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Human Molecular Genetics Advance Access originally published online on February 1, 2006
Human Molecular Genetics 2006 15(6):943-952; doi:10.1093/hmg/ddl011
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Aggregate formation and phosphorylation of neurofilament-L Pro22 Charcot–Marie–Tooth disease mutants

Takahiro Sasaki1,{dagger}, Takahiro Gotow2, Motoko Shiozaki2, Fumika Sakaue1, Taro Saito1, Jean-Pierre Julien3, Yasuo Uchiyama4 and Shin-Ichi Hisanaga1,*

1Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minami-ohsawa, Hachiohji, Tokyo 192-0397, Japan, 2Laboratory of Cell Biology, College of Nutrition, Koshien University, Takarazuka, Hyogo 665-0006, Japan, 3Centre Hospitalier de l'Universite Laval Research Center, Quebec City, Quebec, Canada and 4Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan

* To whom correspondence should be addressed. Tel: +81 426772577; Fax: +81 426772559; Email: hisanaga-shinichi{at}c.metro-u.ac.jp

Received December 4, 2005; Accepted January 30, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Charcot–Marie–Tooth disease (CMT) is the most common inherited peripheral nerve disorder. The causative gene for axonal type CMT2E has been identified as neurofilament light (NF-L) chain. Using cultured cells and in vitro assays, we analyzed the filament formation ability of Pro22 CMT mutant proteins of NF-L, P22S and P22T. NF-L Pro22 mutant proteins formed large aggregates in SW13– cells and cortical neurons and assembled into short twisty threads thinner than 10 nm filaments in vitro. Those threads associated with each other at their ends and entangled into large aggregates, also abnormalities, were detected at steps in oligomer formation. Pro22 mutations abolished Thr21 phosphorylation by cyclin-dependent kinase 5 and external signal regulated kinase, which suppressed filament assembly, but phosphorylation by protein kinase A (PKA) inhibited aggregate formation in vitro and alleviated aggregates in cortical neurons. These results indicate that the Pro22 CMT mutation induces abnormal filament aggregates by disrupting proper oligomer formation and the aggregates are mitigated by phosphorylation with PKA, which makes it a viable target for the development for therapeutics.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Charcot–Marie–Tooth disease (CMT) is the most common inherited peripheral nerve disease (1Go–3Go). CMT is classified into two main types on the basis of nerve conduction velocity, demyelinating CMT1 and axonal CMT2. Each class of CMT is further divided into several subgroups by genetic locus. Pathological features of CMT1 are characterized by segmental demyelination and remyelination, and proteins involved in myelin formation have been identified as gene products for CMT1. Mutations in CMT2, which are accompanied by axonal degeneration, are found in proteins involved in axonal transport and integrity.

The neurofilament light (NF-L) chain gene has been identified as the causative gene for CMT2E (4Go). Neurofilaments (NFs) are the most abundant cytoskeletal element in axons (5Go,6Go) and NF-L is the basic subunit of NFs, which are heteropolymers composed of neurofilament middle (NF-M) chain and neurofilament heavy (NF-H) chain (7Go,8Go). The three NF subunits consist of the N-terminal head, {alpha}-helical central rod and C-terminal tail domains. The rod domain is involved in the formation of coiled-coil dimers, an essential step for filament assembly. The head domain is a region regulating filament assembly through phosphorylation by second-messenger-dependent protein kinases (5Go). NF-M and NF-H have a longer C-terminal tail domain protruding from the core filaments (9Go) with multiple Lys–Ser–Pro repeat sequences which can be phosphorylated by proline-directed protein kinases (PDPKs).

Several missense mutations of NF-L have been reported for CMT (4Go,10Go–15Go). There have been four reports describing the effects of NF-L CMT mutations on filamentous network formation in cultured cells. Those mutations are E7K, P8L, P8Q, P8R, P22S, P22T, E89K, N97S, Q333P and D469N (16Go–19Go). All the mutants, except for E7K and D469N, display aggregate formation when expressed in cultured cells, but it is not known how the mutations affect filament assembly. We have an interest in mutations at Pro22, as mutation at this site abolishes the Thr–Pro PDPK consensus phosphorylation sequence in the head domain of NF-L. We expect that this mutation would disrupt the regulation of filament assembly by phosphorylation.

In this study, we analyzed the assembly and phosphorylation of NF-L P22S and P22T mutants in cultured cells and in vitro. The mutants formed large aggregates in cells and abnormal filaments in vitro. They were not phosphorylated at Thr21 by cyclin-dependent kinase 5 (Cdk5) or external signal regulated kinase 2 (ERK2), which inhibits assembly of the filaments. However, they retained the phosphorylation site for protein kinase A (PKA), which diminished aggregates in vitro and in neurons. Thus, the phosphorylation in the head domain could be used as a therapeutic strategy for CMT2E patients.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Aggregate formation of NF-L mutants, P22S and P22T, in SW13– cells
SW13– cl.2/Vim– cells (here referred to as SW13–), which lack endogenous cytoplasmic intermediate filaments, are often used to assess the assembly of intermediate filament proteins. Several CMT mutants of NF-L, including the mutation at Pro22, have been reported to display defects in filamentous network formation in transfected SW13– cells (16Go–19Go). We first tested the filament formation ability of Pro22 mutants in SW13– cells in our experimental system. Human NF-L wild-type (WT) assembled into a network of filaments (Fig. 1A). Both NF-L P22S and P22T formed large irregular aggregates in SW13– cells (Fig. 1F and K), confirming the previous results by Perez-Olle et al. (19Go).


