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© The Rockefeller University Press,
0021-9525/1999//531 $5.00
The Journal of Cell Biology, Volume 147, Number 3,
, 1999 531-544
Original Article |
Late Onset Death of Motor Neurons in Mice Overexpressing Wild-Type Peripherin
mdju{at}musica.mcgill.ca
Peripherin, a type III intermediate filament (IF) protein, upregulated by injury and inflammatory cytokines, is a component of IF inclusion bodies associated with degenerating motor neurons in sporadic amyotrophic lateral sclerosis (ALS). We report here that sustained overexpression of wild-type peripherin in mice provokes massive and selective degeneration of motor axons during aging. Remarkably, the onset of peripherin-mediated disease was precipitated by a deficiency of neurofilament light (NF-L) protein, a phenomenon associated with sporadic ALS. In NF-L null mice, the overexpression of peripherin led to early- onset formation of IF inclusions and to the selective death of spinal motor neurons at 6 mo of age. We also report the formation of similar peripherin inclusions in presymptomatic transgenic mice expressing a mutant form of superoxide dismutase linked to ALS. Taken together, these results suggest that IF inclusions containing peripherin may play a contributory role in motor neuron disease.
Key Words: peripherin • neurofilament • ALS • motor neuron • transgenic
© 1999 The Rockefeller University Press
AMYOTROPHIC lateral sclerosis (ALS)1 is an adult-onset neurological disorder that affects primarily motor neurons in the brain and spinal cord. The degeneration of motor neurons leads to atrophy of skeletal muscles and, ultimately, to paralysis and death. Approximately 10% of the ALS cases are familial with the disease inherited in an autosomal dominant manner. Mutations in the copper/zinc superoxide dismutase (SOD1) gene have been found in 
20% of familial cases (2% of total ALS cases) (Rosen et al. 1993). Whereas transgenic mouse studies provided compelling evidence that SOD1 mutations cause ALS through a gain of deleterious activities (Gurney et al. 1994; Wong et al. 1995; Ripps et al. 1995; Tu et al. 1996; Bruijn et al. 1997, Bruijn et al. 1998), the etiology of ALS remains unknown for the vast majority of sporadic and familial cases. One hallmark of ALS is the presence of cytoplasmic inclusions of disorganized proteins in the cytoplasm of motor neurons (reviewed in Chou 1995). Some inclusions have been shown to contain mutant SOD1 protein (Kato et al. 1996; Shibata et al. 1996; Bruijn et al. 1998), whereas others contain neuronal intermediate filament (IF) proteins (Carpenter 1968; Tu et al. 1996).
Peripherin is a type III neuronal IF protein of 57 kD which is found with neurofilament (NF) proteins in the majority of IF inclusions (89%) in motor neurons of ALS patients (Corbo and Hays 1992; Migheli et al. 1993; Tu et al. 1996). In adults, peripherin is mostly expressed in autonomic nerves and in peripheral sensory neurons with only low levels detectable in spinal motor neurons (Parysek and Goldman 1988; Brody et al. 1989; Escurat et al. 1990; Troy et al. 1990a,Troy et al. 1990b). However, peripherin gene expression is increased by up to 300% in spinal motor neurons after injury of the sciatic nerve (Troy et al. 1990a), whereas expression of all three NF subunits mRNAs is reduced (Muma et al. 1990). Inflammation is one mechanism that can explain the upregulation of peripherin in injured neurons. In fact, peripherin gene expression is upregulated by the inflammatory cytokines interleukin-6 (IL-6) and leukemia inhibitory factor (LIF; Djabali et al. 1993; Sterneck et al. 1996) through the action of a JAK/STAT signaling pathway activating the STAT 1 and STAT 3 transcription factors (Lecomte et al. 1998). The action of cytokines on peripherin expression suggests that an upregulation of peripherin may be part of a general response of motor neurons to noxious stress that may explain the presence of numerous peripherin inclusion bodies in motor neurons of ALS patients.
