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Human Molecular Genetics Advance Access originally published online on September 19, 2007
Human Molecular Genetics 2007 16(24):3103-3116; doi:10.1093/hmg/ddm272
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Disruption of neurofilament network with aggregation of light neurofilament protein: a common pathway leading to motor neuron degeneration due to Charcot–Marie–Tooth disease-linked mutations in NFL and HSPB1

Jinbin Zhai1,*, Hong Lin1, Jean-Pierre Julien2 and William W. Schlaepfer1

1 Division of Neuropathology, Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA, 2 Department of Anatomy and Physiology, Laval University Research Centre, Centre Hospitalier de l’Université Laval, Québec, QC G1V 4G2, Canada

* To whom correspondence should be addressed at: Division of Neuropathology, Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, 606C Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, PA 19104-6100, USA. Tel: +1-215-662-6696; Fax: +1-215-573-2059; Email: jinbin{at}mail.med.upenn.edu

Received April 19, 2007; Revised August 24, 2007; Accepted September 17, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCE
 
Mutations in neurofilament light (NFL) subunit and small heat-shock protein B1 (HSPB1) cause autosomal-dominant axonal Charcot–Marie–Tooth disease type 2E (CMT2E) and type 2F (CMT2F). Previous studies have shown that CMT mutations in NFL and HSPB1 disrupt NF assembly and cause aggregation of NFL protein. In this study, we investigate the role of aggregation of NFL protein in the neurotoxicity of CMT mutant NFL and CMT mutant HSPB1 in motor neurons. We find that expression of CMT mutant NFL leads to progressive degeneration and loss of neuronal viability of cultured motor neurons. Degenerating motor neurons show fragmentation and loss of neuritic processes associated with disruption of NF network and aggregation of NFL protein. Co-expression of wild-type HSPB1 diminishes aggregation of CMT mutant NFL, induces reversal of CMT mutant NFL aggregates and reduces CMT mutant NFL-induced loss of motor neuron viability. Like CMT mutant NFL, expression of S135F CMT mutant HSPB1 also leads to progressive degeneration of motor neurons with disruption of NF network and aggregation of NFL protein. Further studies show that wild-type and S135F mutant HSPB1 associate with wild-type and CMT mutant NFL and that S135F mutant HSPB1 has dominant effect on disruption of NF assembly and aggregation of NFL protein. Finally, we show that deletion of NFL markedly reduces degeneration and loss of motor neuron viability induced by S135F mutant HSPB1. Together, our data support the view that disruption of NF network with aggregation of NFL is a common triggering event of motor neuron degeneration in CMT2E and CMT2F disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCE
 
Charcot–Marie–Tooth disease (CMT) is the most common inherited peripheral neuropathies affecting motor and sensory nerves of the peripheral nervous system (1). The disease has been classified as demyelinating (CMT1), axonal (CMT2) and intermediate forms of CMT based on nerve conduction velocities. Mutations in >36 genes have been identified in different forms of CMT (http://www.molgen.ua.ac.be/CMTMutations/). Length-dependent peripheral neuropathy in CMT1 due to mutations in genes expressed in myelinating Schwann cells have led to an appreciation of the importance of Schwann cells in maintaining axonal transport and axonal homeostasis (2). However, it is less clear how mutations in neuronal genes in CMT2 lead to peripheral neuropathy, especially since most disease-causing genes are widely expressed and the deleterious effects are limited to motor and/or sensory neurons.

Mutations of neurofilament light (NFL) subunit in an autosomal-dominant axonal form of CMT type 2E (CMT2E) were first described in 2000 (3). Since then, 13 additional mutations in NFL have been identified, most are missense mutations in the assembly-sensitive head and central rod domains of the NFL subunit (4–9). Functional studies have shown that CMT2E mutations disrupt assembly of mutant NFL with itself, with wild-type NFL and with the mid-sized (NFM) and heavy (NFH) NF subunits, and lead to aggregation of mutant protein in SW13Vim– cells, neuronal cell lines, cultured rat cortical neurons and DRG neurons (10–14). Expression of CMT mutant NFL is also disruptive to translocation of NFs, mitochondria and components of anterograde and retrograde rapid axonal transport (10,13). The latter studies have led to the view that motor neuron degeneration may arise from impairment of axonal transport due to NFL protein aggregation. However, it is also possible that aggregation of NFL protein may have direct neurotoxic properties to which motor neurons are particularly susceptible. The latter interpretation is strengthened by the findings that abnormal protein aggregation is a common and widely recognized pathogenic mechanism leading to selective degeneration of neurons-at-risk in other neurodegenerative diseases (15).

More recently, mutations in small heat-shock proteins (sHSPs) B1 (HSPB1, Hsp27) and B8 (HSPB8, Hsp22) have been identified in CMT2F (16–18) and CMT2L (16,19,20). As members of the sHSP family, HSPB1 and HSPB8 share a conserved {alpha}-crystallin domain of approximately 90 amino acid residues, flanked by variable amino- and carboxyl-terminal extensions (21–23). sHSPs reside as dynamic oligomers of 12–43 kDa monomeric units that undergo partial disassembly as they interact with substrates and impart their chaperone activities. They interact widely in the crowded microenvironment of the cell, including with subunits of microfilaments, intermediate filaments (IFs) and microtubules (22). Their interactions with IFs are important for maintaining the integrity of IF networks (24). However, interactions between IFs and associated sHSPs can be disrupted by disease-causing mutations in either IFs or sHSPs, leading to signature pathological aggregates of mutant IFs or sHSP in degenerative diseases of muscle (25), lens (26) and glial cells (27,28).