Figure 0111
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  		Figure 1. Aggregate formation of NF-L CMT mutants, P22S and P22T, in SW13– cells. Immunofluorescent staining of SW13– cells expressing human NF-L WT (A–E), P22S (F–J) or P22T (K–O) alone or in combination with the other NF subunit of NF-M or NF-H. The cells were transfected with pCMV5 vectors encoding NF-L (A, F and K), NF-L and NF-M (B, C, G, H, L and M), NF-L and NF-H (D, E, I, J, N and O) and then were cultured for 24 h. The expression of NF-L (A, B, D, F, G, I, K, L and N), NF-M (C, H and M) and NF-H (E, J and O) was detected by immunostaining with the anti-NF-L rabbit polyclonal antibody, the anti-NF-M mouse monoclonal antibody NN18 and the anti-NF-H mouse monoclonal antibody SMI32, respectively. Scale bar represents 10 µm.

 
NFs are obligate heteropolymers of NF-L, NF-M and NF-H in neurons (7Go,8Go). To examine whether co-expression of NF-M or NF-H suppresses aggregate formation or whether they are also incorporated into the aggregates, NF-L WT, P22S or P22T was cotransfected with NF-M or NF-H in SW13– cells. NF-L WT co-assembled into filaments with NF-M or NF-H (Fig. 1B–E). NF-L P22S and P22T mutants not only formed aggregates when they were co-expressed with NF-M or NF-H but also incorporated NF-M or NF-H into the aggregates (Fig. 1G–J and L–O). We did not notice any difference in aggregates formed between NF-L/NF-M and NF-L/NF-H at least under fluorescent microscopy. These results suggest that NF-L P22S and P22T can form aggregates even in the presence of NF-M and NF-H in neurons. In contrast, Perez-Olle et al. (19Go) describe that co-transfection with NF-M results in heteropolymeric filamentous networks in some cells. This difference may be due to differences in expression levels of proteins and using different expression vectors.

Aggregate formation of NF-L P22S and P22T in cultured cortical neurons
To examine aggregate formation in a neuronal cellular environment, we expressed NF-L P22S and P22T in cultured cortical neurons. NF-L WT, P22S or P22T was expressed in cultured cortical neurons together with enhanced green fluorescent protein (EGFP) to help identify transfected neurons (Fig. 2A, E and I). NF-L WT was observed as a network of filaments in the cell body (Fig. 2D) and its staining was also detected in neurites (Fig. 2B). However, the distribution of NF-L P22S and P22T was restricted to the cell body (Fig. 2F and J), and those in the cell body formed large aggregates, as was seen in SW13– cells (Fig. 2H and L). EGFP was incorporated into these aggregates of NF-L P22S and P22T (arrows in Fig. 2G and K), in contrast to the case of NF-L WT, which did not show colocalization with EGFP even in a highly concentrated region of NF-L WT filaments (Fig. 2C).


Figure 0112
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  		Figure 2. Aggregate formation of NF-L CMT mutants, P22S and P22T, in rat cortical neurons. Human NF-L WT (A–D), P22S (E–H) or P22T (I–L) was expressed in rat cortical neurons. Transfected neurons were identified by fluorescence of co-expressed EGFP (A, C, E, G, I and K). NF-L was stained by anti-human NF-L-specific antibody 24 h after transfection (B, D, F, H, J and L). NF-L WT was detected in the cell body and neurites (A and B), whereas NF-L P22S (E and F) and P22T (I and J) were localized only in the cell body. The higher magnification micrographs of the cell body revealed a filamentous network of NF-L WT (D), but large aggregates for both P22S (H) and P22T (L). Incorporation of EGFP in the aggregates was observed in these mutants (arrows in G, H, K and L). Scale bars represent 10 µm.

 
Large aggregates were grown from smaller punctate structures in SW13– cells
How NF-L P22S or P22T form large aggregates was next examined by observing NF-L staining in SW13– cells at an earlier time after transfection (Fig. 3). At 10 h after transfection, the cells were fixed and stained with anti-NF-L antibody. These cells exhibited various stages of filament assembly, which was most likely dependent upon the expression levels of NF-L protein. Three images of NF-L WT and P22S are shown in Figure 3 and may represent the process of filamentous network formation (Fig. 3A–C) and aggregate growth (Fig. 3D–F), respectively. In the case of NF-L WT, fibrils appeared to elongate gradually from smaller punctate structures, as has been reported for other IF proteins (20Go). Aggregates of NF-L P22S appear to become larger directly from the dotted structures. The results indicate that the large aggregates of NF-L P22S and P22T were formed by growth of the smaller aggregates rather than bundling of previously formed intermediate-size filaments.


