Skip Navigation


Human Molecular Genetics Advance Access originally published online on October 19, 2005
Human Molecular Genetics 2005 14(23):3643-3659; doi:10.1093/hmg/ddi392
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Material
Right arrow All Versions of this Article:
14/23/3643    most recent
ddi392v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (12)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lin, H.
Right arrow Articles by Schlaepfer, W. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, H.
Right arrow Articles by Schlaepfer, W. W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

RNA-binding protein is involved in aggregation of light neurofilament protein and is implicated in the pathogenesis of motor neuron degeneration

Hong Lin, Jinbin Zhai and William W. Schlaepfer*

Division of Neuropathology, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA

* To whom correspondence should be addressed at: 609C Stellar-Chance Laboratories, Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 422 Curie Boulevard, Philadelphia, PA 19104, USA. Tel: +1 2156627372; Fax: +1 2155732059; Email: wws435jp{at}mail.med.upenn.edu

Received August 18, 2005; Accepted October 13, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Abnormal protein aggregation is emerging as a common theme in the pathogenesis of neurodegenerative disease. Our previous studies have shown that overexpression of untranslated light neurofilament (NF-L) RNA causes motor neuron degeneration in transgenic mice, leads to accumulation of ubiquitinated aggregates in degenerating cultured motor neurons and triggers aggregation of NF-L protein and co-aggregation of mutant SOD1 protein in neuronal cells. Here, we report that p190RhoGEF, an RNA-binding protein that binds to a destabilizing element in NF-L mRNA, is involved in aggregation of NF-L protein and is implicated in the pathogenesis of motor neuron degeneration. We show that p190RhoGEF co-aggregates with unassembled NF-L protein and that co-aggregation is associated with down-regulation of parent NF-L mRNA in neuronal cells. Co-expression of NF-M increases NF assembly and reduces RNA-triggered aggregation as well as loss of solubility of NF-L protein. siRNA-induced down-regulation of p190RhoGEF not only reduces aggregation and promotes assembly of NF-L and NF-M, but also causes reversal of aggregation and recovery of NF assembly in transfected cells. Examination of transgenic models of motor neuron disease shows that prominent aggregates of p190RhoGEF and NF-L and down-regulation of NF-L expression occur in degenerating motor neurons of mice expressing untranslated NF-L RNA or a G93A mutant SOD1 transgene. Moreover, aggregates of p190RhoGEF and NF-L appear as early pathological changes in presymptomatic G93A mutant SOD1 transgenic mice. Together, the findings indicate that p190RhoGEF is involved in aggregation of NF-L protein and support a working hypothesis that aggregation of p190RhoGEF and NF-L is an upstream event triggering neurotoxicity in motor neuron disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
There is a growing consensus that misfolding and aggregation of proteins are a common underlying mechanism of neurotoxicity in neurodegenerative diseases (1Go–7Go). Although intracellular and extracellular end products of protein aggregation are neuropathological landmarks of disease, the toxicity of protein aggregation is believed to be due to soluble intermediate forms rather than the insoluble end product inclusions that accumulate in the tissues (8Go–12Go). Hence, neurotoxic interactions may precede the formation of inclusions, and the presence and distribution of inclusions may not correlate with the progression and extent of disease.

Protein aggregation is also strongly implicated in the pathogenesis of motor neuron degeneration due to mutant SOD1 (13Go–16Go). A common feature among more than 100 different disease-causing mutations is that they destabilize SOD1 protein (17Go–24Go), an event that may be enhanced by oxidative damage in the cell (25Go). Although there are multiple and diverse adverse effects of mutant SOD1 aggregation, much less is known about the upstream events that promote preferential aggregation of a widely expressed mutant SOD1 protein in motor neurons in spinal cord and brain. Recent studies suggest that accumulations of mutant protein may result from diminished turnover in disease-afflicted tissues (26Go–28Go). Impairment of the ubiquitin–proteosome system by protein aggregation (29Go) may contribute but does not necessarily account for the preferential involvement of motor neurons in the disease process. Instead, the triggering events may be further upstream, such as the chaperone proteins and other components that interact with and determine the structure and fate of nascent mutant SOD1 proteins, as they emerge from polysomes in motor neurons.

The light neurofilament (NF-L) subunit is a highly expressed gene product of the neuronal cytoskeleton that may be instrumental in the formation of neurotoxic aggregates in motor neuron disease. NF inclusions in motor neurons have long been recognized as common pathological features of familial and sporadic ALS (30Go–33Go). In presymptomatic G86R mutant SOD1 transgenic mice, the formation of NF inclusions is preceded by aggregation and accumulation of NF-L, lacking NF-M or NF-H, in perikarya and neurites of motor neurons in the lumbar spinal cord (34Go). Genetic evidence of NF-L involvement in motor neuron disease is now provided by the discovery of missense mutations in NF-L, which give rise to an autosomal dominant phenotype in Charcot–Marie–Tooth (CMT) disease (35Go,36Go). Several disease-causing mutations disrupt NF-L assembly and lead to aggregation of NF-L in neuronal cells (37Go–39Go). Similar disruptions of assembly with aggregation of NF-L are reproduced by expression of mutant HSP27, a chaperone protein that causes an autosomal recessive form of distal motor neuropathy in CMT2F (40Go).

In previous studies, we have identified a major destabilizing element in the proximal 3'-UTR of the NF-L transcript (41Go,42Go). Mutation of this regulatory element alters the stability of the transcript and creates a mutant NF-L transgene with potent neuropathic effects on motor neurons of transgenic mice (43Go). Expression of NF-L RNA containing this regulatory element in the 3'-UTR of a green fluorescent protein (GFP) reporter gene gives rise to a motor neuron phenotype in transgenic mice (44Go) and has neuropathic effects on primary motor neurons in culture (45Go). The regulatory element in the 3'-UTR of the NF-L transcript also triggers aggregation of NF-L and mutant SOD1 proteins in Neuro 2a cells (46Go), raising the possibilities that neurotoxic protein aggregation is somehow linked to the properties of RNA in the destabilizing element or to cognate RNA-binding proteins.

In the present study, we examine the role of a cognate RNA-binding protein in RNA-triggered aggregation of NF-L protein. We herein report that p190RhoGEF, an RNA-binding protein that binds to the destabilizing element in NF-L mRNA (47Go), co-aggregates with unassembled NF-L protein and that co-aggregation of p190RhoGEF and NF-L protein is associated with loss of NF-L mRNA. We further show that similar aggregates of p190RhoGEF and NF-L and loss of NF-L expression occur in different transgenic models of motor neuron disease. The findings raise the possibility that aggregation of unassembled NF-L protein may be a triggering event promoting neurotoxic aggregation in motor neuron disease and, possibly, in other neurodegenerative conditions as well.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
p190RhoGEF co-localizes with RNA-triggered NF-L protein aggregates but not with NF-L subunits which are assembled in filamentous arrays
To investigate the role of cognate RNA-binding proteins in RNA-triggered NF-L protein aggregation, we used our established cellular model in Neuro 2a cells (46Go). Endogenous mouse NF-L and NF-M genes are methylated and not expressed in undifferentiated Neuro 2a cells (48Go). This enabled transfection of NF-L to provide a single source of NF-L mRNA and NF-L protein for comparing the disposition of NF-L protein and expression of parent NF-L mRNA without interference from endogenous NF-L subunits. In this model, Neuro 2a cells are transfected with a GFP-tagged NF-L transgene with (pGFP/NF+3'-UTR) or without (pGFP/NF–3'-UTR) NF-L 3'-UTR sequence (Fig. 1A). When expressed by a transgene lacking NF-L 3'-UTR sequence (pGFP/NF–3'-UTR), GFP-tagged NF-L protein remains diffusely distributed throughout the cytoplasm of transfected cells (Fig. 1B). When expressed by a transgene with NF-L 3'-UTR sequence (pGFP/NF+3'-UTR), the same GFP-tagged NF-L protein undergoes aggregation in the cytoplasm of transfected cells (Fig. 1E).



View larger version (70K):
[in this window]
[in a new window]
  		Figure 1. p190RhoGEF co-localizes with RNA-triggered NF-L protein aggregates but not with NF-L subunits which are assembled in filamentous arrays. (A) Schematic diagram of GFP-tagged NF-L transgenes with (pGFP/NF+3'-UTR) and without (pGFP/NF–3'-UTR) NF-L 3'-UTR (red). (B–D) Confocal microscopy of GFP fluorescence (B), anti-p190RhoGEF immunoreactivity (C) and the merged image with DAPI-stained nuclei (D) showing diffuse distribution of endogenous p190RhoGEF in cells expressing GFP-tagged NF-L protein from pGFP/NF–3'-UTR transgene and in an untransfected cell. (E–G) Confocal microscopy of GFP fluorescence (E), anti-p190RhoGEF immunoreactivity (F) and the merged image with DAPI-stained nuclei (G) showing co-localization of p190RhoGEF in large protein aggregates in cells transfected with pGFP/NF+3'-UTR. (H–J) Confocal microscopy of GFP fluorescence (H), anti-p190RhoGEF immunoreactivity (I) and the merged image with DAPI-stained nuclei (J) showing filamentous arrays of GFP-tagged NF-L subunit in cells expressing low levels of pGFP/NF+3'-UTR transgene. p190RhoGEF does not co-localize with filamentous arrays of GFP-tagged NF-L (I and J). Scale bars, 10 µm.

 
At low-level expression of pGFP/NF+3'-UTR, NF-L subunits do not aggregate but assemble in filamentous arrays (Fig. 1H) with low-levels of endogenous NF-H in Neuro 2a cells (46Go). Comparable levels of NF-L are present in lysates of cells transfected with pGFP/NF–3'-UTR or pGFP/NF+3'-UTR (46Go).