Disruption of assembly and aggregation of NFL not only occur from mutations of NFL in CMT2E (4–9), but also from mutations of HSPB1 in CMT2F (18,29) and of HSPB8 in CMT2L (19). Also, disease-causing mutations enhance the interactions between HSPB1 and HSPB8, suggesting that they may be part of a common pathway (30). In this study, we analyze and compare the pathological effects from expression of CMT mutants of NFL and HSPB1 in primary cultured motor neurons. We show that CMT mutations of NFL and HSPB1 have similar disruptive effects on NF network, leading to aggregation of NFL protein with progressive degeneration and loss of viability of motor neurons. While co-expression of wild-type HSPB1 diminishes the toxic effects of CMT mutant NFL, expression of S135F mutant HSPB1 has a dominant effect on disruption of NF assembly and leads to progressive degeneration and loss of motor neurons. Furthermore, we show that deletion of NFL markedly reduces degeneration and loss of motor neuron viability induced by S135F mutant HSPB1. The findings indicate that disruption of NF network with aggregation of NFL may represent a common upstream pathway in motor neuron disease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCE
 
Expression of CMT mutant NFL causes degeneration and loss of viability of primary cultured motor neurons
Primary cultured motor neurons derived from E13 mouse spinal cord were analyzed for pathological changes and loss of viability following microinjection of human wild-type or CMT mutant NFL cDNAs. Co-expression of a GFP reporter transgene enables live motor neurons to be monitored for up to one week following cDNA microinjection (31). On day 1 after cDNA microinjection, targeted motor neurons injected with wild-type or with P8R or Q333P mutant NFL are clearly outlined by GFP fluorescence throughout nuclei, perikarya and neuritic processes (Fig. 1A, E and I). The distribution of GFP fluorescence remains unaltered in motor neurons expressing human wild-type NFL on day 3 (Fig. 1B) and day 4 (Fig. 1C). However, motor neurons microinjected with human P8R or Q333P mutant NFL cDNAs show fragmentation of neurites and loss of targeted motor neurons during the same interval of time (Fig. 1E–G and I–K). Fragmentation starts in distal neurites (Fig. 1F and J) and proceeds in a proximal direction leaving behind linear rows of fluorescent punctate remnants (Fig. 1G and K). Fragmentation of neurites is then followed by loss of GFP fluorescence in neurites, perikarya and nuclei of parent motor neurons (Fig. 1G and K). Examination of the targeted motor neurons by phase contrast microscopy shows that fragmentation of neurites and loss of GFP fluorescence are accompanied by degenerative changes in perikarya and nuclei of targeted motor neurons, including shrinkage and loss of nuclear and nucleolar structures (Fig. 1D, H and L). The results demonstrate that expressions of P8R and Q333P CMT mutant NFL have neurotoxic effects on cultured motor neurons.


Figure 1
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  		Figure 1. Expression of human CMT mutant NFL has neurotoxic effects on motor neurons. (A–L) Successive observations of individual targeted motor neurons on days 1–4 following co-microinjection of wild-type (A–D), P8R (E–H) or Q333P (I–L) mutant NFL cDNAs and GFP reporter transgene show progressive fragmentation of neurites and loss of fluorescent neurons injected with P8R (E–G) or Q333P (I–K) mutant NFL, but not following injection of wild-type NFL cDNAs (A–C). (M) A progressive loss of fluorescent motor neurons occurs following co-injection of GFP reporter transgene with P8R or Q333P mutant NFL, but not following co-injection with wild-type NFL. Phase-contrast microscopy of targeted motor neurons (circles) are also shown (D, H and L). (N) Motor neurons microinjected with 50 ng/µl of P8R mutant NFL cDNAs show progressive loss of fluorescent motor neurons, similar to the loss of motor neurons injected with 200 ng/µl of P8R mutant NFL cDNA. Experiments were performed in triplicate with an average of 20 microinjected motor neurons per coverslip. Quantitation of targeted motor neurons was normalized to numbers of fluorescent neurons present on day 1. Scale bars: 50 µm.

 
We next assessed the viability of microinjected motor neurons by quantitating the numbers of GFP fluorescent motor neurons and normalizing to the numbers present on day 1. Motor neurons co-injected with 200 ng/µl of human wild-type NFL cDNA and 100 ng/µl of GFP transgene show minimal loss of motor neurons following microinjection (Fig. 1M). However, motor neurons co-injected with 200 ng/µl of human P8R or Q333P mutant NFL cDNAs and 100 ng/µl of GFP reporter transgene reveal early and progressive decline in the numbers of fluorescent motor neurons during the same post-injection interval (Fig. 1M). Expression of P8R mutant NFL causes an earlier and larger loss of injected motor neurons compared with Q333P mutant NFL (Fig. 1M). By day 5, there is >60% loss of motor neurons expressing P8R or Q333P mutant NFL (Fig. 1M).

To investigate the effects of levels of CMT mutant NFL expression on motor neuron viability, cultured motor neurons were microinjected with different amounts of P8R mutant NFL cDNAs. Motor neurons injected with 50 ng/µl of P8R mutant NFL show early and progressive loss of fluorescent motor neurons in cultures, similar to the loss of motor neurons injected with 200 ng/µl of P8R mutant NFL cDNA (Fig. 1N). The findings indicate that low-level expression of CMT mutant NFL is able to impair motor neuron viability, compared to the viability of motor neurons expressing much higher levels of wild-type NFL (Fig. 1M).

Expression of P8R and Q333P CMT mutant NFL causes disruption of NF network and aggregation of NFL in primary cultured motor neurons
To investigate the effects of CMT mutant NFL on NF network, motor neurons were fixed and examined for NFL immunoreactivity following co-injection of wild-type or CMT mutant NFL with GFP cDNAs. When motor neurons are microinjected with wild-type NFL cDNA, NFL immunoreactivity is enhanced throughout perikarya and neuritic processes of targeted neurons (Fig. 2B) and, like non-injected motor neurons, is present in filamentous arrays (Fig. 2B). The findings are consistent with the incorporation of human wild-type NFL into the endogenous NF network of mouse motor neurons. When motor neurons are injected with P8R or Q333P mutant NFL and GFP cDNAs, GFP immunoreactivity is largely restricted to perikarya and proximal neurites (Fig. 2D and G) and there is fragmentation and loss of distal neurites, similar to the distribution of GFP fluorescence in live motor neurons (Fig. 1G and K). NFL immunoreactivity appears as irregular floccular aggregates in perikarya and proximal neurites of targeted motor neurons (Fig. 2E and H). Aggregation of NFL is associated with shrinkage and condensation (Fig. 2F, insert) or loss (Fig. 2I, insert) of DAPI-stained nuclei, in contrast to the preservation of nuclei in motor neurons overexpressing wild-type NFL (Fig. 2C, insert). We also found that aggregation of P8R mutant NFL is associated with co-aggregation of endogenous NFM in targeted motor neurons (Supplementary Fig. S1). The findings indicate that expression of CMT mutant NFL leads to disruption of NF network, formation of NFL protein aggregates and degeneration of motor neurons, and suggest that disruption of NF network and aggregation of NFL protein may play a role in conferring the neurotoxicity from expression of CMT mutant NFL.