In contrast to NFs, which are obligate heteropolymers of three subunits (Ching and Liem 1993; Lee et al. 1993), peripherin can self-assemble to establish an IF network in cultured cells (Cui et al. 1995; Ho et al. 1995; Beaulieu et al. 1999). Peripherin can also interact in vivo with NF proteins (Parysek et al. 1991) and can form heterodimers with each one of the three NF subunits in vitro (Athlan and Mushynski 1997). We have recently studied the interactions of peripherin with NF proteins in cultured SW13 cells devoid of endogenous cytoplasmic IF proteins (Beaulieu et al. 1999). Our results showed that peripherin can assemble with NFs as well as with the light NF subunit (NF-L) protein alone to form an IF network. Unexpectedly, the expression of peripherin with the heavy NF subunits (NF-H or NF-M) in the absence of NF-L prevented the formation of an IF network in transfected cells (Beaulieu et al. 1999).
We have used a transgenic mouse approach to investigate the potential detrimental effects of a sustained peripherin overexpression in motor neurons. Our results show that an upregulation of peripherin in mice causes a late-onset and selective motor neuron disease characterized by the formation of IF inclusions similar to those found in human ALS and in mice expressing mutant form of SOD1 linked to ALS. Moreover, the formation of IF inclusion bodies and the onset of disease were precipitated by a deficiency in NF-L protein probably through a deleterious action of the large NF subunits on peripherin organization.
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Materials and Methods |
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The constructs were isolated from their vector sequences by digestion with endonuclease restriction enzymes followed by purification on agarose gels using a Gene Clean II KitTM (Bio101). The resulting linear DNA fragments were microinjected into one-cell mouse embryos of C57Bl/C3H genetic background according to standard procedures (Brinster et al. 1981). The integration of the transgenes into mouse genomic DNA was confirmed by Southern blot analysis as described below.
Mice carrying the transgenes in NF-L null background were obtained by breeding transgenic mice with NF-L knockout mice (L–/– mice) (Zhu et al. 1997). The breeding was performed following a two-step procedure. In the first step, F1 transgenic mice were bred with NF-L knockout mice to generate mice carrying the transgene in an L+/– background. These mice were then backcrossed with L–/– mice leading to the production of mice carrying the transgene in L–/– background. All peripherin transgenic mice used in this study were heterozygous for the peripherin transgenes.
The SOD1G37R mice (Wong et al. 1995) were a kind gift of Drs. Donald L. Price (Johns Hopkins Medical School, Baltimore, MD) and Don Cleveland (Ludwig Institute, San Diego, CA).
Southern Blot Analysis
Southern blot analysis of genomic DNA isolated from mouse tails was carried out following procedures described previously (Côté et al. 1993). A 1.8-kb peripherin probe was generated by digestion of the Per DNA construct with EcoRI (see Fig. 1 A). This probe was designed for detection of a 1.8-kb band corresponding to the endogenous peripherin gene while the TPer transgene was detected as a 4.9-kb band. The probe and enzymatic digestion protocols used to detect the NF-L knockout genotype have been described previously (Zhu et al. 1997). The number of transgene copies inserted in the genome was estimated by Southern blotting with serial dilution of DNA extracted from F1 transgenic and nontransgenic mice from each line. The relative intensity of the bands was compared with the signal detected for the endogenous peripherin gene using Gel-pro 2.0.1 (Media Cybernetic) with a Power Macintosh computer.
Northern Blot Analysis
6-mo-old animals were killed by injection of an overdose of chloral hydrate. Tissues were collected, frozen in liquid nitrogen, and kept at –80°C before extraction of total RNA using the Trizol reagentTM (GIBCO-BRL). Total RNA (10 µg except 5 µg for dorsal root ganglia) were fractionated on 1% (wt/vol) agarose-formaldehyde gels, transferred to Gene screen plusTM membranes (NEN Life Science), hybridized, and washed according to standard procedures (Sambrook et al. 1989). The mRNA species were detected by autoradiography using Biomax MRTM film (Kodak) in the presence of an intensifying screen.