RNA-triggered aggregation of NF-L protein maps to the proximal 45 nt of NF-L 3'-UTR (46Go), the site of a destabilizing element and binding site of p190RhoGEF (47Go). To probe whether p190RhoGEF is involved in RNA-triggered NF-L protein aggregation, we examined the distribution of endogenous p190RhoGEF in transfected cells. Whereas p190RhoGEF is diffusely distributed in untransfected Neuro 2a cells and in cells transfected with pGFP/NF–3'-UTR (Fig. 1C and D), p190RhoGEF aggregates in cells with high-level expression of pGFP/NF+3'-UTR (Fig. 1F) and co-localizes with aggregates of GFP-tagged NF-L protein (Fig. 1E–G). When GFP-tagged NF-L is assembled with endogenous NF-H in filamentous arrays (Fig. 1H), p190RhoGEF remains diffusely distributed in the cytoplasm (Fig. 1I) and does not co-localize with NF-L in filamentous arrays (Fig. 1H–J). The findings indicate that p190RhoGEF associates with RNA-triggered aggregates of NF-L protein but not with NF-L subunits assembled in filamentous arrays.

Similar co-aggregation of NF-L and p190RhoGEF occurs when Neuro 2a cells are transfected with untagged NF-L transgene containing NF-L 3'-UTR (Supplementary Material, Fig.S1 A–C). High-level expression of GFP-tagged NF-L protein also undergoes co-aggregation with p190RhoGEF when transfected in cell lines expressing varying levels of endogenous NF subunits, such as motor neuron-like NSC-34 cells (Supplementary Material, Fig. S1D–F) and mouse embryonic carcinoma P19 cells (Supplementary Material, Fig. S1G–I). These findings indicate that RNA-triggered co-aggregation of p190RhoGEF with NF-L protein is not due to the GFP tag or restricted to a line of transfected neuronal cells.

RNA-triggered aggregation of NF-L protein is associated with loss of NF-L mRNA
Because p190RhoGEF binds to the destabilizing element in NF-L mRNA (47Go), we next examined the effects of co-aggregation of p190RhoGEF with NF-L protein on levels of parent NF-L mRNA. In situ hybridizations were conducted on cells transfected with pGFP/NF+3'-UTR using antisense (Fig. 2A–C) and sense (Fig. 2D–F) probes to NF-L mRNA. Sense probes are non-reactive (Fig. 2E), whereas antisense probes readily detect NF-L mRNA diffusely distributed in the cytoplasm of transfected Neuro 2a cells (Fig. 2B). When individual cells are analyzed, prominent NF-L mRNA expression is only observed in cells expressing very low-to-medium-levels of NF-L protein (Fig. 2B, arrowheads). High-level NF-L expression leads to large aggregates of NF-L protein (Fig. 2A, arrows). These cells contain very low levels of NF-L mRNA (Fig. 2B and C, arrows). Extensive loss of NF-L mRNA was observed in all cells containing large aggregates of NF-L protein. A transitional phase with co-localization of NF-L mRNA in NF-L protein aggregates was never observed.



View larger version (65K):
[in this window]
[in a new window]
  		Figure 2. RNA-triggered aggregation of NF-L protein is associated with loss of NF-L mRNA. (A–F) In situ hybridization analysis of NF-L mRNA expression in Neuro 2a cells transfected with pGFP/NF+3'-UTR showing hybridization signals using antisense (B) and sense (E) probes and the merged images with DAPI-stained nuclei (C and F). Transfected cells with large aggregates of GFP-tagged NF-L (A, arrows) show reduced levels of NF-L mRNA (B and C, arrows), whereas transfected cells with very low-to-medium levels of GFP-tagged NF-L (A, arrowheads) have higher levels of NF-L mRNA (B and C, arrowheads). Hybridization with sense probes is negative (E). (G–I) Populations of Neuro 2a cells transfected with pGFP/NF+3'-UTR, separated by FACS on the basis of GFP fluorescence into cells expressing low, medium and high levels of GFP-tagged NF-L and analyzed by western blot for GFP-tagged NF-L protein (G) and by RT–PCR for NF-L mRNA (H). Relative levels of NF-L mRNA and NF-L protein in cells expressing low, medium and high levels of GFP-tagged NF-L (I) show that cells expressing high-level GFP-tagged protein have markedly reduced levels of NF-L mRNA. Western blots and RT–PCR are normalized to levels of anti-ß-actin and GAPDH mRNA, respectively. Mock-transfected cells lack NF-L expression. Scale bars, 10 µm.

 
To further assess the level of NF-L mRNA in relation to the level of NF-L protein expression and extent of NF-L protein aggregation, Neuro 2a cells were transfected with pGFP/NF+3'-UTR and separated by FACS into low-, medium- and high-level expression on the basis of GFP fluorescence. Lysates of the respective populations of cells were assessed by western blots for expression of NF-L protein (Fig. 2G), by RT–PCR for expression of NF-L mRNA (Fig. 2H) and aliquots were examined for GFP fluorescence in cytospin preparations. The presence of low-, medium- and high-level expressions of GFP-tagged NF-L protein in the respective populations of transfected cells was confirmed by western blot (Fig. 2G). Examination of cytospin preparations showed that >95% of high-expressing cells contain large aggregates of NF-L protein, whereas most medium-expressing cells contain diffusely distributed or multiple small punctate forms of GFP-tagged NF-L protein (data not shown). Cells expressing low and medium levels of GFP-tagged NF-L protein show corresponding levels of NF-L mRNA (Fig. 2H), whereas cells expressing high-level GFP-tagged NF-L protein contain low levels of NF-L mRNA (Fig. 2H). Comparative analyses of NF-L mRNA and protein levels in the different populations showed that high-level NF-L protein expression is associated with a markedly reduced level of NF-L mRNA (Fig. 2I). The findings confirm our in situ observations that high-level expression with aggregation of NF-L and co-aggregation of p190RhoGEF is associated with loss of parent NF-L mRNA.

Co-expression of NF-M increases NF assembly and reduces RNA-triggered aggregation and loss of solubility of NF-L protein
To explore the role of p190RhoGEF in aggregation of NF-L protein, we first set out to modify our cellular model of RNA-triggered NF-L protein aggregation by co-transfecting NF-L and NF-M transgenes. Co-expression of NF-M results in co-localization of NF-L and NF-M in a spectrum of filamentous and aggregative forms, including fine filamentous arrays (Fig. 3A and B), thin and thick filamentous bundles (Fig. 3C and D) and cytoplasmic aggregates (Fig. 3E–H). Over time and with increasing levels of expression, there is a progression from finer to courser arrays and from filamentous to aggregative forms. However, co-expression of NF-M leads to an overall increase of cells with filamentous forms and decrease of cells with aggregative forms when compared with cells transfected with NF-L alone (Fig. 3K).



View larger version (46K):
[in this window]
[in a new window]
  		Figure 3. Co-expression of NF-M increases NF-L assembly and reduces RNA-triggered aggregation and loss of solubility of GFP-tagged NF-L protein. (A–H) Confocal microscopy of GFP fluorescence (A, C, E and G) and anti-NF-M immunofluorescence (B, D, F and H) in Neuro 2a cells co-transfected with GFP-tagged NF-L and NF-M transgenes showing co-localization of GFP-tagged NF-L and NF-M in fine filamentous array (A and B), thin-to-thick filamentous bundles (C and D) and irregular aggregates (E–H). (I) Western blots of NF-L and NF-M in cells co-transfected with NF-L (pGFP/NF-L+3'-UTR) and NF-M (pHM6/NF-M) or empty expression vector (pHM6) following fractionation of cell lysates in buffers of increasing solvent strengths (left-hand boxes) or in total Triton-insoluble fraction (right-hand boxes). Lanes show equivalent amounts of respective fractions. (J) Quantitation of GFP-tagged NF-L protein solubilized in 2 M urea in lysates of cells co-transfected with pGFP/NF+3'-UTR and pHM6/NF-M or empty pHM6 expression vector. (K) Quantitation of transfected cells with aggregates of GFP-tagged NF-L subunits when pGFP/NF+3'-UTR is co-transfected with pHM6/NF-M or empty pHM6 expression vector. Scale bars, 10 µm.

 
Because RNA-triggered aggregation of NF-L protein is associated with loss of protein solubility (46Go), we also tested the effects of co-expressing NF-M on the solubility of NF-L protein by examining lysates of cells co-transfected with pGFP/NF+3'-UTR and either a pHM6/NF-M or an empty pHM6 expression vector. Lysates of co-transfected cells were fractionated in buffers of increasing solvent strength to assess the relative solubilities of NF-L protein. Immunoblots of NF-L recovered in the separate fractions show that co-transfection with NF-M increases the percentage of NF-L proteins in more soluble fractions (Fig. 3I). For example, co-expression of NF-M results in a 2–3-fold increase in solubility of NF-L protein in 2 M urea (Fig. 3J). Co-expression of NF-M also increases the total amount of NF-L protein (Fig. 3I, Triton-insoluble lanes), suggesting that co-localization with NF-M may also stabilize NF-L protein. Together, the findings indicate that co-transfection of NF-M not only increases filamentous forms and decreases aggregative forms of NF-L, but also decreases the insolubility of NF-L protein that occurs when NF-L transgene is transfected alone.

We then examined the disposition of p190RhoGEF in the modified model of NF-L protein aggregation. In co-transfected cells, p190RhoGEF does not co-localize with filamentous forms of GFP-tagged NF-L protein including fine filamentous arrays (Fig. 4A–C) or thin-to-thick filamentous bundles (Fig. 4D–F), but still co-localizes with aggregative forms of GFP-tagged NF-L protein (Fig. 4G–L). In many cells where there is non-uniform density of protein aggregates (Fig. 4G), p190RhoGEF co-localizes with the denser regions of NF-L protein aggregates, usually in the interior of the aggregates (Fig. 4G–I, arrowheads). The findings indicate that co-expression of NF-M alters the distribution but does not prevent co-localization of p190RhoGEF in protein aggregates. However, the findings confirm our previous results that p190RhoGEF associates with aggregative forms but not with filamentous forms of NF-L.



View larger version (87K):
[in this window]
[in a new window]
  		Figure 4. p190RhoGEF associates with aggregative forms but not with filamentous forms of NF-L in co-transfected cells. (A–L) Confocal microscopy of GFP fluorescence (A, D, G and J), anti-p190RhoGEF immunofluorescence (B, E, H and K) and merged images with DAPI-stained nuclei (C, F, I and L) in cells co-transfected with GFP-tagged NF-L and NF-M. GFP-tagged NF-L in fine (A) or thin-to-thick bundled (D) filamentous forms does not co-localize with anti-p190RhoGEF (B, C, E and F). Aggregates of GFP-tagged NF-L (G and J) in co-transfected cells are associated with focal aggregation of p190RhoGEF (H, I, K and L). p190RhoGEF tends to co-aggregate within selected regions of NF-L protein aggregation showing strong and dense anti-NF-L immunoreactivity, often in central regions of NF-L protein aggregates (H, I, K and L, arrowheads). Scale bars, 10 µm.