Figure 0113
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  		Figure 3. Aggregates of NF-L P22S are observed soon or shortly after transfection in SW13– cells. The SW13– cells were transfected with NF-L WT (A–C) or P22S (D–F), and their immunofluorescent images were obtained at 10 h after transfection, that is, 14 h earlier than those shown in Figure 1. Cells displayed various assembly or aggregation stages of NF-L. Three typical micrographs are shown to represent the growing process of filaments (A–C) or aggregates (D–F). Higher magnification micrographs are shown in inset. Scale bars in (F) and its inset represent 1 and 10 µm, respectively.

 
In vitro assembly of NF-L P22S and P22T
The Pro22 mutation of NF-L seemed to affect filament assembly rather than interfilament interactions. Thus, to understand the molecular mechanism of aggregate formation, we examined the assembly of NF-L in vitro with purified proteins. NF-L WT, P22S and P22T, which had been tagged with a histidine hexamer at the C-terminus, were purified from Escherichia coli inclusion bodies (Fig. 4A). Purified NF-L proteins were assembled by dialyzing against the assembly buffer at 37°C for 3 h. Filament assembly was first checked by a sedimentation assay (Fig. 4B). NF-L P22S and P22T, as well as NF-L WT, were found in the pellets, indicating that NF-L P22S and P22T can form aggregates that sediment during ultracentrifugation at 100 000g for 20 min. Polymers could be observed by negative staining electron microscopy (Fig. 4C–H). NF-L WT polymerized into long intermediate-size filaments (Fig. 4C and D); however, no such long filaments were observed with NF-L P22S and P22T (Fig. 4E and G). Instead, short, thinner filaments and various sizes of intertwined filaments were observed with both NF-L P22S and P22T (Fig. 4F and H).


Figure 0114
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  		Figure 4. Assembly of NF-L P22S and P22T mutants in vitro. (A) Coomassie brilliant blue (CBB) staining of an SDS–PAGE gel of NF-L proteins purified from E. coli. (B) The sedimentation assay of assembled NF-L proteins. NF-L WT, P22S or P22T was assembled in vitro by dialysis against the assembly buffer at 37°C for 3 h. Assembled NF-L was sedimented by centrifugation at 100 000g for 20 min and the supernatant and pellet were subjected to SDS–PAGE. (C–H) Negative staining electron micrographs of assembled NF-L proteins. Micrographs are NF-L WT in (C and D), P22S in (E and F) and P22T in (G and H), respectively, in low and higher magnifications. Scale bars represent 200 nm.

 
A more detailed analysis of the filamentous structures was observed by low-angle rotary-shadowing electron microscopy. Long, straight filaments, typical for the intermediate filament, were observed with NF-L WT (Fig. 5A–C). However, no such filaments were found in the samples assembled from NF-L P22S and P22T, which produced only mixtures of short threads and masses of intertwined filaments (Fig. 5D and I). Higher magnification views are shown in Fig. 5E–H and J–M for NF-L P22S and P22T, respectively. Similar structures were observed with NF-L P22S and P22T. Some of the short threads appeared to be helical (Fig. 5E and J) and some appeared to be beaded and attached at their ends to each other (Fig. 5F and K). Larger masses were composed of several intertwined longer threads (Fig. 5G, H, l and m). The size of the aggregates was smaller than those observed by negative staining electron microscopy, but this was due to fragmentation of the aggregates when the samples were sprayed onto mica. The diameter of NF-L P22S or P22T threads was smaller than that of NF-L WT filaments. To verify this, assemblies of NF-L WT and Pro22 mutants were observed in identical specimens (Fig. 5 N–Q). The threads of Pro22 mutants were evidently thinner than NF-L WT filaments. The diameter of NF-L P22S and P22T threads was 18 nm, three-fourths of the wild-type filaments (24 nm), when measured with rotary-shadowed samples.


Figure 0115
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  		Figure 5. Low-angle rotary-shadowing electron micrographs of NF-L P22S and P22T polymers. NF-L assemblies were observed by low-angle rotary-shadowing electron microscopy. Lower magnification views are shown in (A) for NF-L WT, in (D) for NF-L P22S and in (I) for NF-L P22T. NF-L WT formed long linear filaments, whereas NF-L P22S and P22T formed short threads or their intertwined aggregates. Higher magnification views are shown in (B and C) for NF-L WT, (E–H) for P22S and (J–M) for P22T. Short threads of P22S and P22T appeared helical or beaded, and the intertwined aggregates appeared to be composed of a number of threads associated irregularly at their ends. The threads of NF-L P22S and P22T were thinner than those of NF-L WT filaments. The mixtures of WT and P22S and WT and P22T are shown in (N and O) and (P and Q), respectively, for direct comparison of their diameters. Scale bars represent 200 nm.