The actin probe corresponded to the complete cDNA from the rat β-actin. The peripherin probe corresponded to the first 538 bp of the peripherin transcript (GenBank/EMBL/DDBJ accession number X59840). This probe was derived by PCR from mouse genomic DNA using the (ATGAGCCATCATCACTCGTCGGGCC) 5' primer and the (TCTGCTTGAGCGCCGCTAGGTCCT) 3' primer. The NF-L probe was derived by PCR of the mouse NF-L first exon (GenBank/EMBL/DDBJ accession number M20480) using the (ATGAGTTCGTTCGGCTCGGATCCGAT) 5' primer and the (CCTCATAGCGAGCCTGCAGGTTGCG) 3' primer. The primers used to derive these two probes were purchased from GIBCO-BRL and the PCR reactions were carried out using Taq polymerase (Pharmacia). All probes were purified on agarose gels and labeled using the random primer method before hybridization (Feinberg and Vogelstein 1983).
Antibodies
The anti-peripherin monoclonal antibody (MAB1527), the anti-peripherin polyclonal antibody (AB1530), and the anti 
-internexin polyclonal antibody (AB1515) were from Chemicon. The anti-peripherin monoclonal antibody (NCL-Periph) was from Novocastra Laboratories Ltd. The anti–NF-H polyclonal antibody (N-4142) is from Sigma. The anti–NF-M monoclonal antibody (NN-18), the anti–NF-L monoclonal antibody (NR-4), the anti-hyperphosphorylated NF-H monoclonal antibody (RT-97), and the anti-actin clone c4 monoclonal antibody were from Boehringer Mannheim. The anti-hypophosphorylated NF-H monoclonal antibody (Smi-32) was from Steinberger Monoclonal Inc.
SDS-PAGE and Western Blot Analysis
6–10-mo-old mice (except if otherwise mentioned in figure legend) were killed and the tissues collected as for Northern blot analysis. For total protein extraction, tissues were homogenized in 0.5% (wt/vol) SDS, and 8 M urea (SDS/urea buffer). Preparation of the cytoskeletal insoluble proteins was carried out using a modified version of the method described by Ching and Liem 1993. Tissues were first homogenized at 48°C in a buffer containing Tris (10 mM), NaCl (150 mM), EDTA (1 mM), Triton X-100 (1% wt/vol), PMSF (2 mM), leupeptin (2 mg/ml), pepstatin (1 mg/ml), and aprotinin (10 mg/ml). The homogenates were then centrifuged at 14,000 g for 15 min at 4°C using a Sorvall MC-12V centrifugator (Dupont). The supernatants (soluble fraction) were collected and kept for further use. The pellets (insoluble fraction) were resuspended in a volume of SDS/urea buffer equivalent to the volume of the soluble fraction. The protein concentration in the different extracts was measured using the DC-Protein assayTM, (BioRad). The protein extracts were diluted in 2x sample buffer (30% [wt/vol] glycerol, 4% [wt/vol] SDS, 160 mM Tris HCl [pH 6.8], 10% [vol/vol] β-mercaptoethanol and 0.02% [wt/vol] bromophenol blue) and boiled for 3 min. Proteins were separated on 8% or 10% SDS-PAGE and either transferred to a nitrocellulose membrane or stained with 0.2% (wt/vol) Coomassie blue-R250 in a 10% (vol/vol) acetic acid and 45% (vol/vol) methanol solution. Nitrocellulose membranes were blocked in PBS containing 5% (wt/vol) skimmed milk powder (PBS/milk) before incubation with the primary antibodies diluted as follows: MAB1527, NR-4, and NN-18, 1:1,000 dilution; AB1515 and N-4142, 1:2,000; anti-actin, 1:5,000. After incubation, the blots were rinsed in PBS containing 0.1% (wt/vol) of Tween-20 (PBS/Tween) and incubated for 1 h with the appropriate secondary antibody (anti–mouse IgG peroxidase or anti–rabbit IgG peroxidase from the Jackson ImmunoResearch Laboratories) diluted 1:1,000 in PBS/milk. The immune complexes were revealed using the RenaissanceÙ chemiluminescence reagents (NEN Life Science).