Figure 2
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  		Figure 2. NFL undergoes aggregation in degenerated motor neurons due to expression of CMT mutant NFL. Motor neurons were fixed, immunostained and examined by fluorescence microscopy on day 5 after cDNA microinjection. (A–C) Motor neuron co-injected with human wild-type NFL cDNA and GFP reporter transgene shows widespread GFP fluorescence (A) and extensive filamentous arrays of NFL in perikaryon and neurites (B) with normal appearance of DAPI-stained nucleus (C, insert). (D–I) Motor neurons co-injected with P8R (D–F) or Q333P (G–I) CMT mutant NFL cDNA and GFP reporter gene show irregular floccular aggregates of GFP (D, G) and NFL (E, H) immunoreactivities in perikarya (arrows) and proximal neurites (arrowheads) as well as shrinkage and fragmentation (F, insert, arrow) or loss (I, insert) of DAPI-stained nuclei. Scale bars: 25 µm.

 
HSPB1 associates with wild-type and CMT mutant NFL
To further address the role of altered assembly and aggregation of NFL in motor neuron degeneration, we examined the properties of HSPB1 as a potential modulator of assembly and aggregation of NFL. We first studied the interactions of HSPB1 with NFL by examining the distribution of HSPB1 and NFL in SW13Vim– cells. When SW13Vim– cells were co-transfected with human wild-type NFL and HSPB1 transgenes, human wild-type NFL forms fine filamentous networks in the cells (Fig. 3A), and HSPB1 is widely distributed in the cytoplasm (Fig. 3B). When SW13Vim– cells were co-transfected with P8R or Q333P CMT mutant NFL and HSPB1 transgenes, P8R and Q333P mutant NFLs form multiple protein aggregates in the cytoplasm of SW13Vim– cells (Fig. 3D and G, arrows), and HSPB1 co-localizes with aggregates of P8R (Fig. 3D–F, arrows) and Q333P (Fig. 3G–I, arrows) mutant NFL.


Figure 3
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  		Figure 3. HSPB1 associates with NFL in vivo. (A–I) When wild-type NFL is co-transfected with HSPB1 into SW13Vim– cells, NFL forms filamentous network (A), and HSPB1 is diffusely distributed in the cytoplasm of transfected cells (B). When cells are co-transfected with P8R or Q333P CMT mutant NFL and HSPB1, HSPB1 undergoes aggregation (E, H) and co-localizes with aggregates of CMT mutant NFL (F, I). (J) HSPB1 is found mostly in Triton-soluble fraction of cell lysates when expressed alone in SW13Vim– cells (lanes 1 and 5), but is enriched in Triton-insoluble pellets when co-expressed with wild-type NFL (lanes 2 and 6), P8R (lanes 3 and 7) or Q333P (lanes 4 and 8) CMT mutant NFL and assessed at 24 h after co-transfection. (K) Wild-type and P8R or Q333P CMT mutant NFL proteins co-precipitate with HSPB1 when Neuro2a cells are co-transfected with HA-tagged HSPB1 and NFL cDNAs and cell lysates incubated with anti-HA antibodies 48 h following transfection. (L) HSPB1 co-precipitates with wild-type and P8R or Q333P CMT mutant NFL when cell lysates are incubated with anti-NFL antibodies. Scale bars: 20 µm.

 
To further study the interaction of HSPB1 with wild-type and CMT mutant NFL, co-fractionation studies were undertaken on SW13Vim– cells that were transfected with HA-tagged HSPB1 alone or together with wild-type or CMT mutant NFL and separated into Triton-soluble and Triton-insoluble fractions. When expressed alone, most HSPB1 is found in Triton-soluble supernatants (Fig. 3J, upper panel lane1), compared with Triton-insoluble pellets (Fig. 3J, upper panel lane 5). Co-expression of wild-type and CMT mutant (P8R or Q333P) NFL diminishes Triton-soluble (Fig. 3J, upper panel lanes 2–4) and increases Triton-insoluble HSPB1 (Fig. 3J, upper panel lanes 6–8). The resulting partitioning of HSPB1 in Triton-insoluble fractions associated with NFL expression could reflect interaction of HSPB1 with wild-type NFL or entrapment in aggregates of CMT mutant NFL.

Co-immunoprecipitation assays were then undertaken to further investigate the interactions of HSPB1 with wild-type and CMT mutant NFL. As shown in Fig. 3K, both wild-type and CMT mutant NFL are co-precipitated with HSPB1 by HA antibodies. Similarly, HSPB1 is also co-precipitated with wild-type and CMT mutant NFL by NFL antibodies (Fig. 3L). Together, the data demonstrate that HSPB1 interacts with both wild-type and CMT mutant NFL in vivo and suggest that HSPB1 could play a role in modulating the aggregative properties of CMT mutant NFL.

HSPB1 reduces aggregation and induces reversal of CMT mutant NFL aggregates
To study the role of HSPB1 in modulating the aggregative properties of CMT mutant NFL, SW13Vim– cells were transfected with P8R or Q333P mutant NFL alone or with HSPB1, and the effects of HSPB1 on CMT mutant NFL protein aggregation were analyzed. While co-expression of HSPB1 does not alter the expression levels of P8R or Q333P mutant NFL in transfected SW13Vim– cells (Fig. 4A), it reduces the percentages of cells containing CMT mutant NFL protein aggregates (Fig. 4B and C). At 24 and 48 h, co-expression of HSPB1 reduces the numbers of cells containing aggregates of P8R mutant NFL from 73 and 79% to 49 and 51%, respectively (Fig. 4B). Similarly, at 24 and 48 h after co-transfection, expression of HSPB1 reduces the percentage of cells containing Q333P mutant NFL aggregates from 68 and 67% to 51 and 28%, respectively (Fig. 4C). It was noted that, at 48 h after transfection, the inhibition of NFL protein aggregation by HSPB1 is more apparent in cells expressing Q333P than those expressing P8R mutant NFL (compare Fig. 4B and C). Together, the findings demonstrate that co-expression of HSPB1 reduces the aggregation of CMT mutant NFL proteins in SW13Vim– cells.