 
Small interference RNA-induced silencing of p190RhoGEF expression increases NF assembly and reduces RNA-triggered aggregation of NF-L protein
The role of p190RhoGEF in RNA-triggered NF-L protein aggregation was further studied by silencing endogenous p190RhoGEF using small interference RNA (siRNA). Western blot shows that a 48 h transfection of Neuro 2a cells with siRNA D-01, D-02 or D-03 reduces p190RhoGEF by >80%, whereas siRNA D-04 reduces p190RhoGEF by ~50%, compared with untreated and mock-transfected cells (Fig. 5A). We then examined the effects of siRNA-induced down-regulation of p190RhoGEF on the disposition of NF-L when co-transfected with NF-M. We first examined co-transfections of NF-L and NF-M in Neuro 2a cells following siRNA-induced down-regulation of p190RhoGEF. At 24 h, there is a marked increase of GFP-tagged NF-L in filamentous forms (Fig. 5C) when compared with co-transfections in mock-treated cells (Fig. 5B). Quantitation of these effects shows that down-regulation of p190RhoGEF significantly reduces the percentage of cells bearing aggregative form and increases the percentage of cells containing filamentous forms of NF-L (Fig. 5E). Similar levels of NF-L protein are observed in co-transfected cells with and without down-regulation of p190RhoGEF (Fig. 5D). We further examined whether siRNA-induced down-regulation of p190RhoGEF could recover the disposition of NF-L subunits from aggregative to filamentous forms. Neuro 2a cells were first co-transfected with NF-L and NF-M for 24 h and then subjected to siRNA-induced down-regulation of p190RhoGEF. Interestingly, down-regulation of p190RhoGEF reduces the percentage of cells bearing aggregative form and increases the percentage of cells containing filamentous forms of NF-L (Fig. 5F), very similar to the effects observed when cells are co-transfected following siRNA-induced down-regulation of p190RhoGEF (Fig. 5E). The findings indicate that silencing of p190RhoGEF expression reduces aggregation and promotes assembly of NF-L subunits and suggest that p190RhoGEF may associate with NF-L in a manner that promotes aggregation and reduces assembly of NF-L subunits.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 5. siRNA-induced silencing of p190RhoGEF expression reduces aggregation and increases assembly of NF-L subunits. (A) Western blot of endogenous p190RhoGEF in Neuro 2a cells transfected with siRNA (D-01, D-02, D-03 and D-04) to p190RhoGEF compared with untreated or mock-transfected cells. (B and C) GFP fluorescence of GFP-tagged NF-L in cells co-transfected with pGFP/NF+3'-UTR and pHM6/NF-M without (mock) or with (siRNA-p190RhoGEF) siRNA-induced down-regulation of p190RhoGEF. (D) Western blot of NF-L in total lysates of Neuro 2a cells co-transfected with pGFP/NF-L+3'-UTR and pHM6/NF-M in untreated cells, in mock-treated cells and in cells treated with siRNA to p190RhoGEF. (E and F) Quantitation of co-transfected cells with NF-L and NF-M in filamentous and aggregative forms that were untreated, mock-transfected or co-transfected with individual siRNAs (D-01 or D-02) or pooled siRNAs (D-01, D-02, D-03 and D-04). Transfection of NF-L and NF-M transgenes for a 24 h period after down-regulation of endogenous p190RhoGEF (E) or prior to down-regulation of endogenous p190RhoGEF (F). *P<0.05 compared with untreated or mock-treated and **P<0.01 compared with untreated or mock-treated. Scale bars, 10 µm.

 
The co-localization of p190RhoGEF and NF-L in protein aggregates and the ability of siRNA-induced down-regulation of p190RhoGEF to reduce aggregation of NF-L suggest that p190RhoGEF may associate with unassembled NF-L protein. To explore this possibility, we prepared unassembled NF-L protein from Triton X-100 soluble supernatants of motor neuron-like NSC-34 cells and from mouse spinal cord homogenates, as described (49Go), and conducted co-precipitation assays on these fractions. Following incubation of supernatants with anti-p190RhoGEF, immunoprecipitates were immunoblotted with anti-NF-L. NF-L is present in immunocomplexes precipitated by agarose beads conjugated with anti-p190RhoGEF but not by the agarose beads alone (Supplementary Material, Fig. S2A, upper panel). Re-probing shows that p190RhoGEF is present in the anti-p190RhoGEF precipitates but not in the agarose bead controls (Supplementary Material, Fig. S2A, lower panel). Similar studies show that both NF-L and p190RhoGEF are present in the supernatants fractions of mouse spinal cord homogenates (Supplementary Material, Fig. S2B, lane 1) and that NF-L is co-precipitated with p190RhoGEF (Supplementary Material, Fig. S2B, upper panel) by anti-p190RhoGEF agarose beads. The findings indicate that p190RhoGEF associates with unassembled NF-L protein in neuronal cells and in mouse spinal cord. The interactions of p190RhoGEF and NF-L may be indirect, as we have been unable to demonstrate direct binding by overlay assay (data not shown).

p190RhoGEF and NF-L are altered in transgenic mouse models of motor neuron disease
Because overexpression of untranslated NF-L RNA triggers motor neuron degeneration in transgenic mice (44Go) and in cultured motor neurons (45Go) and expression of NF-L 3'-UTR causes aggregation of NF-L and mutant SOD1 proteins in neuronal cells (46Go), interactions of p190RhoGEF with NF-L mRNA and NF-L protein could be instrumental in leading to the pathological changes in motor neurons of mice expressing untranslated NF-L sequence in the 3'-UTR of a GFP reporter transgene (44Go) or a human G93A mutant SOD1 transgene. To explore these possibilities, we re-examined our transgenic model of NF-L RNA-triggered motor neuron degeneration (44Go). We detect expression of the GFP reporter transgene in a population of motor neurons of lumbar spinal cord with condensed nuclei and cytoplasm (Fig. 6A, dark arrows), but not in non-condensed motor neurons (Fig. 6A, white arrows). Similar groups of condensed motor neurons show enhanced anti-ubiquitin (Fig. 6B, dark arrows), increased anti-p190RhoGEF (Fig. 6C, dark arrows) and loss of anti-NF-L (Fig. 6D, dark arrows) immunoreactivities when compared with non-condensed motor neurons (Figs. 6B–D, white arrows). In addition, strong anti-p190RhoGEF and anti-NF-L immunoreactivities occur in discrete punctate inclusions within the neuropil in the vicinity of motor neurons (Fig 6C and D, arrowheads). These inclusions are suggestive of dystrophic axons or dendrites in RNA-triggered motor neuron degeneration in mice and may occur in the neurites of degenerating motor neurons. Accumulations of ubiquitinated aggregates in neurites appear as early pathological changes in RNA-triggered degeneration of cultured motor neurons (45Go).



View larger version (152K):
[in this window]
[in a new window]
  		Figure 6. p190RhoGEF and NF-L are altered in RNA-triggered transgenic model of motor neuron disease. (A–D) Light microscopic examination of anti-GFP (A), anti-ubiquitin (B), anti-p190RhoGEF (C) and anti-NF-L (D) immunoreactivities in motor neurons in the lumbar spinal cord of transgenic mouse expressing a GFP reporter transgene with NF-L sequence in the 3'-UTR (pGFP/NF-L RNA) and exhibiting motor neuron phenotype (44Go). GFP-expressing motor neurons show condensed perikarya and nuclei (A, dark arrows) compared with non-expressing motor neurons (A, white arrows). Similar populations of condensed motor neurons show enhanced anti-ubiquitin (B, dark arrows) and p190RhoGEF (C, dark arrows) and reduced NF-L (D, dark arrows) compared with non-condensed motor neurons in the same sections (B–D, white arrows). Punctate inclusions containing p190RhoGEF (C, arrowheads) and NF-L (D, arrowheads) are present in the neuropil in the vicinity of motor neurons. Scale bars, 25 µm.

 
We then examined p190RhoGEF immunoreactivity in the lumbar spinal cord of symptomatic (120 days) mice bearing a human G93A mutant SOD1 transgene. These sections show extensive vacuolization of neuropil surrounding motor neurons, as described (50Go). Strong anti-p190RhoGEF immunoreactivity is present in numerous small and large punctate inclusions (Fig. 7A, arrowheads) and linear profiles (Fig. 7A, arrows) within the vacuolated neuropil. Punctate inclusions (arrowheads) and linear profiles (arrows) in vacuolated neuropil also contain strong anti-NF-L (Fig. 7B) as well as anti-SOD1 immunoreactivities (Fig. 7C). Focal aggregates of anti-ubiquitin immunoreactivity are also present in dystrophic neuritic processes (Fig. 7D, arrows) and in punctate inclusions (Fig. 7D, arrowheads) in symptomatic mice. Interestingly, we also observed loss of anti-NF-L immunoreactivities in perikarya of degenerating motor neurons (Fig. 7B, arrows with open arrowheads).



View larger version (180K):
[in this window]
[in a new window]
  		Figure 7. p190RhoGEF and NF-L are altered in G93A SOD1 transgenic model of motor neuron disease. (A–D) Immunohistochemical examination of p190RhoGEF (A), NF-L (B), human SOD1 (C) and ubiquitin (D) in ventral horn of lumbar spinal cord of symptomatic (120 days) G93A mutant SOD1 transgenic mice showing punctate inclusions (arrowheads) and linear profiles (dark arrows) in vacuolated neuropil containing anti-p190RhoGEF (A), anti-NF-L (B), anti-SOD1 (C) and anti-ubiquitin (D) immunoreactivities. Some degenerating motor neurons also reveal loss of perikaryal NF-L immunoreactivity (B, white arrows). (E and F) Immunohistochemical examination of p190RhoGEF (E) and NF-L (F) in ventral horn of lumbar spinal cord of presymptomatic (80 days) G93A mutant transgenic mice showing punctate inclusions (arrowheads) containing p190RhoGEF (E) and NF-L (F) in the neuropil surrounding motor neurons. Scale bar, 25 µm.