Immunohistological Analysis
Immunohistochemical analysis was carried out as described by Jacomy and Bosler 1995. Mice were anesthetized by injection of chloral hydrate and perfused with a 16-g/liter sodium cacodylate buffer (pH 7.5) followed by fixative (3% [vol/vol] glutaraldehyde in sodium cacodylate buffer). 50-µm tissue sections were prepared using a vibratome. Floating sections were rinsed in PBS and treated for 30 min with a 1% (wt/vol) sodium borohydride solution to reduce epitope masking by glutaraldehyde. Sections were then blocked for 1 h in a PBS solution containing 3% (wt/vol) BSA, 0.5% (vol/vol) Triton X-100, and 0.03% (wt/vol) hydrogen peroxide. Incubation with the various antibodies was performed overnight at room temperature with agitation in a PBS solution containing 3% (wt/vol) BSA and 0.05% (vol/vol) Triton X-100. The antibody labeling was developed using a Vector ABC kit (Vector Laboratories Ltd.) and SigmafastTM tablets (Sigma).
For indirect double immunofluorescence analysis, mice were perfused with PBS (pH 7.5) followed by fixative (4% vol/vol paraformaldehyde in a phosphate buffered solution). Tissues were postfixed for 2 h in paraformaldehyde, rinsed in PBS, and incubated overnight in a 20% (wt/vol) phosphate-buffered sucrose solution. 20-µm tissue sections were prepared using a cryostat. Sections were then blocked for 30 min in a PBS solution containing 3% (wt/vol) BSA and 0.5% (vol/vol) Triton X-100. Incubation with mixtures of primary antibodies was performed overnight at room temperature in a PBS solution containing 3% (wt/vol) BSA and 0.05% (wt/vol) Triton X-100. Incubation with a mixture of the anti–mouse-FITC and anti–rabbit rhodamine-conjugated antibody (Jackson ImmunoResearch Laboratory) was carried out for one hour under the same conditions. The samples were finally mounted in ProlongTM (Molecular Probe, Inc.) and examined with a fluorescence microscope.
Morphology and Electron Microscopy
Tissue sections were prepared for embedding in Epon as described in Zhu et al. 1997. Thin sections were stained with Toluidine blue and examined under a light microscope. The counting of axons in the L5 ventral root was performed manually from photographs. For electron microscopy, ultrathin sections were stained with a lead citrate solution and examined with a Philips CM10 transmission electron microscope.
To evaluate the survival of spinal motor neurons, a portion of the spinal cord spanning 1-mm rostral to 1-mm caudal of the L5 ventral root entry point was dissected out from two 14-mo-old mice of each genotype. The spinal cord was then cut in 50-µm sections using a vibratome and stained with cresyl violet according to standard procedures (Bancroft and Stevens 1990). The large motor neurons in the ventral horn were then counted with the help of a technician who ignored the genotypes of the mice. Since this method did not produce an equal number of sections from each sample, the mean number of motor neurons per ventral horn section was calculated and compared for each genotype.
Motor Dysfunction
To evaluate motor dysfunction in the mice, we simply measured their capacity to grasp a vertical grid. The mice were placed at the center of a horizontal grid. The grid was then moved slowly to a vertical position. The time spent by the mice on the vertical grid was measured for a maximum of 2 min. This test was performed using five mice of each genotype and was repeated three times on separate days for a total of 15 tests per genotype.
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Six founders were obtained for each DNA construct. After Northern blot analysis of nervous tissues, we focused our efforts on the characterization of two lines of transgenic mice, the TPer line carrying 4 integrated copies of the TPer construct, and the Per line bearing 20 copies of the Per construct (Fig. 1 B).
Northern blot analysis was carried out to study the expression pattern of peripherin mRNA in normal and transgenic mice. Peripherin mRNA was detected in dorsal root ganglia (DRG) of normal (WT) mice, TPer transgenic mice, and Per transgenic mice (Fig. 1 C). Peripherin mRNA was also detected in the brain but not in the cerebellum of the TPer mice (Fig. 1 C). No peripherin mRNA was detected in nonnervous tissues of transgenic mice (Fig. 1 C). A combination of immunohistochemistry and Western blot analysis using anti-peripherin monoclonal antibodies was carried out to further analyze the peripherin expression patterns in the transgenic lines. Our analysis of normal mice allowed the detection of peripherin in DRG, spinal cord, and some sensory fibers of the brain stem. The TPer line showed a larger expression pattern with the detection of peripherin in the brain, in the optic nerve, in some reticular neurons of the brainstem, in the mesencephalic trigeminal nucleus, in the motor nucleus of the trigeminal and facial nerves, in the nucleus ambiguous, in the ventral and dorsal columns of the spinal cord, in spinal motor neurons, and in DRG. The Per mice overexpressed peripherin in sensory and motor neurons from the spinal cord and brain stem but they did not express detectable levels of peripherin in the brain.