 
Several intermediate assemblies of NF-L can be formed stably through the choice of dialysis solution (21Go). After dialysis against the low-salt assembly buffer, human NF-L WT formed the short intermediate-size filaments consisting of four or five beaded segments (Fig. 6A). NF-L P22S displayed structures of two or three beads connected by a thin fibril (Fig. 6B). Some of them appeared to be boomerang-like structures, with a bend at the middle bead (Fig. 6B, arrow). When dialyzed against the low salt, alkaline assembly solution, NF-L WT assembled into short intermediate-size filaments and thin rods (arrows in Fig. 6C). Under the same conditions, NF-L P22S showed globular particles with several whisker-like short filaments around them (Fig. 6D). Although we do not know how these structures are constructed from mutant NF-L, these results indicate that disruption in filament assembly of the Pro22 mutant resides in a step of oligomer formation.


Figure 0116
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  		Figure 6. Rotary-shadowing electron micrographs of oligomers of NF-L P22T. NF-L WT (A and C) or P22T (B and D) was dialyzed against the low-salt assembly buffer at 4°C for 3 h (A and B) or the low-salt alkaline assembly buffer at 4°C for 3 h (C and D). Oligomeric structures of NF-L were observed by low-angle rotary-shadowing electron microscopy. NF-L WT formed the intermediate-size short filaments with four or five beaded segments in a low-salt assembly buffer (A), and the mixture of the intermediate-size short filaments and rods of octamer (or tetramer) complexes (arrows) in the low-salt alkaline assembly buffer (C). NF-L P22T formed two or three beaded segments often bending at the middle in the low-salt assembly buffer (B, arrow) and globular particles with some whiskers in the low-salt alkaline assembly buffer (D). The size of those beads was not constant, but was larger than the beads in the intermediate-size short filaments of NF-L WT. Scale bar represents 200 nm.

 
Thr21 phosphorylation by PDPKs and its loss in NF-L P22S and P22T mutants
Pro22 is the amino acid C-terminal next to Thr21, constituting the Thr–Pro consensus phosphorylation sequence for PDPKs. The mutation of Pro22 results in the abolition of this consensus sequence in NF-L. As there has been no report on Thr21 phosphorylation, we examined phosphorylation at Thr21 using Cdk5-p35 as a PDPK. Phosphorylation of NF-L WT and T21A is shown in Figure 7A. NF-L WT was phosphorylated by Cdk5-p35, and this phosphorylation was markedly decreased in the T21A mutant. A single major spot on the 2D-phosphopeptide map disappeared with T21A phosphorylated by Cdk5-p35 (Fig. 7B). These results indicate that Thr21 is the major Cdk5 phosphorylation site in NF-L.


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  		Figure 7. Phosphorylation of NF-L at Thr21 by PDPKs. (A) Phosphorylation of NF-L WT and T21A by Cdk5-p35. NF-L WT and T21A (CBB) were phosphorylated in vitro by Cdk5-p35 using [{gamma}-32P]ATP. Phosphorylation was detected by autoradiograph after SDS–PAGE (ARG). (B) Phosphorylated NF-L was subjected to 2D-phosphopeptide mapping after trypsin digestion. The major spot (arrow in WT) disappeared in the T21A mutant. (C) Sedimentation assay of assembled filaments from NF-L phosphorylated by Cdk5-p35. Rat NF-L WT or T21A was incubated with Cdk5-p35 in the presence or absence of ATP. The reaction was stopped by addition of 6 M urea and then the NF-L proteins were assembled into filaments by dialysis against the assembly buffer at 37°C for 3 h. The assembled filaments were pelleted by centrifugation at 100 000g for 20 min and the supernatant and pellet were examined by SDS–PAGE. (D–I) The assembled filaments were observed by negative staining electron microscopy; unphosphorylated NF-L WT in (D and E), phosphorylated NF-L WT in (F and G) and phosphorylated NF-L T21A in (H and I), respectively, in low and higher magnifications. Scale bars represent 200 nm. (J) Phosphorylation of NF-L WT, P22S and P22T by PDPKs in vitro. NF-L proteins were phosphorylated by GSK3ß, ERK2 or Cdk5-p35 using [{gamma}-32P]ATP. The time course of phosphorylation detection was 0, 5, 15, 30 and 60 min, shown by autoradiography after SDS–PAGE.

 
Phosphorylation at the head domain of NF-L is known to induce filament disassembly or prevent filament assembly (22Go–24Go). We examined whether Thr21 phosphorylation also affects filament assembly. NF-L WT or T21A phosphorylated by Cdk5-p35 was assembled by dialyzing against the assembly buffer and assembled filaments were examined by sedimentation assay (Fig. 7C) and electron microscopy (Fig. 7D–I). Unphosphorylated NF-L was sedimented by centrifugation (Fig. 7C) and formed long filaments (Fig. 7D and E) (see also Supplementary Material, Fig. S1). However, about a half of phosphorylated NF-L WT remained in the supernatant after centrifugation (Fig. 7C), and only short filaments were detected under electron microscopy (Fig. 7F and G), indicating that the phosphorylation at Thr21 suppresses filament assembly. This was further demonstrated by the decreased effect of phosphorylation on the filament assembly of NF-L T21A (Fig. 7C and F–I).