 
To assess the role of punctate inclusions containing p190RhoGEF and NF-L in mutant SOD1 toxicity, we examined the lumbar spinal cord of presymptomatic (80 days) mice prior to appearance of a vacuolated neuropil. Multiple punctate inclusions containing p190RhoGEF are present in the neuropil surrounding motor neurons in the ventral horn of lumbar spinal cord from presymptomatic transgenic mice (Fig. 7E, arrowheads). Similar punctate inclusions containing NF-L are very prominent in the neuropil in serial sections of lumbar spinal cord from the presymptomatic transgenic mice (Fig. 7F, arrowheads). The findings suggest that punctate accumulations of p190RhoGEF and NF-L in neurites (axons or dendrites) of degenerating motor neurons may be an early pathological change in transgenic models of motor neuron disease due to overexpression of untranslated NF-L RNA (44Go) or a G93A mutant SOD1 transgene.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Our previous studies have shown that overexpression of untranslated NF-L RNA causes motor neuron degeneration in transgenic mice (44Go), leads to accumulation of ubiquitinated aggregates in degenerating cultured motor neurons (45Go) and triggers aggregation of NF-L protein and co-aggregation of mutant SOD1 protein in neuronal cells (46Go). We now show that RNA-triggered aggregation of NF-L protein is associated with co-aggregation of p190RhoGEF and that co-aggregation of p190RhoGEF and NF-L protein can lead to down-regulation of NF-L mRNA in neuronal cells. We also show that aggregates of p190RhoGEF and NF-L protein and loss of NF-L expression occur in degenerating motor neurons of different transgenic models of motor neuron disease expressing untranslated NF-L RNA or a G93A mutant SOD1 transgene. Loss of NF-L mRNA occurs in degenerating motor neurons of sporadic and familial ALS (51Go–53Go) and is even more severe in neurons containing NF inclusions (53Go). Loss of NF-L expression has also been reported in neurons-at-risk in other neurodegenerative diseases (54Go–58Go).

The salient feature of our study is the involvement of p190RhoGEF in a pathway linking loss of NF-L mRNA and aggregation of NF-L protein. However, neither the linkage nor the interactions of p190RhoGEF with NF-L RNA and with NF-L protein are straightforward. For example, cross-linking of p190RhoGEF to the destabilizing element in NF-L mRNA is altered by admixing p190RhoGEF with brain extracts (47Go), suggesting that additional factors modify the RNA–protein interaction. Likewise, our ability to demonstrate interactions of p190RhoGEF and NF-L protein by co-precipitation but not by gel overlay assay suggests that the interactions of p190RhoGEF and NF-L protein may be indirect and dependent on additional factors. We therefore favor the view that p190RhoGEF interacts with NF-L mRNA in an RNA–protein complex and with unassembled NF-L protein in a multi-protein complex. Aggregation of NF-L protein and destabilization of NF-L mRNA result from altered interactions of p190RhoGEF in the respective complexes. A cartoon of our working hypothesis is shown in Figure 8.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 8. A model of p190RhoGEF involvement in linking aggregation of NF-L protein with loss of NF-L mRNA in motor neuron degeneration. p190RhoGEF is a component of an RNA–protein complex that regulates the stability of the NF-L mRNA. p190RhoGEF is also a component of a multi-protein complex that interacts with unassembled NF-L protein. In the presence of NF-M or NF-H subunits, unassembled NF-L subunits dissociate from the multi-protein complex and undergo assembly with NF-M or NF-H. However, alterations of p190RhoGEF interactions in the RNA–protein complex can affect the interactions of p190RhoGEF in the multi-protein complex and trigger co-aggregation of p190RhoGEF and NF-L protein. Likewise, altered interactions of p190RhoGEF in the multi-protein complex can affect the interactions of p190RboGEF in the RNA–protein complex and link aggregation of NF-L protein with loss of NF-L mRNA. In this model, overexpression of NF-L RNA or NF-L protein can affect the interactions of p190RhoGEF in the RNA–protein complex and in the multi-protein complex and thereby lead to aggregation of NF-L and p190RhoGEF and loss of NF-L expression in degenerating motor neurons of motor neuron disease.

 
The model is consistent with observations that aggregation of NF-L protein and loss of NF-L mRNA are dependent on the presence and interactions of p190RhoGEF in an RNA–protein complex on NF-L mRNA and in a multi-protein complex on NF-L protein. For example, aggregation of NF-L protein in neuronal cells is dependent on the presence of NF-L 3'-UTR in the transgene (46Go), thereby enabling a complex containing p190RhoGEF to form on the destabilizing element in the transcript. Conversely, loss of NF-L mRNA is dependent on co-aggregation of NF-L protein and p190RhoGEF protein which could reflect the formation of a complex containing p190RhoGEF on unassembled NF-L. Hence, loss of NF-L mRNA occurs following co-aggregation of p190RhoGEF and NF-L but not after siRNA-induced down-regulation of p190RhoGEF. In addition, loss of NF-L mRNA does not occur when NF-L co-aggregates with NF-M. In the latter instance, co-localization of NF-M with NF-L in protein aggregates may alter the interactions of p190RhoGEF in the multi-protein complex and the ability of the multi-protein complex to trigger down-regulation of NF-L mRNA.

Our working model also provides novel perspectives as to the nature of motor neuron degeneration resulting from overexpression of untranslated NF-L RNA in mice (44Go) and in cultured motor neurons (45Go). Excess of untranslated NF-L RNA could alter the interactions of p190RhoGEF in the RNA–protein complex and thereby modify the interactions of the p190RhoGEF in the multi-protein complex on unassembled NF-L protein. The latter modifications could lead to aggregation of p190RhoGEF and NF-L in degenerating motor neurons of mice. The presence of NF-L protein aggregates in neurites and the loss of NF-L protein in neuronal perikarya suggest that loss of NF-L expression occurs subsequent to aggregation of NF-L protein. Moreover, the prominence of NF-L protein as aggregates in degenerating motor neurons would not be expected to result from a loss of endogenous NF-L expression, but could be instrumental in triggering down-regulation of NF-L, as observed in transfected cells (46Go). It is also possible that the up-regulation of p190RhoGEF in degenerating motor neurons is a reactive change due to formation of complexes containing p190RhoGEF on NF-L protein or on NF-L mRNA.

The presence of similar aggregates of NF-L and p190RhoGEF in neurites of motor neurons in presymptomatic mice expressing a G93A mutant SOD1 transgene suggests that these accumulations may also be instrumental in the aggregation of mutant SOD1 protein. Proximal neurites of motor neurons are sites where aggregates of G93A mutant SOD1 protein are reported to arise (50Go,59Go), raising the possibility that aggregation of mutant SOD1 protein may be promoted by aggregates of NF-L protein, as demonstrated in transfected neuronal cells (46Go). It is also possible that aggregates of NF-L and mutant SOD1 protein have mutually synergistic effects (60Go) and that aggregates of mutant SOD1 may also enhance the aggregation of NF-L protein and p190RhoGEF and loss of NF-L expression. Hence, p190RhoGEF may be involved in the pathogenesis as well as loss of NF-L mRNA in mutant SOD1-induced motor neuron degeneration. Moreover, a lack of synergistic effects of NF-L and mutant SOD1 protein aggregates may help explain the ameliorating effects of deleting NF-L on the toxicity of a mutant SOD1 transgene (61Go).

The prospective role of NFs in the pathogenesis of motor neuron disease derives from alterations of NF expression in mice that reproduce the pathology and phenotype of human disease. Interestingly, the disease state is not reproduced by deletion of one or more NF genes (62Go), but rather by overexpressing an NF transgene (63Go–65Go) that alters NF subunit stoichiometry and leads to accumulation of NFs in motor neurons. Accumulations of NFs, however, are not necessarily neurotoxic because overexpression of a mutant NF-H with a lacZ replacement of C-terminal sequence leads to massive accumulations of NFs without the phenotype of disease (66Go) and without altering the toxicity of mutant SOD1 (67Go). Instead, the neurotoxicity of NF transgenes appears to be associated with alterations of NF subunit stoichiometry that might alter NF-L assembly. Expression of a mutant NF-L reported to disrupt NF assembly causes severe motor neuron degeneration in transgenic mice (68Go). Correction of subunit stoichiometry rescues motor impairment caused by overexpression of an NF-H transgene (69Go).

Further evidence that alterations in NF-L assembly cause motor neuron disease is provided by the assembly-disrupting mutations in NF-L that give rise to a distal motor and sensory axonal neuropathy in CMT2E (37Go–39Go). Although the pathological aggregates of NF-L are associated with defective axonal transport (38Go,70Go), it is unclear whether altered transport is directly related to aggregation of NF-L subunit. Defects in axonal transport are common phenomena in neurodegenerative diseases due to neurotoxic protein aggregates (71Go) and may account for the preferential susceptibility of distal axons in motor neuron disease (72Go,73Go). Recent genetic screens of ALS patients have identified risk factor mutations in peripherin that disrupt NF-L assembly in transfected SW13Vim- cells (74Go,75Go) and may disrupt NF-L assembly in motor neuron disease. Genetic screens have also identifed mutations in the KSP repeat phosphorylation domain of NF-H as risk factors in sporadic ALS (76Go–78Go), although it is unclear whether the mutations affect NF-L assembly.

Our studies indicate that the putative complexes containing p190RhoGEF not only link NF-L protein aggregation with loss of NF-L expression but also regulate NF-L assembly. siRNA-induced silencing of p190RhoGEF reduces aggregation and increases assembly of NF-L and NF-M. It also promotes their re-assembly when siRNAs are added after cells have been co-transfected with NF-L and NF-M. Because the complex containing p190RhoGEF has the unique property of interacting with unassembled, but not with assembled NF-L, we propose that the complex acts as an interactive component that is only dissociated by assembly of NF-L with heterologous subunits. By preventing NF-L self-assembly, the p190RhoGEF-containing complex may help assure a heterologous composition of assembled NFs in vivo.