Figure 4
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  		Figure 4. HSPB1 reduces aggregation and induces reversal of aggregation of CMT mutant NFL. (A) Co-expression of HSPB1 does not alter the expression level of P8R or Q333P mutant NFL in SW13Vim– cells when the whole cell lysates were analyzed by western blot. (B and C) Co-transfection of HSPB1 with P8R (B) or Q333P (C) mutant NFL cDNAs reduces the percentage of SW13Vim– cells containing aggregates of CMT mutant NFL at 24 and 48 h after transfection when compared with cells transfected with P8R or Q333P mutant NFL alone. The percentage of cells expressing mutant NFL as aggregates were determined by blinded observers from three independent experiments. (D–F) When cultured motor neurons were co-injected with HSPB1 cDNA, there is a decline in the percentage of neurons with aggregative forms, as depicted (E), of mutant P8R NFL at 24 and 48 h (D) and corresponding increase of neurons bearing filamentous arrays, as depicted (F), of mutant P8R NFL. Identical levels of P8R NFL cDNA were co-injected with HSPB1 cDNA or empty expression vectors. Data were calculated from three separated experiments. (G–L) Motor neurons co-injected with GFP-tagged P8R mutant NFL and HSPB1 cDNAs reveal aggregates of mutant NFL in perikarya (G and J) on day 1. On days 2 and 3, the same motor neurons reveal disappearance of perikaryal aggregates of mutant NFL with the appearance of mutant NFL in filamentous arrays in perikarya and neuritic processes (H-I and K-L). Scale bars: 25 µm.

 
We next examined the effects of HSPB1 on aggregation of CMT mutant NFL in motor neurons by monitoring fluorescence of GFP-tagged P8R mutant NFL in live motor neurons. When motor neurons were co-injected with P8R mutant NFL cDNA and an empty expression vector, 52 and 48% of injected motor neurons contain aggregates of GFP-tagged NFL protein at 24 and 48 h (Fig. 4D), as depicted in Fig. 4E. When motor neurons were co-injected with P8R mutant NFL and HSPB1 cDNAs, however, the percentage of aggregate-bearing motor neurons is reduced to 31 and 24% at 24 and 48 h, respectively (Fig. 4D). There is a corresponding increase of GFP fluorescence in filamentous arrays, as depicted in Fig. 4F. These findings demonstrate that co-expression of HSPB1 reduces aggregation of CMT mutant NFL in motor neurons.

Interestingly, successive examination of motor neurons containing aggregates of GFP-P8R mutant NFL on day 1 (Fig. 4G and J) shows that the NFL aggregates are no longer present in the same neurons on day 2 (Fig. 4H and K) or day 3 (Fig. 4I and L). Instead, the GFP-P8R NFL protein becomes redistributed in filamentous forms in the perikarya and neuritic processes of motor neurons (Fig. 4H, I, K and L). This phenomenon is readily observed following co-injection of HSPB1 but not following injection of P8R mutant NFL cDNA alone. The findings suggest that HSPB1 not only alters the aggregative properties of CMT mutant NFL but also induces the reversal of CMT mutant NFL protein aggregates and promotes their incorporation into filamentous arrays in motor neurons.

HSPB1 reduces P8R CMT mutant NFL-induced loss of motor neuron in cultures
To investigate the role of HSPB1 in modulating the neurotoxicity of CMT mutant NFL, we next assessed the effects of HSPB1 expression on viability of motor neurons expressing CMT mutant NFL. When motor neurons were microinjected with GFP-tagged P8R mutant NFL cDNA, expression of P8R mutant NFL causes progressive decline in the number of fluorescent motor neurons (Fig. 5), similar to the loss of motor neurons injected with untagged P8R mutant NFL cDNA (Fig. 1). However, co-injection of HSPB1 cDNA markedly reduces the loss of motor neurons expressing GFP-P8R mutant NFL (Fig. 5). The findings demonstrate that HSPB1 reduces the neurotoxicity of CMT mutant NFL in motor neurons and suggest that HSPB1 is an important factor regulating the aggregation and neurotoxicity of CMT mutant NFL.


Figure 5
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  		Figure 5. HSPB1 reduces P8R mutant NFL-induced loss of motor neurons. Viability of motor neurons was assessed by quantitating the numbers of fluorescent motor neurons and normalizing to the numbers present on day 1 after cDNA microinjection. Motor neurons were co-microinjected with GFP-tagged P8R NFL and HSPB1 cDNAs or empty expression vector. Experiments were performed in triplicate with a minimum of 60 microinjected motor neurons.