To examine whether the Pro22 mutation effects Thr21 phosphorylation and which PDPK phosphorylates the site, P22S or P22T was phosphorylated by three different PDPKs: glycogen synthase kinase 3ß (GSK3ß), ERK and Cdk5-p35 (Fig. 7J). ERK and Cdk5-p35 phosphorylated NF-L WT strongly, and the phosphorylation decreased considerably with NF-L P22S and P22T mutants. In contrast, a slight phosphorylation of NF-L WT was observed when GSK3ß was used, and the extent of phosphorylation was not significantly changed by mutation at Pro22. These results suggest that Pro22 mutation abolishes a regulatory mechanism of filament disassembly, i.e. the phosphorylation at Thr21 by Cdk5-p35 or ERK.

Alleviation of NF-L P22S and P22T aggregates by PKA-dependent phosphorylation
Aggregates of NF-L P22S and P22T could be induced by abnormal interaction between mutant subunits during assembly (Figs 4–6), and the loss of Cdk5-p35 or ERK-dependent phosphorylation at Thr21 in the head domain could enhance aggregate formation by eliminating a disassembly pathway in neurons. However, other phosphorylation sites besides Thr21 in the head domain might still function to regulate the assembly of mutant NF-L. PKA is a well-known head domain kinase (22Go,23Go,25Go–27Go) and its phosphorylation, for example, at Ser41, Ser55 and Ser62, may not be affected by the Pro22 mutation. This was confirmed by examining phosphorylation of NF-L Pro22 mutants by PKA. NF-L P22S and P22T were phosphorylated to the same extent as NF-L WT and were disassembled as efficiently as NF-L WT in vitro by PKA phosphorylation (Fig. 8A–G).


Figure 0118
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  		Figure 8. Phosphorylation of NF-L P22S or P22T by PKA and its effect on aggregate alleviation in neurons. (A) Phosphorylation of NF-L WT, P22S or P22T by PKA in vitro. NF-L proteins were phosphorylated by the catalytic subunit of PKA. The time course of phosphorylation detection was 0, 5, 15, 30 and 60 min, shown by autoradiography after SDS–PAGE. (B–G) Electron micrographs of NF-L WT, P22S or P22T before (B–D) and after (E–G) phosphorylation. Scale bar in (G) represents 200 nm. (H) The Triton solubility of phosphorylated NF-L Pro22 mutants in cortical neurons. Rat cortical neurons were transfected with myc-tagged human NF-L WT (Myc-WT), P22S (Myc-P22S) or P22T (Myc-P22T) and were treated with 15 µM forskolin and 0.1 µM okadaic acid for 2 h. Mock transfection (Mock) is shown in right side four lanes. The Triton-soluble (S) and -insoluble (P) fractions were subjected to western blotting with anti-NF-L rabbit polyclonal antibody. Myc-NF-L is an upper band indicated by white arrowhead, and endogenous NF-L is a lower band indicated by black arrowhead. The soluble Myc-NF-L proteins are indicated by arrows in the Triton-soluble fractions (lanes 5, 11 and 17). Smeared bands may represent highly phosphorylated NF-L species. (I–T) The forskolin and okadaic acid treatment alleviated aggregates formed from NF-L P22S or P22T mutants in cultured neurons. NF-L WT (I–L), P22S (M–P) or P22T (Q–T) was expressed in rat cortical neurons. The neurons were treated with 15 µM forskolin and 0.1 µM okadaic acid for 2 h (K, L, O, P, S and T). As a control, untreated neurons are shown in (I, M and Q). Neurons treated with DMSO are shown in (J, N and R). Human NF-L exogenously expressed was detected by immunostaining with anti-human NF-L-specific monoclonal antibody. Note that diffuse staining in the cytoplasm was increased and the aggregates of NF-L P22S or P22T appeared to be dispersed after the treatment with forskolin and okadaic acid. Scale bar represents 10 µm.

 
Next, we examined the effect of PKA-dependent phosphorylation on alleviation of the aggregates of NF-L Pro22 mutants in cultured neurons. We first made sure that the activation of PKA by forskolin and okadaic acid increased the Triton solubility of NF-L WT when transfected into cultured neurons. Myc-tagged NF-L WT was detected in the pellet fraction in control experiments (Fig. 8H, lanes 2 and 4), but was recovered in a Triton-soluble fraction after treatment with forskolin and okadaic acid (Fig. 8H, arrow in lane 5). The immunostaining of exogenously expressed myc-tagged NF-L WT revealed an increase in diffuse staining in the cell body after the treatment with forskolin and okadaic acid (Fig. 8I–L). The Triton solubility of NF-L P22S and P22T was also increased by forskolin and okadaic acid treatments (Fig. 8H, arrow in lanes 11 and 17). This was confirmed by immunostaining of the aggregates of NF-L P22S and P22T. The fluorescent staining of the aggregates appeared diffuse after the treatment, compared with those in the untreated neurons (Fig. 8M–T). Although the dissolution was not complete, longer treatment resulted in fragmentation of neurites and neurons not adhering to the culture dish. These results suggest that phosphorylation by PKA is effective in dissociating the aggregates of NF-L Pro22 mutants in cultured neurons.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In this study, we investigated the mechanism of aggregate formation of NF-L CMT mutants, P22S and P22T, and the effect of phosphorylation at the head domain on aggregate alleviation. Pro22 mutants of NF-L formed large aggregates in cultured SW13– cells and cortical neurons. We found that NF-L P22S and P22T assembled into twisty threads that were thinner than NF-L WT filaments in vitro. These threads were joined at the ends and entangled, resulting in massive aggregates. Structural disturbance was observed during the steps of oligomer formation, indicating that Pro22 mutations induce the abnormal interaction between NF-L subunits in tetramers or octamers. Thr21 was found to be a novel phosphorylation site in the NF-L head domain for Cdk5-p35 or ERK, which inhibited filament assembly, and the phosphorylation was abolished by the Pro22 mutation, suggesting the possibility that the Pro22 mutation causes more severe symptoms by disrupting a phosphorylation-dependent aggregate eliminating pathway in neurons. However, phosphorylation by PKA was not affected by the Pro22 mutation. The increased PKA-phosphorylation of NF-L Pro22 mutants in cultured neurons reduced the aggregates.