Interactions of the p190RhoGEF-containing complex with NF-L subunits promote aggregation of unassembled subunit and permit assembly with NF-M or NF-H. This is reminiscent of the obligate heteropolymer behavior of NF-L subunits when transfected in SW13Vim- cells (79Go,80Go). However, the obligate heteropolymer requirement of NF-L assembly in vivo contrasts with the ready self-assembly property of NF-L in vitro (81Go,82Go) and in the yeast two-hybrid system (83Go). This raises the question as to the factors or conditions that restrict self-assembly in vivo. The p190RhoGEF-containing complex may be a factor restricting self-assembly of NF-L in vivo.

Because large neurons express an abundance of all three NF subunits, the complex containing p190RhoGEF would not interfere in assembly of NF-L with NF-M and NF-H, but could serve as a gatekeeper of NF-L assembly, preventing self-assembly and providing a feedback mechanism to down-regulate NF-L mRNA in response to an excess or persistence of unassembled NF-L subunits in the cell. The pathway could also serve to regulate levels of NF-L mRNA expression in accordance with levels of NF-L assembly in the cell. Feedback regulation of NF-L expression is necessary for maintaining a steady-state level of NF metabolism and, in turn, for preserving the size and shape of the axonal cytoskeleton of large NF-rich neurons. The ability to co-precipitate low levels of p190RhoGEF and NF-L from detergent-soluble supernatants is consistent with the existence of a complex containing p190RhoGEF and NF-L protein in normal adult mouse spinal cord.

The possibility that complexes containing p190RhoGEF operate in feedback pathways linking NF-L expression and NF assembly provides additional perspectives as to the nature of pathological aggregates of NF-L and p190RhoGEF in motor neuron disease. First, it suggests that the multi-component complexes containing p190RhoGEF that interact with NF-L mRNA and NF-L protein are important for maintaining neuronal homeostasis and that alterations of these complexes can give rise to a pathological state. Hence, accumulations of NF-L and p190RhoGEF aggregates in degenerating motor neurons result from an alteration in a pathway intended to maintain neuronal homeostasis. Moreover, the upstream triggering events could arise from alteration of individual components within each complex or from their altered interactions in the complexes or with components in other pathways in the cell. Finally, the interactions of multi-component complexes could be influenced by specific neuronal microenvironments, thereby contributing to the vulnerabilities of different subsets of large NF-rich neurons-at-risk in neurodegenerative diseases.

It is noteworthy that p190RhoGEF is a highly interactive protein (84Go–88Go) with potential activities in multiple neuronal pathways. The involvement of p190RhoGEF in aggregation of NF-L in motor neuron degeneration may be of particular interest in view of the causative role of alsin (89Go,90Go), especially its RhoGEF component (91Go), and of senataxin (92Go), another RNA-binding protein, in familial ALS. Finally, stabilization of NF-L mRNA may also be a pivotal upstream regulatory event that is not only linked to the disposition of NF-L protein but also regulated by glycolytic enzymes and is thereby associated with the high-energy requirements of large NF-rich neurons (93Go).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Expression vectors
Full-length NF-L coding region with and without 3'-UTR were amplified by PCR from a mouse brain cDNA library (Stratagene, La Jolla, CA, USA) and fused in-frame to the C-terminus of EGFP coding region in pEGFP/C2 expression vector (BD Biosciences Clontech, Palo Alto, CA, USA) to generate pGFP/NF+3'-UTR and pGFP/NF–3'-UTR transgenes (46Go), as shown schematically in Figure 1A. NF-M was amplified by PCR from a mouse spinal cord cDNA library (Stratagene) and cloned into pHM6 (Roche Applied Sciences, Indianapolis, IN, USA). GFP-tagged NF-L and hemagglutinin (HA)-tagged NF-M proteins were expressed as 97 and 160 kDa proteins in transfected cells (Figs 2G and 3I). The integrity of all DNA constructs was verified by DNA sequencing.

Neuronal cultures and transfection
Mouse Neuro 2a neuroblastoma and motor neuron-like NSC-34 cells (provided by Dr Neil Cashman, University of Toronto, Canada) were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA)with 10% fetal calf serum. Cells were transfected with FuGENE6 according to manufacturer's instructions (Roche Applied Sciences). Co-transfections were performed with 1:1 ratio of each expression vector unless otherwise specified. Cells were collected for western blots at 24 or 48 h following transfection.

Immunocytochemistry and confocal microscopy
Cells were fixed in 4% paraformaldehyde for 1 h at 4°C or in acetone/methanol for 3–5 min at room temperature and permeabilized in 0.5% Triton X-100. Fixed cells were incubated overnight at 4°C with the following primary antibodies: goat polyclonal anti-NF-L (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat polyclonal anti-NF-M (1:100 dilution, Santa Cruz Biotechnology) or rabbit polyclonal anti-p190RhoGEF antibody (1:100 dilution) (85Go). Secondary antibody incubations were conducted with Alexa Fluor® 594 chicken anti-rabbit IgG or Alexa Fluor 594 donkey anti-goat IgG (Molecular Probes, Eugenes, OR, USA), and cover slips were mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). GFP fluorescence, anti-NF-L, anti-NF-M and anti-p190RhoGEF immunofluorescence were visualized in DAPI-stained cells by multi-track sequential scanning using a Zeiss LSM510 META laser scanning confocal microscope.

In situ hybridization analysis
Neuro 2a cells were washed and fixed with freshly made 4% paraformaldehyde in DEPC-treated phosphate-buffered saline (PBS) for 1 h at 4°C, then washed once with 50% formamide and 5xSSC, incubated in hybridization buffer (50% formamide, 5xSSC, 1 mg/ml yeast tRNA, 200 mg/ml heparin, 1xDenhardt's solution, 0.1% Tween-20, 0.1% Triton X-100 and 5 mM EDTA, pH 8.0) for 1 h at room temperature and then hybridized with 50 ng/ml sense or antisense NF-L oligonuclotide probe labeled by tetramethylrhodamine-5-dUTP using DIG oligonucleotide tailing kit 2nd generation (Roche Applied Sciences) in hybridization buffer at 37°C overnight. Neuro 2a cells were then washed with 0.2xSSC and mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories). GFP fluorescence and red fluorescence for mRNA signals were visualized in DAPI-stained cells by multi-track sequential scanning using a Zeiss LSM510 META laser scanning confocal microscope.

Western blot analyses of protein solubility
Neuro 2a cells were lysed on ice in lysis buffer containing 10 mM Tris–HCl, pH 7.0, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1xproteinase inhibitor cocktail (Sigma, St Louis, MO, USA) and 1% Triton X-100. Total lysates were fractionated by centrifugation at 13 500g to obtain detergent-soluble supernatantsand detergent-insoluble pellets. To further assess protein solubilities, detergent-insoluble pellets were washed with lysis buffer and solubilized by vortexing and sonification in Tris buffer (10 mM Tris–HCl, pH 7.5, 125 mM NaCl, 10 mM EDTA) containing 2 M urea and centrifuged at 13 500g to obtain supernatant fractions of proteins solubilized in 2 M urea. Pellets were washed and solubilized in Tris buffers containing 4 and 8 M urea, as described earlier, to obtain supernatant fractions solubilized in 4 and 8 M urea, respectively. Pellets were then washed with 8 M urea buffer and solubilized by sonification and boiling in SDS–PAGE sample buffer to obtain fractions of proteins solubilized in 1% SDS. Aliquots of proteins in supernatant fractions were resolved on denaturing 4–12% NuPAGE gel (Invitrogen) and transferred to Immobilon-P (Millipore, Billerica, MA, USA). Amounts of protein in each fraction were assessed by densitometric scanning and normalized to the respective dilution factors. Membranes were blocked overnight at 4°C in TBST–milk (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20, 5% non-fat milk powder) and incubated sequentially in primary andsecondary antibodies (anti-rabbit or anti-goat IgG conjugated with HRP) for 1 h at room temperature. Labeled proteins were detected using western blot chemiluminescence reagent (Roche Applied Sciences).

siRNA to p190RhoGEF
Four siRNA sequences to p190RhoGEF, D-01, D-02, D-03 and D-04, were designed and synthesized by Dharmacon (Lafayette, CO, USA). For siRNA transfection, Neuro 2a cells were plated into 12-well plates at 1x105 cells/ml in antibiotic-free medium and incubated at 37°C with 5% CO2 overnight and then transfected with siRNAs using Liopectamine 2000 (Invitrogen) for 48 h according to manufacturer's instructions. To assess the effects of siRNA on endogenous p190RhoGEF expression, Neuro 2a cells were lysed on ice in lysis buffer containing 10 mM Tris–HCl, pH 7.0, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM PMSF, mammalian protease inhibitor cocktail (Sigma) and 1% Triton X-100. Total lysates were centrifuged at 13 500g and supernatants were analyzed by western blot. To assess the effects of siRNA-induced silencing of p190RhoGEF on aggregation and assembly of NF-L subunits, Neuro 2a cells were co-transfected with GFP-tagged NF-L and HA-tagged NF-M transgenes using Lipofectamine 2000 (Invitrogen) at 48 h after transfections with p190RhoGEF siRNAs. At 24 h following co-transfection, Neuro 2a cells were fixed and mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories). The number of co-transfected cells with filamentous arrays and with aggregates was counted under the fluorescence microscopy and the percentages were calculated. Three independent observations were conducted on each preparation.

Light microscopic and immunohistochemistry
Transgenic mice bearing a human G93A mutant SOD1 were purchased from Jackson's Laboratory (Bar Harbor, MI, USA). Transgenic mice were anesthetized with CO2 and cardiac perfused with 10 ml of PBS, followed by 20 ml of PBS containing 4% paraformaldehyde. Brains and spinal cords were excised and immersed overnight in 4% paraformaldehyde, washed in PBS, dehydrated and embedded in paraffin. Spinal cords were cut and positioned inparaffin blocks to obtain representative cross-sections from cervical to lumbar cord. Other spinal cord sections were obtained from transgenic mice expressing untranslated NF-L RNA (44Go). Paraffin-embedded sections of spinal cord sections were deparaffined, rehydrated, antigen-retrieved in Antigen Unmasking Solution (Vector Laboratories) and then treated with 3% H2O2 to block endogenous peroxidase. After incubation with normal rabbit serum (1:10 in PBS), the sections were incubated with goat polyclonal anti-NF-L antibody (1:100 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-p190RhoGEF antibody (1:100 dilution) (85Go), rabbit polyclonal anti-GFP antibody (1:200 dilution, Clontech) or rabbit polyclonal anti-ubiquitin (1:500 dilution, Santa Cruz Biotechnology) overnight at 4°C, washed and then stained with VECTASTAIN Elite ABC Kit (Vector Laboratories). The immunoreactivity was revealed using DAB Substrate Kit for peroxidase (Vector Laboratories) and observed under light microscopy.