 
S135F mutant HSPB1 has dominant effects on disruption of assembly and aggregation of NFL protein
The ability of CMT mutant HSPB1 to disrupt assembly and promote aggregation of NFL in transfected cells (18) raises the possibility that altered assembly and aggregation of NFL play a role in the pathogenesis of CMT2F. To investigate this possibility, we first analyzed the deleterious effects of disease-causing S135F mutant HSPB1 on NFL assembly. We found that, when transfected alone into SW13Vim– cells, human wild-type NFL assembles into filamentous network (Fig. 6A) and undergoes minimal aggregation (Fig. 6B), S135F mutant HSPB1 is diffusely distributed in the cytoplasm (Fig. 6C) of transfected cells. However, when human wild-type NFL was co-transfected with S135F mutant HSPB1 into SW13Vim– cells, co-expression of S135F mutant HSPB1 causes formation of NFL protein aggregates (Fig. 6D), which co-aggregate with mutant HSPB1 (Fig. 6E and F). Co-expression of S135F mutant HSPB1 also causes aggregation of wild-type NFL in motor neuron-like NSC34 cells (Fig. 6G–I). Quantitative assays show that, while human wild-type NFL undergoes minimal aggregation when transfected alone in SW13 Vim– and NSC34 cells (Fig. 6J and K), co-expression of S135F mutant HSPB1 causes massive aggregation of NFL protein (Fig. 6J and K). Interestingly, co-expression of wild-type HSPB1 does not overcome the ability of S135F mutant HSPB1 to disrupt assembly and cause aggregation of NFL protein in SW13Vim– (Fig. 6J) and NSC34 cells (Fig. 6K). Together, the data demonstrate that S135F mutant HSPB1 has a dominant disruptive effect on NF assembly in non-neuronal and neuronal cells, i.e. not overcome by wild-type HSPB1. Similar phenomena were observed with GFP-tagged human wild-type NFL (data not shown).


Figure 6
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  		Figure 6. S135F CMT mutant HSPB1 has dominant effects on disruption of assembly with aggregation of NFL protein. (A and B) Wild-type NFL is expressed mainly as filamentous arrays (A) and to a much lesser extent, as protein aggregates (B) when expressed in SW13Vim– cells. (C) S135F mutant HSPB1 is diffusely distributed in the cytoplasm when transfected alone into SW13Vim– cells. (D–F) Expression of S135F mutant HSPB1 disrupts assembly and causes aggregation of NFL when SW13Vim– cells are co-transfected with human wild-type NFL with S135F mutant HSPB1. (G–I) Co-expression of S135F mutant HSPB1 with human wild-type NFL disrupts assembly and causes aggregation of NFL in motor neuron-like NSC34 cells. (J) Quantitative assay shows that expression of S135F mutant HSPB1 causes massive aggregates formation of human wild-type NFL in SW13Vim– cells, and that co-expression of wild-type HSPB1 does not prevent the aggregation of NFL caused by S135F mutant HSPB1. (K) Similar quantitative assay shows that expression of wild-type HSPB1 fails to prevent the aggregation of NFL caused by S135F mutant HSPB1 in NSC34 cells expressing endogenous NFs. Scale bars: 20 µm.

 
S135F mutant HSPB1 associates with wild-type and CMT mutant NFL
To investigate the biochemical basis underlying the dominant effects of S135F mutant HSPB1 on disruption of NF assembly, we examined the interactions of S135F mutant HSPB1 with wild-type and CMT mutant NFL by co-immunoprecipitation assay. We found that both wild-type and CMT mutant (P8R and Q333P) NFL are co-precipitated with S135F mutant HSPB1 (Fig. 7A and B). Co-immunoprecipitation assay further shows that wild-type and S135F mutant HSPB1 associate with endogenous NFL (Fig. 7C). The results demonstrate that wild-type and S135F mutant HSPB1 interact with NFL and suggest that disruption of NF assembly and aggregation of NFL is a pathway underlying the neurotoxicity of CMT mutant HSPB1.


Figure 7
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  		Figure 7. S135F mutant HSPB1 associates with wild-type and CMT mutant NFL. (A) Co-immunoprecipitation assay of Neuro2a cells co-transfected with HA-tagged S135F mutant HSPB1 and NFL cDNAs shows that wild-type and P8R or Q333P CMT mutant NFL proteins are co-precipitated with S135F mutant HSPB1 by anti-HA antibodies. (B) Similar co-IP assay shows that S135F mutant HSPB1 is co-precipitated with wild-type and P8R or Q333P CMT mutant NFL by anti-NFL antibodies. (C) Co-IP assay of NSC34 cells transfected with HA-tagged wild-type or S135F mutant HSPB1 shows that wild-type and S135F mutant HSPB1 are co-precipitated with endogenous NFL by anti-NFL.

 
Expression of CMT mutant HSPB1 causes degeneration and loss of viability of cultured motor neurons
In order to address the toxicity of mutant HSPB1 on motor neurons, we first established a cellular model of CMT mutant HSPB1 toxicity in motor neurons by cDNA microinjection. Primary cultured motor neurons were microinjected with 200 ng/µl of wild-type or S135F mutant HSPB1 cDNA and 100 ng/µl of GFP reporter transgene. On day 1 after microinjection, GFP fluorescence outlines the cell bodies and neurites of injected motor neurons (Fig. 8A and D). On days 2 and 3, GFP fluorescence in targeted motor neurons injected with wild-type HSPB1 remains unaltered (Fig. 8B and C). However, motor neurons injected with S135F mutant HSPB1 cDNA show fragmentation of neurites (Fig. 8E, neuron 1) and loss of fluorescent motor neurons (Fig. 8E, neuron 2) on day 2. Fragmentation of neurites and loss of targeted motor neurons become widespread by day 3 (Fig. 8F).


Figure 8
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  		Figure 8. Expression of S135F CMT mutant HSPB1 has neurotoxic effects on motor neurons. (A–F) Successive observation of individual targeted motor neurons on days 1-3 following co-injection of wild-type or S135F CMT mutant HSPB1 cDNAs and GFP reporter transgene show progressive fragmentation of neurites and loss of fluorescent neurons injected with S135F mutant HSPB1 cDNA (D–F). Motor neurons injected with wild-type HSPB1 cDNAs show minimal morphological changes (A–C). (G) An early and severe loss in numbers of fluorescent motor neurons occurs following co-injection of GFP reporter transgene with 200 ng/µl of S135F mutant HSPB1 cDNA, but not following co-injection with 200 ng/µl of wild-type HSPB1 cDNA. Motor neurons microinjected with 50 ng/µl of S135F mutant HSPB1 cDNAs show reduced loss of fluorescent motor neurons in cultures, compared with motor neurons injected with 200 ng/µl of S135F mutant HSPB1 cDNA. Experiments were performed in triplicate with 15–60 injected motor neurons per coverslip. Quantitation was normalized to numbers of fluorescent neurons present on day 1. Scale bars: 50 µm.