Abnormal filament assembly of NF-L P22S or P22T mutants
In this study, NF-L P22S and P22T mutants formed large aggregates in SW13– cells (19Go) and cortical neurons. Other CMT mutants, E7K, P8L, P8Q, P8R, E89K, N97S, Q333P and D469N, had also been analyzed for aggregate formation in the same or similar types of cells (16Go–19Go). These mutants, except for E7K and D469N, were not able to form filamentous networks, but instead formed aggregates, although the aggregated structures differed depending on the mutation sites. NF-L Q333P formed small dot-like aggregates with a disruption of filaments. The mutants at Pro8 showed a similar phenotype to P22S and P22T, which gave one huge aggregate. And NF-L E89K and N97S showed ‘sheet-like’ non-filamentous structures (18Go). Q333P is located in the rod domain, E89K and N97S are at the border between the head and rod and Pro8 and Pro22 mutations are in the N-terminal region of the head domain of NF-L. Thus, the position of the mutation may determine the phenotype of the assembly defect. By carrying out a thorough study of all the mutants, it could be possible to predict the CMT phenotypes from the position and amino acid of mutation.

Pro8 and Pro22 have been suggested to be hot spots for CMT2E mutations (11Go–13Go,15Go). The head domain of NF-L, where both residues are located, is the domain that regulates filament assembly through its phosphorylation. The effect of phosphorylation could result in the introduction of negative charges rather than conformation changes, because the head domain is assumed to be globular with no secondary structure (28Go). However, proline is a unique amino acid residue that disrupts secondary structures by providing the fixed bending angle to a polypeptide chain. The fact that mutations at Pro residues make NF-L unable to form proper filaments indicates that there might be some structural entities required for proper assembly of filaments.

Low-angle rotary-shadowing electron microscopy is a useful method to analyze the assembly process at a molecular level (21Go). We found several structural abnormalities in polymers formed from Pro22 mutants using this technique. NF-L P22S and P22T assembled into short threads that were thinner than NF-L WT filaments (32 subunits per cross-section) and thicker than protofibrils (eight subunits per cross-section). Two types of short threads were observed, helical and beaded. The massive aggregates appeared to be composed of intertwined threads attached to each other at their ends. The diameter of the threads was about half the diameter of the intermediate-size filaments of NF-L WT when observed by negative staining electron microscopy. Assuming that the subunit arrangement in the threads is similar to that of the normal filaments, the threads of Pro22 mutants might be composed of around 16 subunits per cross-section. The structural abnormality of NF-L Pro22 mutants was also detected in 4- or 8-mer complexes, which are mainly formed by the coiled-coil interaction of the {alpha}-helical rod domain between NF subunits, indicating that the structural abnormality of the head domain would affect the coiled-coil interaction in the 4- or 8-mer complex. This may be crucial to determine what role the head domain plays during filament assembly.

The large aggregates detected in SW13– cells and neurons may correspond to the mass of the intertwined threads observed under electron microscopy, although the exact relationship must await further analysis of the intracellular aggregates. The aggregates appeared to grow directly from smaller punctate structures, but not from an accumulation of intermediate-size filaments. This was consistent with our in vitro observation that aggregates were not made of packed intermediate-size filaments, but comprised many threads associated mainly in an end-to-end fashion, indicating the gradual growth of aggregates without assembling into intermediate-size filaments. Thus, the mutation at Pro22 is the primary cause for aggregate formation of NF-L.