Co-immunoprecipitation assays
NSC-34 cells were lysed in cold lysis buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100, 1 mM PMSF and mammalian protease inhibitor (Sigma)] and clarified by centrifugation at 13 500g for 10 min at 4°C. Spinal cords from normal adult mice were homogenized in ice-cold homogenization buffer containing 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and a cocktail of protease inhibitor (Sigma) using a Teflon-on-glass homogenizer and centrifuged at 13 500g for 15 min at 4°C, as described by Nguyen et al. (49Go). Supernatants from cell lysates or spinal cord homogenate were subjected to co-immunoprecipitation (94Go). About 500 µg of total protein extracts were incubated overnight at 4°C with unconjugated beads or agarose beads conjugated with anti-p190RhoGEF (85Go). The beads were washed four times, boiled in SDS loading buffer and eluted proteins analyzed by western blot with anti-NF-L antibody (Santa Cruz Biotechnology).


   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 thank Rafaela Cañete-Soler for sharing insights into the regulation of NF-L mRNA stability and for critical evaluations of anonymous reviewers that have led to the development of our working hypothesis. This work was supported by NIH grants (W.W.S.) and Research Development Grant MDA3833 (H.L.) from the MDA association.

Conflict of Interest statement. None declared.


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

  1. Glatzel, M., Stoeck, K., Seeger, H., Luhrs, T. and Aguzzi, A. (2005) Human prion diseases: molecular and clinical aspects. Arch. Neurol., 62, 545–552.[Abstract/Free Full Text]

  2. Goedert, M. and Jakes, R. (2005) Mutations causing neurodegenerative tauopathies. Biochim. Biophys. Acta, 1739, 240–250.[Medline]

  3. Kuret, J., Chirita, C.N., Congdon, E.E., Kannanayakal, T., Li, G., Necula, M., Yin, H. and Zhong, Q. (2005) Pathways of tau fibrillization. Biochim. Biophys. Acta, 1739, 167–178.[Medline]

  4. Muchowski, P.J. and Wacker, J.L. (2005) Modulation of neurodegeneration by molecular chaperones. Nat. Rev. Neurosci., 6, 11–22.[CrossRef][Web of Science][Medline]

  5. von Bergen, M., Barghorn, S., Biernat, J., Mandelkow, E.M. and Mandelkow, E. (2005) Tau aggregation is driven by a transition from random coil to beta sheet structure. Biochim. Biophys. Acta, 1739, 158–166.[Medline]

  6. Ross, C.A. and Poirier, M.A. (2004) Protein aggregation and neurodegenerative disease. Nat. Med., 10 (suppl), S10–S17.[CrossRef][Medline]

  7. Stoppini, M., Andreola, A., Foresti, G. and Bellotti, V. (2004) Neurodegenerative diseases caused by protein aggregation: a phenomenon at the borderline between molecular evolution and ageing. Pharmacol. Res., 50, 419–431.[CrossRef][Web of Science][Medline]

  8. Glabe, C.C. (2005) Amyloid accumulation and pathogensis of Alzheimer's disease: significance of monomeric, oligomeric and fibrillar Abeta. Subcell. Biochem., 38, 167–177.[Medline]

  9. Arrasate, M., Mitra, S., Schweitzer, E.S., Segal, M.R. and Finkbeiner, S. (2004) Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature, 431, 805–810.[CrossRef][Medline]

  10. Caughey, B. and Lansbury, P.T. (2003) Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu. Rev. Neurosci., 26, 267–298.[CrossRef][Web of Science][Medline]

  11. Sanchez, I., Mahlke, C. and Yuan, J. (2003) Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders. Nature, 421, 373–379.[CrossRef][Medline]

  12. Walsh, D.M., Klyubin, I., Fadeeva, J.V., Cullen, W.K., Anwyl, R., Wolfe, M.S., Rowan, M.J. and Selkoe, D.J. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature, 416, 535–539.[CrossRef][Medline]

  13. Strong, M.J. and Gordon, P.H. (2005) Primary lateral sclerosis, hereditary spastic paraplegia and amyotrophic lateral sclerosis: discrete entities or spectrum? Amyotroph. Lateral Scler. Other Motor Neuron Disord., 6, 8–16.[Web of Science][Medline]

  14. Bruijn, L.I., Miller, T.M. and Cleveland, D.W. (2004) Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu. Rev. Neurosci., 27, 723–749.[CrossRef][Web of Science][Medline]

  15. Wood, J.D., Beaujeux, T.P. and Shaw, P.J. (2003) Protein aggregation in motor neurone disorders. Neuropathol. Appl. Neurobiol., 29, 529–545.[CrossRef][Web of Science][Medline]

  16. Robertson, J., Kriz, J., Nguyen, M.D. and Julien, J.P. (2002) Pathways to motor neuron degeneration in transgenic mouse models. Biochimie, 84, 1151–1160.[Medline]

  17. Antonyuk, S., Elam, J.S., Hough, M.A., Strange, R.W., Doucette, P.A., Rodriguez, J.A., Hayward, L.J., Valentine, J.S., Hart, P.J. and Hasnain, S.S. (2005) Structural consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg. Protein Sci., 14, 1201–1213.[CrossRef][Web of Science][Medline]

  18. Furukawa, Y. and O'Halloran, T.V. (2005) Amyotrophic lateral sclerosis mutations have the greatest destabilizing effect on the apo- and reduced form of SOD1, leading to unfolding and oxidative aggregation. J. Biol. Chem., 280, 17266–17274.[Abstract/Free Full Text]

  19. Lindberg, M.J., Bystrom, R., Boknas, N., Andersen, P.M. and Oliveberg, M. (2005) Systematically perturbed folding patterns of amyotrophic lateral sclerosis (ALS)-associated SOD1 mutants. Proc. Natl Acad. Sci. USA, 102, 9754–9759.[Abstract/Free Full Text]

  20. Arnesano, F., Banci, L., Bertini, I., Martinelli, M., Furukawa, Y. and O'Halloran, T.V. (2004) The unusually stable quaternary structure of human Cu, Zn-superoxide dismutase 1 is controlled by both metal occupancy and disulfide status. J. Biol. Chem., 279, 47998–48003.[Abstract/Free Full Text]

  21. Hough, M.A., Grossmann, J.G., Antonyuk, S.V., Strange, R.W., Doucette, P.A., Rodriguez, J.A., Whitson, L.J., Hart, P.J., Hayward, L.J., Valentine, J.S. et al. (2004) Dimer destabilization in superoxide dismutase may result in disease-causing properties: structures of motor neuron disease mutants. Proc. Natl Acad. Sci. USA, 101, 5976–5981.[Abstract/Free Full Text]

  22. Khare, S.D., Caplow, M. and Dokholyan, N.V. (2004) The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis. Proc. Natl Acad. Sci. USA, 101, 15094–15099.[Abstract/Free Full Text]

  23. Lindberg, M.J., Normark, J., Holmgren, A. and Oliveberg, M. (2004) Folding of human superoxide dismutase: disulfide reduction prevents dimerization and produces marginally stable monomers. Proc. Natl Acad. Sci. USA, 101, 15893–15898.[Abstract/Free Full Text]

  24. Rakhit, R., Crow, J.P., Lepock, J.R., Kondejewski, L.H., Cashman, N.R. and Chakrabartty, A. (2004) Monomeric Cu, Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic lateral sclerosis. J. Biol. Chem., 279, 15499–15504.[Abstract/Free Full Text]

  25. Valentine, J.S. and Hart, P.J. (2003) Misfolded CuZnSOD and amyotrophic lateral sclerosis. Proc. Natl Acad. Sci. USA, 100, 3617–3622.[Abstract/Free Full Text]

  26. Wang, J., Xu, G., Li, H., Gonzales, V., Fromholt, D., Karch, C., Copeland, N.G., Jenkins, N.A. and Borchelt, D.R. (2005) Somatodendritic accumulation of misfolded SOD1-L126Z in motor neurons mediates degeneration: {alpha}B-crystallin modulates aggregation. Hum. Mol. Genet., 14, 2335–2347.[Abstract/Free Full Text]

  27. Watanabe, Y., Yasui, K., Nakano, T., Doi, K., Fukada, Y., Kitayama, M., Ishimoto, M., Kurihara, S., Kawashima, M., Fukuda, H. et al. (2005) Mouse motor neuron disease caused by truncated SOD1 with or without C-terminal modification. Brain Res. Mol. Brain Res., 135, 12–20.[Medline]

  28. Jonsson, P.A., Ernhill, K., Andersen, P.M., Bergemalm, D., Brannstrom, T., Gredal, O., Nilsson, P. and Marklund, S.L. (2004) Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis. Brain, 127, 73–88.[Abstract/Free Full Text]

  29. Bennett, E.J., Bence, N.F., Jayakumar, R. and Kopito, R.R. (2005) Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol. Cell, 17, 351–365.[CrossRef][Web of Science][Medline]

  30. Corbo, M. and Hays, A.P. (1992) Peripherin and neurofilament protein coexist in spinal spheroids of motor neuron disease. J. Neuropathol. Exp. Neurol., 51, 531–537.[Web of Science][Medline]

  31. Murayama, S., Bouldin, T.W. and Suzuki, K. (1992) Immunocytochemical and ultrastructural studies of upper motor neurons in amyotrophic lateral sclerosis. Acta Neuropathol. (Berl.), 83, 518–524.[CrossRef][Medline]