 
Analyses of viability of targeted motor neurons reveal minimal loss of targeted motor neurons following co-injection of 200 ng/µl of wild-type HSPB1 cDNA with GFP transgene (Fig. 8G). However, motor neurons co-injected with 200 ng/µl of S135F mutant HSPB1 cDNA and GFP reporter gene show early and rapid loss of motor neuron during the same post-injection interval (Fig. 8G). Co-injection of 50 ng/µl of S135F mutant HSPB1 cDNA with GFP transgene shows reduced rapidity and severity in loss of motor neurons (Fig. 8G). The results demonstrate that S135F mutant HSPB1 has severe neurotoxic effects on cultured motor neurons and the effects are dose-dependent.

Expression of S135F mutant HSPB1 causes disruption of NF network and aggregation of NFL in motor neurons
To investigate the effects of CMT mutant HSPB1 on NF network in motor neurons, we examined NFL immunoreactivity in targeted motor neurons. When motor neurons are injected with wild-type HSPB1, HSPB1 immunoreactivity is widely distributed in the perikarya and neurites (Fig. 9A), and the endogenous NFL appears as filamentous arrays (Fig. 9B and C), similar to the NFL network in uninjected motor neurons. When motor neurons are injected with S135F mutant HSPB1, the perikarya of targeted neurons have an abnormally rounded contour and contain irregular aggregates of HSPB1 (Fig. 9D and G, inserts) that co-localize with NFL (Fig. 9E and F) and NFH (Fig. 9H and I, inserts), thereby revealing disruption of endogenous NF network in targeted neurons. HSPB1 immunoreactivity also appears as punctate aggregates in fragmented neurites (Fig. 9D and G, arrows) and co-localizes with NFL (Fig. 9E and F, arrows) and NFH (Fig. 9H and I, arrows) immunoreactivities. The data indicate that the neurotoxic effects of S135F mutant HSPB1 are associated with disruption of NF network and co-aggregation of NFL and HSPB1 in motor neurons.


Figure 9
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  		Figure 9. Disruption of NF network with aggregation of NFL and NFH proteins in degenerated motor neurons due to expression of S135F mutant HSPB1. (A–C) Co-injection of wild-type HSPB1 cDNA with GFP reporter transgene does not alter the extensive filamentous arrays of NFL in neurites and perikarya (B) of motor neurons when immunostained for NFL on day 5 after injection. (D–F) Injection of S135F mutant HSPB1 cDNA leads to extensive fragmentation of neurites that are outlined by HSPB1 (D, arrows) and NFL (E, arrows) immunoreactivities and to co-aggregation of HSPB1 and NFL in neurites (D–F, arrows) and perikarya (D–F, inserts). (G–I) Expression of S135F mutant HSPB1 cDNA leads to disruption of NF with aggregation of NFH in perikarya (H, insert) and neurites (H, arrows) and to co-aggregation of HSPB1 with NFH in perikarya (I, insert) and neurites (I, arrows). Scale bars: 25 µm.

 
NFL-null motor neurons are resistant to S135F mutant HSPB1-induced neurotoxicity
We next set out to probe the role of NFL in CMT mutant HSPB1 toxicity using NFL-null motor neurons prepared from NFL-knockout mice (32). Deletion of NFL does not alter the growth and development of motor neurons in cultures (Supplementary Fig. S2). When NFL-null motor neurons were microinjected with 200 ng/µl of S135F mutant HSPB1 cDNA and 100 ng/µl of GFP reporter transgene, GFP fluorescence outlines the neurites and perikarya of targeted neurons (Fig. 10A). GFP fluorescence remains unaltered in the same neuron on day 3 (Fig. 10B) and day 5 (Fig. 10C) after microinjection. Examination of the same neuron by phase contrast microscopy reveals preservation of perikarya, nucleus and nucleolus structures (Fig. 10D, insert), confirming the lack of degenerative changes in targeted NFL-null motor neurons expressing S135F mutant HSPB1. Examination of HSPB1 and NFM immunoreactivities shows that neither S135F mutant HSPB1 (Supplementary Fig. S3A) nor endogenous NFM (Supplementary Fig. S3B) undergoes aggregation in targeted NFL-null motor neurons. The data indicate that the presence of NFL is important for the toxicity of CMT mutant HSPB1 in motor neurons.


Figure 10
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  		Figure 10. NFL-null motor neurons are resistant to S135F mutant HSPB1-induced neurotoxicity. (A–C) Successive observation of GFP fluorescence of NFL-null motor neurons over a 5-day interval following co-microinjection of S135F mutant HSPB1 cDNA and GFP transgene show minimal morphological change. (D) Examination of the same NFL-null motor neuron by phase contrast microscopy shows preservation of characteristic large perikarya, nucleus and prominent nucleolus on day 5 after cDNA microinjection. (E) NFL-null motor neurons microinjected with 200 ng/µl of S135F mutant HSPB1 cDNAs show minimal loss of GFP fluorescent motor neurons in cultures over a 5-day interval, compared with NFL (+/+) motor neurons injected with same concentration of S135F mutant HSPB1 cDNA. Experiments were performed in triplicate with an average of 20 injected motor neurons per coverslip. Quantitation of fluorescent neurons was normalized to numbers of fluorescent neurons present on day 1. Scale bar: 50 µm.

 
Next we compared the toxicity of S135F mutant HSPB1 in NFL (+/+) and NFL-null motor neurons. Whereas microinjection of 200 ng/µl of S135F mutant HSPB1 cDNA causes early and severe loss of NFL (+/+) motor neurons in cultures (Fig. 10E), NFL-null motor neurons microinjecting with the same concentration of S135F mutant HSPB1 show minimal loss of viability (Fig. 10E). The data demonstrate that deletion of NFL markedly reduces the toxicity of S135F mutant HSPB1 in motor neurons. Together, these studies indicate that NFL is instrumental in the neurotoxicity of CMT mutant HSPB1 and that disruption of NF network and aggregation of NFL is an important upstream pathway leading to motor neuron degeneration.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCE
 
Modeling of disease in primary cultures of motor neurons has enabled a direct examination of the neurotoxic effects of disease-causing mutations in neurons-at-risk. In this study, we show that expression of CMT mutant NFL or CMT mutant HSPB1 has neurotoxic effects on cultured motor neurons and that CMT mutations in NFL and in HSPB1 cause the same pattern of neurodegenerative changes in motor neurons. Whereas expression of wild-type NFL is incorporated into the endogenous NF network with enhancement of NFL immunoreactivity throughout perikarya and neurites of targeted neurons, expression of CMT mutant forms of NFL and HSPB1 causes disruption of NF network, aggregation of NFL protein and progressive degeneration of motor neurons. The findings suggest that the neurotoxic effects arise from early and direct disruptive effects of mutant proteins on NF assembly. This view is supported by previous studies showing that CMT mutations in NFL, HSPB1 and HSPB8 disrupt NFL assembly and cause aggregation of neurofilament proteins in neuronal and non-neuronal cells (10–13,18,19,29).

