewed with Olympus BX microscope.

Northern blot analysis
RNA was extracted from rat sciatic nerves and from various tissues by CsCl₂ gradient centrifugation, and from cultured rat Schwann cells or N20 cell line by acid guanidinium thiocyanate–phenol–chloroform extraction method, as previously described (44). Total RNA were analysed by northern blotting as described (44) and hybridized using the rat full-length Mtmr2 cDNA. The presence of RNA in each lane was verified by hybridization with the rat full-length glyceraldehyde-3-phosphate deydrogenase (GAPDH) cDNA.

Yeast two-hybrid screenings and interactions
A first yeast two-hybrid screening was performed using yeast strain MaV203 provided with the ProQuest^TM Two-hybrid System (LifeTechnologies, GIBCO BRL). As a bait, a 2 kb PCR product corresponding to the ORF of human MTMR2 carrying the D320A substitution was amplified from a pCMVtag-2B-MTMR2 (D320A) construct (gift of Dr Jocelyn Laporte) and subcloned into SalI and NotI sites of the pDBLeu vector, in frame with the GAL4 binding domain. A ProQuest two-hybrid human fetal brain cDNA library (LifeTechnologies, GIBCO BRL) was used to transform the yeast strain MaV203 already containing the bait, thus following a sequential transformation protocol. Among more than 6x10⁶ independent clones, 44 clones were selected on plates lacking leucine, tryptophan and histidine, containing 25 mM 3-aminotriazole. These clones have been tested for ß-galactosidase activity by filter-lift assay and for expression of the third reporter gene of MaV203 yeast strain, the URA3 gene. Positive clones were finally confirmed by reassessment of interaction, co-transforming the MaV203 yeast strain with isolated plasmids and testing again the reporter gene expression. In this way, four clones were demonstrating interaction with MTMR2.

A second yeast two-hybrid screening was carried out using as a bait a 1710 bp ORF of rat Mtmr2 cDNA cloned into SalI site of the pAS2.1 vector in frame with GAL4 binding domain. The full-length rat Mtmr2 cDNA was obtained by RT–PCR carried out using diluted cDNA as recommended by the manufacturer's instructions with 2 mM MgCl₂; a mixture of Taq Gold polymerase (Applied Biosystem) and Pfu Turbo polymerase (Stratagene) and 35 cycles of amplification. Total RNA from rat liver was isolated using Trizol (Life Technologies, Invitrogen) and first-strand cDNA was prepared from 1 µg of RNA using the Advantage^TM RT-for-PCR kit (Beckton Dikinson, Clontech). A rat sciatic nerve cDNA library cloned into the pACTII vector carrying the activating domain of GAL4 (22) was used to perform a sequential transformation of yeast strain Y190 (Clontech), previously transformed with the bait. More than 400 positive clones were selected on plates lacking leucine, tryptophan and histidine containing 35 mM 3-aminotriazole. Among them, 333 clones were found to be positive testing their ß-galactosidase activity by filter-lift assay. The corresponding cDNAs cloned into pACTII vector were rescued by colony segregation using a culture medium lacking leucine and containing cicloeximide following manufacturers instructions (Clontech). Their interactions with the bait were then confirmed by yeast mating of Y190 containing the pACTII as the only vector with a second yeast strain Y187 (Clontech), previously transformed with the bait plasmid, cloned into the pAS2.1 vector. In this way, a final number of 118 clones were found to activate expression of both reporter genes HIS3 and LacZ.

cDNAs cloned in the pPC86 vector for the ProQuest Yeast Two Hybrid system (Life Technologies, GIBCO) and in the pACTII vector for MatchMaker II system (Clontech) were isolated by lysis of yeast cells using SDS 1%; Triton X-100 1%; 100 mM NaCl; 10 mM Tris-HCl pH 8.0; 1 mM EDTA, and glass beads acid washed (SIGMA) and transformed into XL2 Blue Ultracompetent cells (Stratagene) following the manufacturer's instructions. Plasmid template for clone sequencing was prepared using a QIAprep spin miniprep kit (Qiagen). A big dye terminator chemistry on a 3100ABI sequencer was used. DNA sequence was analysed using Sequence Analysis (ABI) while sequence assembly was performed using Sequence Navigator (ABI). All constructs generated by PCR were first cloned in the TA vector system (Invitrogen) and then confirmed by sequencing.

Cloning constructs
A human NF-L cDNA was amplified by RT–PCR, using a first-strand cDNA prepared as described above. In this case, total RNA from brain was used for the cDNA synthesis and the RT–PCR reaction was performed using 10% DMSO. The cDNA was first cloned into TA vector system (Invitrogen), confirmed by sequencing and subcloned into both the pcDNA 3.1 and pcDNA 3.1/myc-His vectors (Invitrogen).

Our NF-L cDNA corresponds to the sequence recently reported by Perez-Olle et al. (36), thus differing from the published sequence (GenBank accession no. X05608). Both wild-type MTMR2 and D320A MTMR2 cDNAs cloned into the pCMVTag-2B expression vector (Stratagene) were kindly provided by Dr Jocelyn Laporte.

Co-immunoprecipitation and western blotting
Transiently transfected COS-7 cells were harvested 48 h after transfection, washed twice with cold PBS pH 7.4, pelleted by centrifugation and lysed in buffer containing NP40 1%, 150 mM NaCl, 50 mM Tris–HCl pH 8.0, and protease inhibitor cocktail (Roche). Following centrifugation at 13 000 rpm the lysate was incubated with 6 µg/ml anti-myc antibody (mouse 9E10 monoclonal, Roche) or anti Flag-antibody (mouse M2 monoclonal, Stratagene). After 3 h incubation at 4°C for the anti-Flag antibody or overnight for the anti-Myc antibody, 30 µl of protein A agarose (Amersham-Pharmacia Biotech) was added to precipitate antibodies and the samples were incubated for at least 1 h at 4°C. The agarose was washed three times with cold PBS-Tween 0.1% and the immunoprecipitated was denatured in Laemli buffer with ß-mercaptoethanol and resolved by SDS–PAGE in 7.5% gel. Proteins were then transferred on PVDF (Millipore) membrane and blocked in PBS, 0.1% Tween X-100, and 5% nonfat milk. Western blots were probed with rabbit anti NF-L antibody (Chemicon), 1 : 800 and mouse anti-Flag (Stratagene, 1 : 1500) or with affinity purified M53 anti-MTMR2 (1 : 1500) antibody and revealed using ECL (Amersham-Pharmacia Biotech).

	ACKNOWLEDGEMENTS

 
We thank Ms Cinzia Ferri for her excellent technical assistance. We are also grateful to Dr Carla Taveggia for her precious contribution. We acknowledge the kind collaboration of Dr Molham Al Rayess, for providing nerve specimens; Dr Yan Cheng from the Columbia University, who provided the rabbit anti rat-MTMR2 antibody; and Dr Jocelyn Laporte, from IGBMC, Strasbourg, France for his kind collaboration and useful discussion. A.B. is an Assistant Telethon Scientist from the Dulbecco Telethon Institute. A.P.M. is a Wellcome Trust Principal Research Fellow. M.L.F. and L.W. were supported by grants from the NIH (NS41319 and NS45630), Telethon, Italy, and the Italian and Great Britain Multiple Sclerosis Societies. S.C.P. and A.Q. were supported by grants from AceSM.

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Laboratory of Molecular Genetics, Gaslini Institute, Largo Gaslini 5, 16148 Genova, Italy. Tel: +39 0105636403; Fax: +39 0103779797; Email: bolino{at}unige.it or bolino.alessandra{at}hsr.it

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