  32. Leigh, P.N., Dodson, A., Swash, M., Brion, J.P. and Anderton, B.H. (1989) Cytoskeletal abnormalities in motor neuron disease. An immunocytochemical study. Brain, 112, 521–535.[Abstract/Free Full Text]

  33. Munoz, D.G., Greene, C., Perl, D.P. and Selkoe, D.J. (1988) Accumulation of phosphorylated neurofilaments in anterior horn motoneurons of amyotrophic lateral sclerosis patients. J. Neuropathol. Exp. Neurol., 47, 9–18.[Web of Science][Medline]

  34. Morrison, B.M., Shu, I.W., Wilcox, A.L., Gordon, J.W. and Morrison, J.H. (2000) Early and selective pathology of light chain neurofilament in the spinal cord and sciatic nerve of G86R mutant superoxide dismutase transgenic mice. Exp. Neurol., 165, 207–220.[CrossRef][Web of Science][Medline]

  35. Irobi, J., De Jonghe, P. and Timmerman, V. (2004) Molecular genetics of distal hereditary motor neuropathies. Hum. Mol. Genet., 13 (Spec no. 2), R195–R202.[Abstract/Free Full Text]

  36. Young, P. and Suter, U. (2003) The causes of Charcot–Marie–Tooth disease. Cell. Mol. Life Sci., 60, 2547–2560.[CrossRef][Web of Science][Medline]

  37. Perez-Olle, R., Jones, S.T. and Liem, R.K. (2004) Phenotypic analysis of neurofilament light gene mutations linked to Charcot–Marie–Tooth disease in cell culture models. Hum. Mol. Genet., 13, 2207–2220.[Abstract/Free Full Text]

  38. Brownlees, J., Ackerley, S., Grierson, A.J., Jacobsen, N.J., Shea, K., Anderton, B.H., Leigh, P.N., Shaw, C.E. and Miller, C.C. (2002) Charcot–Marie–Tooth disease neurofilament mutations disrupt neurofilament assembly and axonal transport. Hum. Mol. Genet., 11, 2837–2844.[Abstract/Free Full Text]

  39. Perez-Olle, R., Leung, C.L. and Liem, R.K. (2002) Effects of Charcot–Marie–Tooth-linked mutations of the neurofilament light subunit on intermediate filament formation. J. Cell Sci., 115, 4937–4946.[CrossRef][Web of Science][Medline]

  40. Evgrafov, O.V., Mersiyanova, I., Irobi, J., Van Den Bosch, L., Dierick, I., Leung, C.L., Schagina, O., Verpoorten, N., Van Impe, K., Fedotov, V. et al. (2004) Mutant small heat-shock protein 27 causes axonal Charcot–Marie–Tooth disease and distal hereditary motor neuropathy. Nat. Genet., 36, 602–606.[CrossRef][Web of Science][Medline]

  41. Canete-Soler, R., Schwartz, M.L., Hua, Y. and Schlaepfer, W.W. (1998) Characterization of ribonucleoprotein complexes and their binding sites on the neurofilament light subunit mRNA. J. Biol. Chem., 273, 12655–12661.[Abstract/Free Full Text]

  42. Canete-Soler, R., Schwartz, M.L., Hua, Y. and Schlaepfer, W.W. (1998) Stability determinants are localized to the 3'-untranslated region and 3'-coding region of the neurofilament light subunit mRNA using a tetracycline-inducible promoter. J. Biol. Chem., 273, 12650–12654.[Abstract/Free Full Text]

  43. Canete-Soler, R., Silberg, D.G., Gershon, M.D. and Schlaepfer, W.W. (1999) Mutation in neurofilament transgene implicates RNA processing in the pathogenesis of neurodegenerative disease. J. Neurosci., 19, 1273–1283.[Abstract/Free Full Text]

  44. Nie, Z., Wu, J., Zhai, J., Lin, H., Ge, W., Schlaepfer, W.W. and Canete-Soler, R. (2002) Untranslated element in neurofilament mRNA has neuropathic effect on motor neurons of transgenic mice. J. Neurosci., 22, 7662–7670.[Abstract/Free Full Text]

  45. Lin, H., Zhai, J., Nie, Z., Wu, J., Meinkoth, J.L., Schlaepfer, W.W. and Canete-Soler, R. (2003) Neurofilament RNA causes neurodegeneration with accumulation of ubiquitinated aggregates in cultured motor neurons. J. Neuropathol. Exp. Neurol., 62, 936–950.[Web of Science][Medline]

  46. Lin, H., Zhai, J., Canete-Soler, R. and Schlaepfer, W.W. (2004) 3' untranslated region in a light neurofilament (NF-L) mRNA triggers aggregation of NF-L and mutant superoxide dismutase 1 proteins in neuronal cells. J. Neurosci., 24, 2716–2726.[Abstract/Free Full Text]

  47. Canete-Soler, R., Wu, J., Zhai, J., Shamim, M. and Schlaepfer, W.W. (2001) p190RhoGEF Binds to a destabilizing element in the 3' untranslated region of light neurofilament subunit mRNA and alters the stability of the transcript. J. Biol. Chem., 276, 32046–32050.[Abstract/Free Full Text]

  48. Bruce, J., Schwartz, M.L., Shneidman, P.S. and Schlaepfer, W.W. (1993) Methylation and expression of neurofilament genes in tissues and in cell lines of the mouse. Brain Res. Mol. Brain Res., 17, 269–278.[Medline]

  49. Nguyen, M.D., Shu, T., Sanada, K., Lariviere, R.C., Tseng, H.C., Park, S.K., Julien, J.P. and Tsai, L.H. (2004) A NUDEL-dependent mechanism of neurofilament assembly regulates the integrity of CNS neurons. Nat. Cell Biol., 6, 595–608.[CrossRef][Web of Science][Medline]

  50. Sasaki, S., Warita, H., Murakami, T., Shibata, N., Komori, T., Abe, K., Kobayashi, M. and Iwata, M. (2005) Ultrastructural study of aggregates in the spinal cord of transgenic mice with a G93A mutant SOD1 gene. Acta Neuropathol. (Berl.), 109, 247–255.[CrossRef][Medline]

  51. Menzies, F.M., Grierson, A.J., Cookson, M.R., Heath, P.R., Tomkins, J., Figlewicz, D.A., Ince, P.G. and Shaw, P.J. (2002) Selective loss of neurofilament expression in Cu/Zn superoxide dismutase (SOD1) linked amyotrophic lateral sclerosis. J. Neurochem., 82, 1118–1128.[CrossRef][Web of Science][Medline]

  52. Wong, N.K., He, B.P. and Strong, M.J. (2000) Characterization of neuronal intermediate filament protein expression in cervical spinal motor neurons in sporadic amyotrophic lateral sclerosis (ALS). J. Neuropathol. Exp. Neurol., 59, 972–982.[Web of Science][Medline]

  53. Bergeron, C., Beric-Maskarel, K., Muntasser, S., Weyer, L., Somerville, M.J. and Percy, M.E. (1994) Neurofilament light and polyadenylated mRNA levels are decreased in amyotrophic lateral sclerosis motor neurons. J. Neuropathol. Exp. Neurol., 53, 221–230.[Web of Science][Medline]

  54. Sjogren, M. and Englund, E. (2004) Negative neurofilament light and tau immunostaining in frontotemporal dementia. Dement. Geriatr. Cogn. Disord., 17, 346–349.[Web of Science][Medline]

  55. Kuchel, G.A., Poon, T., Irshad, K., Richard, C., Julien, J.P. and Cowen, T. (1997) Decreased neurofilament gene expression is an index of selective axonal hypotrophy in ageing. Neuroreport, 8, 799–805.[Web of Science][Medline]

  56. Nakamura, Y., Hasimoto, R., Kashiwagi, Y., Miyamae, Y., Shinosaki, K., Nishikawa, T., Hattori, H., Kudo, T. and Takeda, M. (1997) Abnormal distribution of neurofilament L in neurons with Alzheimer's disease. Neurosci. Lett., 225, 201–204.[CrossRef][Web of Science][Medline]

  57. Kittur, S., Hoh, J., Endo, H., Tourtellotte, W., Weeks, B.S., Markesbery, W. and Adler, W. (1994) Cytoskeletal neurofilament gene expression in brain tissue from Alzheimer's disease patients. I. Decrease in NF-L and NF-M message. J. Geriatr. Psychiatry Neurol., 7, 153–158.[Medline]

  58. McLachlan, D.R., Lukiw, W.J., Wong, L., Bergeron, C. and Bech-Hansen, N.T. (1988) Selective messenger RNA reduction in Alzheimer's disease. Brain Res., 427, 255–261.[Medline]

  59. Stieber, A., Gonatas, J.O. and Gonatas, N.K. (2000) Aggregates of mutant protein appear progressively in dendrites, in periaxonal processes of oligodendrocytes, and in neuronal and astrocytic perikarya of mice expressing the SOD1(G93A) mutation of familial amyotrophic lateral sclerosis. J. Neurol. Sci., 177, 114–123.[CrossRef][Web of Science][Medline]

  60. Giasson, B.I., Forman, M.S., Higuchi, M., Golbe, L.I., Graves, C.L., Kotzbauer, P.T., Trojanowski, J.Q. and Lee, V.M. (2003) Initiation and synergistic fibrillization of tau and alpha-synuclein. Science, 300, 636–640.[Abstract/Free Full Text]

  61. Williamson, T.L., Bruijn, L.I., Zhu, Q., Anderson, K.L., Anderson, S.D., Julien, J.P. and Cleveland, D.W. (1998) Absence of neurofilaments reduces the selective vulnerability of motor neurons and slows disease caused by a familial amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutant. Proc. Natl Acad. Sci. USA, 95, 9631–9636.[Abstract/Free Full Text]

  62. Lariviere, R.C. and Julien, J.P. (2004) Functions of intermediate filaments in neuronal development and disease. J. Neurobiol., 58, 131–148.[CrossRef][Web of Science][Medline]

  63. Cote, F., Collard, J.F. and Julien, J.P. (1993) Progressive neuronopathy in transgenic mice expressing the human neurofilament heavy gene: a mouse model of amyotrophic lateral sclerosis. Cell, 73, 35–46.[CrossRef][Web of Science][Medline]