The relationship of in vitro reconstructed abnormal filaments and axonal NFs in a Pro22 CMT patient
Three individual families have been reported to have the Pro22 mutation: P22S for a Slovenian family (12Go), and an Italian family (13Go), and P22T for a Japanese family (11Go). However, pathological features are only available on a sural nerve biopsy sample from the Italian family (13Go). The transverse section of sural nerve showed the loss of large myelinated axons and a few swollen axons encased in a thin myelin sheath. The authors noted that NFs were grouped in a random orientation in swollen axons on electron micrographs. As there was no description of large aggregates or thinner and twisted filaments, such abnormal filaments might not be detected in those axons. NFs in patient axons should be heteropolymers of NF-L P22S with wild-type NF-L, NF-M and NF-H. Assuming that the subunit ratio in NFs is 3:1:1 for NF-L:NF-M:NF-H, as it is for bovine spinal cord NFs (29Go), and that mutant NF-L is also incorporated into NFs in the same manner, mutant NF-L would account only for 30% of total NF subunits. In contrast, the aggregates we observed in vitro and in SW13– cells were composed exclusively of mutant NF-L and aggregates in cortical neurons would also be formed mainly of over-expressed Pro22 mutants. It has been reported that co-expression of other mutants with WT NF proteins in SW13– cells would attenuate aggregate formation (19Go). It would be important to investigate NF assembly at the ratio of mutant NF-L found in the axons of patients.

Phosphorylation of NF-L Pro22 mutants and its possible use as a therapeutic strategy
The relationship between mutation and phosphorylation sites and the effect of phosphorylation on filament assembly or aggregate formation are schematically shown in Figure 9. Thr21–Pro22 is the only PDPK consensus phosphorylation sequence in NF-L. We found Thr21 as a novel phosphorylation site on NF-L for Cdk5 or ERK. The mutation at Pro22 resulted in the loss of phosphorylation at Thr21. Like other phosphorylation sites in the head domain (24Go–27Go), phosphorylation at Thr21 suppressed filament assembly in vitro. Thus, the phosphorylation at the head domain could be a modulator of aggregate formation. The abolition of phosphorylation at Thr21 by Pro22 mutation could enhance aggregate formation of NF-L Pro22 mutants more than other NF-L CMT mutants. The symptoms of Pro22 mutations are indeed more severe than other CMT mutants (11Go–13Go). These results may also suggest that the phosphorylation at Thr21 would work in vivo in regulating NF organization.


Figure 0119
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  		Figure 9. A scheme displaying CMT mutation sites and phosphorylation sites in NF-L and their effects on NF-L assembly. (A) CMT mutation sites whose effects on filamentous network formation in cultured cells have been studied are shown above molecular structure of NF-L. CMT mutations and PKA or Cdk5 (or ERK) phosphorylation sites in the head domain are shown above or below the amino acid sequence of the NF-L head domain. Pro22 mutation studied here is indicated in bold. The Thr21 Cdk5 (or ERK) phosphorylation site is indicated by a thin arrow. The PKA phosphorylation sites are shown by short arrows. (B) Effect of phosphorylation on filament assembly of NF-L WT or aggregate formation of Pro22 CMT mutants. PKA and Cdk5 (or ERK) inhibit assembly of NF-L by phosphorylation of the head domain. Pro22 mutation to Ser or Thr changed the assembly property of NF-L to form aggregates. Abolishment of Thr21 Cdk5 (or ERK1/2) phosphorylation by the mutations may exacerbate aggregate formation, but PKA phosphorylation was not affected by the Pro22 mutation.

 
Defects in axonal transport are thought to be a cause of distal axonal atrophy in CMT patients. The aggregates formed by NF-L CMT mutants inhibit the supply of cytoskeletal proteins of NFs themselves and tubulin (19Go), inhibit other axonal proteins by trapping them into aggregates as was observed with EGFP and perturb the transport of other axonal components such as mitochondria (16Go–19Go). The alleviation of aggregated NFs in the cell body would be one way to resume axonal transport. We showed that NF aggregations were reduced to some extent following head domain phosphorylation by PKA. Looking at the mutation sites and PKA phosphorylation sites, PKA could act as a disassembly factor even for other CMT mutants (Fig. 9A), although this has to be examined in future. Our results may suggest the use of head domain phosphorylation as a potential therapeutic approach to dissociate NF aggregates seen in CMT.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Construction of plasmids
The mammalian expression plasmids of human NF-L, rat NF-M and rat NF-H were constructed by cloning the respective cDNAs into the pCMV5 vector. The bacterial expression plasmids of rat NF-L and human NF-L were constructed by cloning the respective cDNAs into the pET23a (+) vector. To tag a histidine hexamer at the C-terminus of human NF-L, human NF-L expression plasmid was fused to the histidine hexamer of pET23a (+) by deleting the nucleotide sequence of the stop codon from human NF-L cDNA by PCR. NF-L T21A, P22S and P22T mutants were generated using the Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions.

Cell culture and transfection
SW13– cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 100 U/ml penicillin and 0.1 mg/ml streptomycin. Transfection into SW13– cells was performed using the Lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer's instructions. Cerebral cortical neurons were prepared from 17-day-old embryonic rat brains (30Go). Cortical neurons cultured for three days were transfected with a calcium phosphate Profection kit (Promega, Madison, WI, USA).