The ability of HSPB1 to interact with CMT mutant NFL and to reduce aggregation and neurotoxicity of mutant NFL supports a role of NFL aggregation in motor neuron degeneration. sHSPs are molecular chaperones that interact with partially denatured proteins and prevent misfolding and aggregation of the proteins (21). Overexpression of HSPB1 has recently been shown to reduce aggregation and toxicity of {alpha}-synuclein and ß-amyloid proteins (33,34). Additionally, sHSPs have the capacity to regulate the cytoskeletal dynamics (35). For example, HSPB1 can associate with soluble and filamentous forms of IF proteins, control the interactions between filaments and prevent aggregation of IF protein (24). Our findings that HSPB1 interacts with both wild-type and CMT mutant NFL, triggers reversal of CMT mutant NFL aggregation and promotes NFL assembly indicate that the chaperone activity of HSPB1 and ability to stabilize the NF network can contribute to its neuroprotective properties.

The prospective role of NFL protein aggregation in motor neuron disease adds to a growing list of neurodegenerative diseases in which neurotoxicity is associated with abnormal protein aggregation (36–38). In each instance, the role of abnormal protein aggregation in pathogenesis of disease has come from studies of mutations that cause familial disease and the recognition of similar protein aggregates in sporadic forms of disease. In accordance with this general principle, NF inclusions have long been recognized as a hallmark pathological feature of degenerating motor neurons in sporadic and familial forms of ALS (39). Evidence that perturbations in NF assembly and aggregation of NF proteins can have highly selective adverse effects on motor neurons comes from studies of transgenic mice expressing NF transgenes. For instance, altering NF subunit stoichiometry by overexpression of wild-type NF transgenes causes aggregation of NF proteins and motor neuron dysfunction (40–42), adverse pathological effects that can be reversed by correcting the NF subunit stoichiometry (43). Expression of either mutant NFL transgene (44,45) or untranslated NFL RNA (46) leads to a more severe motor phenotype with degeneration of motor neurons in transgenic mice. Moreover, expression of untranslated NFL RNA reproduces the pathology of ALS, including the selective degeneration of motor neurons and aggregation of NFL protein in degenerating motor neurons (47). Thus, experimental models provide additional support for the findings of the present study that aggregation of NFL is an early event in motor neuron disease.

Compelling evidence that alterations in NF assembly with aggregation of NFL protein can lead to motor neuron degeneration is provided by the disruptive effects on the endogenous NF network in motor neurons from expression of S135F mutant HSPB1. Interestingly, the ability of CMT mutant HSPB1 to disrupt NF assembly and cause aggregation of NFL protein cannot be overcome by co-expression of wild-type HSPB1, indicating a toxic dominant effect of S135F mutant HSPB1 on the NF network. Biochemical studies show that S135F mutation does not impair the ability of HSPB1 to interact with NFL proteins. Therefore, it is possible that disruption of NFL assembly by S135F mutation in HSPB1 may be due to increased binding affinity, as suggested by disease-causing mutations in other sHSP (25). Moreover, the dominant toxic effects of CMT mutant HSPB1 on NFL assembly further supports the view that disruption of NF network with aggregation of NF proteins is an underlying mechanism in mutant HSPB1-induce motor neuropathy. In accordance with this view, previous studies have shown that CMT mutant HSPB1 disrupts self-assembly of NFL in transfected Neuro2a cells (18) and blocks incorporation of NFM into endogenous NF network in cortical neurons (29).

Our findings that CMT mutations in NFL and HSPB1 cause motor neuron degeneration through disruption of NF network and aggregation of NFL protein are similar to the mechanism underlying the alterations in assembly and aggregation of IF protein in desmin-related myopathy (DRM). In DRM, missense mutations in either desmin (48), an IF protein in muscles, or {alpha}B-crystallin (HSPB5) (49,50), lead to collapse of the desmin filament network and formation of intracellular protein aggregates that contain desmin and {alpha}B-crystallin (48,49,51,52). The ability of HSPB1 to interact with CMT mutant NFL and to diminish their adverse effects on NF assembly provides additional evidence for the role of sHSPs in maintaining the integrity and homeostasis of IF network (24,28). Together, the findings strongly suggest a pathogenic role of disruption of IF networks with aggregation of the IF proteins in the disease states.

The prospective role of NFL aggregation in the pathogenesis of motor neuron degeneration is also supported by marked reduction of toxicity of S135F mutant HSPB1 in NFL-null motor neurons. Deletion of NFL has previously been shown to delay the onset and slow the progression of disease caused by mutant SOD1 in transgenic mice (53). Moreover, deletion of NFL also markedly reduces the accumulation of abnormal tau in motor neurons and attenuates the associated motor neuron phenotype (54). These studies implicate the role of NFL in the aggregation and neurotoxicity of other widely expressed disease-causing proteins in motor neurons. This prospective may also help to explain selective aggregation with neurotoxic effects of disease-causing mutations in widely expressed proteins like SOD1 on motor neurons. Synergistic aggregation of NFL and mutant SOD1 proteins, as observed in transfected cells (55), could promote aggregation of mutant SOD1, exacerbation of NFL aggregation and the neurotoxic effects of protein aggregation on motor neurons. For example, the synergistic interactions of NFL and mutant SOD1 may promote selective aggregation and neurotoxicity of mutant SOD1 in motor neurons of transgenic mice expressing G86R (56) and G93A (47) mutant SOD1 in which NFL protein aggregates are detected. Similar synergistic effects of NFL protein aggregates may also account for the aggregation and neurotoxicity in motor neurons of other widely expressed mutant proteins that are destabilized by missense mutations and cause autosomal-dominant motor neuron disease (57).