  64. Xu, Z., Cork, L.C., Griffin, J.W. and Cleveland, D.W. (1993) Increased expression of neurofilament subunit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease. Cell, 73, 23–33.[CrossRef][Web of Science][Medline]

  65. Gama Sosa, M.A., Friedrich, V.L., Jr, DeGasperi, R., Kelley, K., Wen, P.H., Senturk, E., Lazzarini, R.A. and Elder, G.A. (2003) Human midsized neurofilament subunit induces motor neuron disease in transgenic mice. Exp. Neurol., 184, 408–419.[CrossRef][Web of Science][Medline]

  66. Eyer, J. and Peterson, A. (1994) Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament-beta-galactosidase fusion protein. Neuron, 12, 389–405.[CrossRef][Web of Science][Medline]

  67. Eyer, J., Cleveland, D.W., Wong, P.C. and Peterson, A.C. (1998) Pathogenesis of two axonopathies does not require axonal neurofilaments. Nature, 391, 584–587.[CrossRef][Medline]

  68. Lee, M.K., Marszalek, J.R. and Cleveland, D.W. (1994) A mutant neurofilament subunit causes massive, selective motor neuron death: implications for the pathogenesis of human motor neuron disease. Neuron, 13, 975–988.[CrossRef][Web of Science][Medline]

  69. Meier, J., Couillard-Despres, S., Jacomy, H., Gravel, C. and Julien, J.P. (1999) Extra neurofilament NF-L subunits rescue motor neuron disease caused by overexpression of the human NF-H gene in mice. J. Neuropathol. Exp. Neurol., 58, 1099–10110.[Web of Science][Medline]

  70. Perez-Olle, R., Lopez-Toledano, M.A., Goryunov, D., Cabrera-Poch, N., Stefanis, L., Brown, K. and Liem, R.K. (2005) Mutations in the neurofilament light gene linked to Charcot–Marie–Tooth disease cause defects in transport. J. Neurochem., 93, 861–874.[CrossRef][Web of Science][Medline]

  71. Roy, S., Zhang, B., Lee, V.M. and Trojanowski, J.Q. (2005) Axonal transport defects: a common theme in neurodegenerative diseases. Acta Neuropathol. (Berl.), 109, 5–13.[CrossRef][Medline]

  72. Fischer, L.R., Culver, D.G., Tennant, P., Davis, A.A., Wang, M., Castellano-Sanchez, A., Khan, J., Polak, M.A. and Glass, J.D. (2004) Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp. Neurol., 185, 232–240.[CrossRef][Web of Science][Medline]

  73. Schaefer, A.M., Sanes, J.R. and Lichtman, J.W. (2005) A compensatory subpopulation of motor neurons in a mouse model of amyotrophic lateral sclerosis. J. Comp. Neurol., 490, 209–219.[CrossRef][Web of Science][Medline]

  74. Gros-Louis, F., Lariviere, R., Gowing, G., Laurent, S., Camu, W., Bouchard, J.P., Meininger, V., Rouleau, G.A. and Julien, J.P. (2004) A frameshift deletion in peripherin gene associated with amyotrophic lateral sclerosis. J. Biol. Chem., 279, 45951–45956.[Abstract/Free Full Text]

  75. Leung, C.L., He, C.Z., Kaufmann, P., Chin, S.S., Naini, A., Liem, R.K., Mitsumoto, H. and Hays, A.P. (2004) A pathogenic peripherin gene mutation in a patient with amyotrophic lateral sclerosis. Brain Pathol., 14, 290–296.[Web of Science][Medline]

  76. Al-Chalabi, A., Andersen, P.M., Nilsson, P., Chioza, B., Andersson, J.L., Russ, C., Shaw, C.E., Powell, J.F. and Leigh, P.N. (1999) Deletions of the heavy neurofilament subunit tail in amyotrophic lateral sclerosis. Hum. Mol. Genet., 8, 157–164.[Abstract/Free Full Text]

  77. Tomkins, J., Usher, P., Slade, J.Y., Ince, P.G., Curtis, A., Bushby, K. and Shaw, P.J. (1998) Novel insertion in the KSP region of the neurofilament heavy gene in amyotrophic lateral sclerosis (ALS). Neuroreport, 9, 3967–3970.[Web of Science][Medline]

  78. Figlewicz, D.A., Krizus, A., Martinoli, M.G., Meininger, V., Dib, M., Rouleau, G.A. and Julien, J.P. (1994) Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis. Hum. Mol. Genet., 3, 1757–1761.[Abstract/Free Full Text]

  79. Ching, G.Y. and Liem, R.K. (1993) Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments. J. Cell Biol., 122, 1323–1335.[Abstract/Free Full Text]

  80. Lee, M.K., Xu, Z., Wong, P.C. and Cleveland, D.W. (1993) Neurofilaments are obligate heteropolymers in vivo. J. Cell Biol., 122, 1337–1350.[Abstract/Free Full Text]

  81. Liem, R.K. and Hutchison, S.B. (1982) Purification of individual components of the neurofilament triplet: filament assembly from the 70 000-dalton subunit. Biochemistry, 21, 3221–3226.[CrossRef][Medline]

  82. Geisler, N. and Weber, K. (1981) Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments. J. Mol. Biol., 151, 565–571.[CrossRef][Web of Science][Medline]

  83. Leung, C.L. and Liem, R.K. (1996) Characterization of interactions between the neurofilament triplet proteins by the yeast two-hybrid system. J. Biol. Chem., 271, 14041–14044.[Abstract/Free Full Text]

  84. Zhai, J., Lin, H., Shamim, M., Schlaepfer, W.W. and Canete-Soler, R. (2001) Identification of a novel interaction of 14-3-3 with p190RhoGEF. J. Biol. Chem., 276, 41318–41324.[Abstract/Free Full Text]

  85. Zhai, J., Lin, H., Nie, Z., Wu, J., Canete-Soler, R., Schlaepfer, W.W. and Schlaepfer, D.D. (2003) Direct interaction of focal adhesion kinase with p190RhoGEF. J. Biol. Chem., 278, 24865–24873.[Abstract/Free Full Text]

  86. Meyer, D., Liu, A. and Margolis, B. (1999) Interaction of c-Jun amino-terminal kinase interacting protein-1 with p190 rhoGEF and its localization in differentiated neurons. J. Biol. Chem., 274, 35113–35118.[Abstract/Free Full Text]

  87. van Horck, F.P., Ahmadian, M.R., Haeusler, L.C., Moolenaar, W.H. and Kranenburg, O. (2001) Characterization of p190RhoGEF, a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules. J. Biol. Chem., 276, 4948–4956.[Abstract/Free Full Text]

  88. Ge, W., Wu, J., Zhai, J., Nie, Z., Lin, H., Schlaepfer, W.W. and Canete-Soler, R. (2002) Binding of p190RhoGEF to a destabilizing element on the light neurofilament mRNA is competed by BC1 RNA. J. Biol. Chem., 277, 42701–42705.[Abstract/Free Full Text]

  89. Hadano, S., Hand, C.K., Osuga, H., Yanagisawa, Y., Otomo, A., Devon, R.S., Miyamoto, N., Showguchi-Miyata, J., Okada, Y., Singaraja, R. et al. (2001) A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat. Genet., 29, 166–173.[CrossRef][Web of Science][Medline]

  90. Yang, Y., Hentati, A., Deng, H.X., Dabbagh, O., Sasaki, T., Hirano, M., Hung, W.Y., Ouahchi, K., Yan, J., Azim, A.C. et al. (2001) The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis. Nat. Genet., 29, 160–165.[CrossRef][Web of Science][Medline]

  91. Kanekura, K., Hashimoto, Y., Niikura, T., Aiso, S., Matsuoka, M. and Nishimoto, I. (2004) Alsin, the product of ALS2 gene, suppresses SOD1 mutant neurotoxicity through RhoGEF domain by interacting with SOD1 mutants. J. Biol. Chem., 279, 19247–19256.[Abstract/Free Full Text]

  92. Chen, Y.Z., Bennett, C.L., Huynh, H.M., Blair, I.P., Puls, I., Irobi, J., Dierick, I., Abel, A., Kennerson, M.L., Rabin, B.A. et al. (2004) DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am. J. Hum. Genet., 74, 1128–1135.[CrossRef][Web of Science][Medline]

  93. Canete-Soler, R., Reddy, K.S., Tolan, D.R. and Zhai, J. (2005) Aldolases A and C are ribonucleolytic components of a neuronal complex that regulates the stability of the light-neurofilament mRNA. J. Neurosci., 25, 4353–4364.[Abstract/Free Full Text]

  94. Zhai, J., Lin, H., Cañete-Soler, R. and Schlaepfer, W.W. (2005) HoxB2 binds mutant SOD1 and is altered in transgenic model ALS. Hum. Mol. Genet., 14, 2692–2640.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 JCBHome page
Y. Lim, S.-T. Lim, A. Tomar, M. Gardel, J. A. Bernard-Trifilo, X. L. Chen, S. A. Uryu, R. Canete-Soler, J. Zhai, H. Lin, et al.
PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility
J. Cell Biol., January 10, 2008; 180(1): 187 - 203.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
J. Zhai, H. Lin, J.-P. Julien, and W. W. Schlaepfer
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
Hum. Mol. Genet., December 15, 2007; 16(24): 3103 - 3116.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
M. F. Mehler and J. S. Mattick
Noncoding RNAs and RNA Editing in Brain Development, Functional Diversification, and Neurological Disease
Physiol Rev, July 1, 2007; 87(3): 799 - 823.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
W. W. Schlaepfer, H. Lin, and J. Zhai
Aggregation of Light Neurofilament Protein has Direct Neurotoxic Effect on Motor Neurons
FASEB J, April 1, 2007; 21(5): A25 - A25.


Home page
 Hum Mol GenetHome page
R. J. Osborne and C. A. Thornton
RNA-dominant diseases
Hum. Mol. Genet., October 15, 2006; 15(suppl_2): R162 - R169.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Material
Right arrow All Versions of this Article:
14/23/3643    most recent
ddi392v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (12)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lin, H.
Right arrow Articles by Schlaepfer, W. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, H.
Right arrow Articles by Schlaepfer, W. W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?