Immunofluorescence staining of transfected cells
SW13– cells and cortical neurons were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min, and then permeabilized in 0.1% Triton X-100 in PBS for 1 min. After blocking with 1% skim milk in PBS for 30 min, cells were probed with anti-NF-L rabbit polyclonal antibody NA1214 (500-fold dilution, Affiniti, Nottingham, UK), anti-human-specific NF-L mouse monoclonal antibody NF70 (200-fold dilution, Chemicon, Temecula, CA, USA), anti-NF-M mouse monoclonal antibody NN18 (500-dilution, Sigma-Aldrich, St Louis, MO, USA) or anti-NF-H mouse monoclonal antibody SMI32 (2000-fold dilution, Sternberger Monoclonal Inc., Baltimore, MD, USA) for 1 h at room temperature. After being washed with PBS, they were stained with FITC-conjugated secondary anti-mouse IgG or TRITC-conjugated secondary anti-rabbit IgG for 1 h at room temperature. After washing, specimens were mounted with 90% glycerol containing 1 mg/ml p-phenylenediamine in PBS. Images were observed under an Axioskop fluorescence microscopy or an LSM 410 laser scanning confocal microscope (Carl Zeiss Inc., Oberkochen, Germany) and processed with Adobe Photoshop software.

Preparation of NF-L proteins expressed in E. coli
Rat NF-L proteins were expressed in E. coli BL21 (DE3) pLysS in the presence of 0.5 mM IPTG for 3 h at 37°C. NF-L recovered in inclusion bodies was solubilized in 8 M urea in 10 mM sodium phosphate (pH 7.5), 1 mM EGTA and 0.5 mM dithiothreitol and was purified by columns of DEAE–cellulose and Mono-Q (Amersham Pharmacia Biotech., Tokyo, Japan) in the presence of 6 M urea. Human NF-L proteins were purified with Ni-NTA agarose beads (Qiagen, Valencia, CA, USA) and Mono-Q and Sephacryl S-200 gel filtration columns (Amersham Pharmacia Biotech.) in the presence of 6 M urea.

Assembly of NF-L proteins in vitro
Assembly of NF-L (0.3 mg/ml) was performed by dialyzing against assembly buffer [20 mM Pipes (pH 6.8), 0.1 mM EDTA, 0.15 M NaCl, 2 mM MgCl2, 0.5 mM dithiothreitol, 0.4 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), 10 µg/ml leupeptin and 1 µg/ml pepstatin A] at 37°C for 3 h. Filament assembly was assessed by centrifugation at 100 000g for 20 min or by electron microscopic observation. Short filaments were reconstituted by dialyzing against the low-salt assembly buffer, the assembly buffer without 0.15 M NaCl, at 4°C for 3 h. NF-L oligomers were reconstituted by dialyzing against the low salt, alkaline assembly buffer [5 mM Tris–HCl (pH 8.5), 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.4 mM AEBSF, 10 µg/ml leupeptin and 1 µg/ml pepstatin A] at 4°C for 3 h.

Electron microscopy analysis
Assembled filaments were diluted to 0.1 mg/ml and stained negatively with 1.5% uranyl acetate or processed for low-angle rotary-shadowing as described previously (31Go). Specimens were examined with a JEM-1010 electron microscope (JEOL, Tokyo, Japan).

Phosphorylation of NF-L proteins
NF-L was phosphorylated by Cdk5-p35, ERK2, GSK3ß or PKA using [{gamma}-32P]ATP (32Go). The reaction was stopped by the addition of 4x Laemmli's sample buffer. After sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on a 10% polyacrylamide gel, phosphorylation was detected with a BAS 2000 Bioimage Analyzer (Fuji Film, Tokyo, Japan). Two-dimensional phosphopeptide map analyses were performed as described previously (32Go).

Triton-solubility assay of NFs in cultured cortical neurons
Cortical neurons were treated with 15 µM forskoline and 0.1 µM okadaic acid for 2 h, collected by brief centrifugation, and suspended in Triton buffer [100 mM Pipes (pH 6.8), 5 mM EGTA, 2 mM MgCl2, 50 mM KCl, 1% Triton X-100, 5 mM NaF, 10 mM ß-glycerophosphate, 0.4 mM AEBSF and 10 µg/ml leupeptin]. Triton-soluble and -insoluble fractions were separated by centrifugation at 100 000g for 20 min.

SDS–PAGE, western blotting and protein concentration determination
SDS–PAGE was performed according to Laemmli (33Go). Separated proteins were transferred to Immobilon membranes (Millipore, Bedford, MA, USA). Blots were probed with anti-NF-L rabbit polyclonal antibody NA1214 (1000-fold dilution), followed by an alkaline phosphatase-conjugated secondary antibody (1000-fold dilution, DAKO, Glostrup, Denmark). The reaction was developed using a BCIP/NBT phosphatase system (KPL, Gaithersburg, MD, USA). Protein concentrations were determined with the Coomassie protein assay reagent using bovine serum albumin as a standard (Pierce Biotechnology, Inc., Rockford, IL, USA).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We would like to express our thanks to Dr Robert Evans at University of Colorado Health Science Centre for providing SW13– cells, Dr Masahiko Yamamoto for discussion and Dr Pavan Krishnamurthy for reading the manuscript. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan to T.G., Y.U. and S.H.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Nathan Kline Institute, New York University School of Medicine, Orangeburg, NY 10962, USA. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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