In summary, our hypothesis that disruption of NF network and aggregation of NFL protein is a triggering mechanism in motor neuron degeneration is supported by direct modeling of CMT mutant NFL and HSPB1-linked disease in motor neurons. As a triggering event of motor neuron dysfunction and degeneration, the changes could begin to explain the specific vulnerability of motor neurons in mice expressing NF transgenes, in transgenic models of disease as well as in sporadic and familial forms of motor neuron disease. As an upstream mechanism, disruption of assembly and aggregation of NFL protein could provoke the widespread degenerative and/or reactive changes in many diverse pathways of motor neurons and surrounding cells that have been well documented in motor neuron disease (58). More importantly, the identification of a prospective, upstream triggering mechanism could provide novel targets and/or strategies for effective treatment and palliation of motor neuron disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCE
 
DNA constructs and reagents
Full-length human NFL cDNA was purchased from GeneCopoeia (Maryland, USA) and was expressed either as untagged (pReceiver-M02/hNFL) or C-terminal GFP-tagged (pReceiver-M03/hNFL-GFP) NFL. Full-length HSPB1 cDNA was amplified by PCR and expressed as HA-tagged protein in pReceiver-M06 mammalian vector. Mutagenesis was performed using the QuikChangeTM Site-directed Mutagenesis Kit (Stratagene). All constructs were verified by DNA sequencing. Primary antibodies used in this study were purchased from Roche Applied Science (anti-HA), Santa Cruz Biotechnology Inc. (anti-GFP, anti-NFL, anti-NFM, anti-NFH and anti-HSPB1) and Upstate (anti-HSPB1). Secondary antibodies were purchased from Molecular Probes.

Primary cultures of motor neurons and cDNA microinjection
Primary cultured motor neurons were prepared from NFL (+/+) or NFL (–/–) embryonic spinal cords as described previously (31). NFL (–/–) mice (32) were gifts from Dr Jean-Pierre Julien (Canada). Briefly, spinal cords were dissected from day 13 (E13) mouse embryos, dissociated with trypsin and plated on poly-L-lysine (Sigma) precoated-12-mm glass coverslips (Fisher) at a density of 2 x 105cells/well in 24-well culture plates. Cells were grown in modified N3 medium and treated with 1.4 µg/ml cytosine arabinoside (Sigma) after 4–6 days culture to minimize growth of non-neuronal cells. Cultures were subjected to cDNA microinjection after 6 weeks culture.

cDNA microinjection was conducted using a Zeiss Axiovert-10 microscope, Eppendorf 5170 micromanipulator and Eppendorf 5242 microinjector. For microinjection, motor neurons were co-injected with 50–200 ng/µl of mammalian expression vectors encoding untagged human NFL or HA-tagged HSPB1 together with 100 ng/µl of GFP reporter cDNAs. For some experiments, motor neurons were microinjected with 50–200 ng/µl of human GFP-tagged NFL cDNA alone or together with 200 ng/µl of HSPB1 cDNA or empty expression vector. Microinjected motor neurons were subsequently identified by GFP fluorescence.

Assessment of motor neuron viability
After cDNA microinjection, the numbers of motor neurons containing GFP fluorescence on each coverslip were counted under fluorescence microscopy during a 5-day interval. The survival of injected motor neurons was confirmed by phase contrast microscopy. Data were presented as percentage of survival motor neurons by normalizing the numbers of motor neurons from different time points to the numbers present on day 1. Each experiment was performed in triplicate and conducted on a minimum of 60 injected motor neurons.

Cell culture and transfection
SW13Vim– cells and neuronal cell lines (NSC34 cells and Neuro2a cells) were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Cells were transfected using FuGene 6 Transfection Reagent (Roche Applied Science) following the manufacture’s instructions.

Co-fractionation assay, co-immunoprecipitation assay and western blot
At 48 h after transfection, SW13Vim– cells were lysed in cold lysis buffer [50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100, 1 mM PMSF and mammalian protease inhibitor cocktail (Sigma)] and centrifuged at 10 000g for 15 min at 4°C to obtain Triton-soluble supernatants and Triton-insoluble pellets. Triton-insoluble pellets were washed with lysis buffer three times and then boiled in Laemmli sample buffer. Similar amounts of supernatant and pellet fractions were analyzed by western blots. For co-immunoprecipitation assay, Triton-soluble supernatants were incubated with antibodies at 4°C overnight. After four washes with lysis buffer, immunoprecipitates were boiled in SDS loading buffer, separated by 4–12% SDS–PAGE (Invitrogen) and subjected to western blot analyses.

Fluorescence microscopy
Motor neurons were visualized by phase and fluorescence microscopy using a Nikon TE300 inverted microscopy. Sequential changes in number and morphology of fluorescent motor neurons were assessed by direct visualization and were also recorded as digital camera images of the same coverslip areas taken daily over a 5-day period. For immunofluorescence analysis, cells were fixed with methanol/acetone (1:1) for 5 min at room temperature. After treatment with 0.1% of Triton X-100 for 10 min, fixed cells were incubated with primary antibodies overnight at 4°C followed by incubation with fluorescence labeled secondary antibodies. After mounting, cells were examined and photographed under fluorescent microscopy (Zeiss LSM 510).


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCE
 
This research was supported by grant from the National Institutes of Health (NIH, USA) to W.W.S. and two Research Development Grants from Muscular Dystrophy Association (MDA4264 to J.Z. and MDA3833 to H.L.).


   ACKNOWLEDGEMENTS
 
We thank Dr Janice Robertson (University of Toronto, CA) for kind gift of SW13Vim– cells.

Conflict of Interest statement. None declared.


   REFERENCE
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCE
 

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