Densitometry of Western blots, made up by the serial dilution of total protein extracts from dorsal and ventral roots (Fig. 1 D), showed that as compared with WT mice the peripherin levels were increased by approximately fourfold in the ventral roots of TPer mice, approximately sevenfold in the ventral roots of Per mice, approximately twofold in dorsal roots of TPer mice, and approximately threefold in dorsal roots of Per mice. An analysis of cytoskeletal Triton-insoluble extracts from the brain and ventral roots revealed a recovery of peripherin in the insoluble fraction (Fig. 1 E). Densitometry of Coomassie blue–stained SDS-PAGE of Triton-insoluble extracts was further carried out to compare the peripherin levels to those of NF-L in ventral and dorsal root axons (Fig. 1 F and Table ). The identification of the bands corresponding to NF-M, NF-L, and peripherin was confirmed by Western blot analysis (Fig. 1 F). The results indicated that the levels of peripherin in ventral roots of transgenic mice overexpressing peripherin remained inferior to NF-L protein levels (Table ).
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35% of motor axons in aged mice overexpressing peripherin when compared with their WT littermates (Fig. 2 and Table ). Note that axonal loss occurred predominantly in the ventral roots and that DRG axons were essentially spared. Unlike spinal motor neurons from WT mice (Fig. 2 C), most spinal motor neurons from transgenic mice were characterized by strong and diffuse peripherin immunoreactivity in their perikarya and neurites without evidence of perikaryal swellings (Fig. 2 D). However, some peripherin inclusion bodies were observed in neurites especially in aged mice (Fig. 2 D).
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Previous results obtained from cell culture studies and from L–/– mice (Beaulieu et al. 1999; Williamson et al. 1998) suggested that the organization of peripherin might be affected by the absence of NF-L. Western blot analysis of spinal cord extracts from the L–/–, Per;L–/–, and TPer;L–/– mice revealed reduced levels of peripherin, NF-H, and NF-M proteins as a consequence of NF-L deficiency (Fig. 3 A). Declines in levels of peripherin were also detected in the brain and optic nerve extracts from TPer;L–/– mice indicating that peripherin produced under the control of the Thy-1 promoter was also affected by the absence of NF-L (Fig. 3 A). Northern blot analysis of RNA from the brain and DRG of mice with various genotypes revealed that the NF-L disruption had no effects on peripherin mRNA levels (Fig. 3 B).
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-internexin (Pachter and Liem 1985). We have therefore examined the levels of -internexin to test whether the downregulation of peripherin in NF-L null mice was due to a general destabilization of neuronal IF proteins. In contrast to peripherin, the levels of -internexin were not affected by the absence of NF-L in L–/– and TPer;L–/– mice as revealed by Western blot analysis of optic nerve extracts (Fig. 3 C). Then we examined whether the peripherin levels in motor and sensory neurons were equally affected by the absence of NF-L (Fig. 3 D). Whereas peripherin was readily detected by Western blot in ventral root extracts of normal mice, the levels of peripherin and NF proteins were at exceedingly low levels in mice of the NF-L null background. It is noteworthy that the levels of peripherin were less affected by the absence of NF-L in dorsal roots than in ventral roots (Fig. 3 D). Electron microscopy of nondegenerating axons from ventral roots of 6-mo-old mice from the Per;L–/– and TPer;L–/– genotypes revealed that the cytoskeletal organization of these axons did not greatly differ from the one of motor axons of L–/– mice. In both cases there was a scarcity of IF structures with an elevated density of microtubules when compared with ventral root axons from mice of the WT and TPer genotypes (Fig. 4, A–D). In contrast to ventral root axons, small unmyelinated axons of dorsal roots in L–/– and TPer;L–/– mice showed little or no changes in the density of IF structures (Fig. 4, E–H). This observation is compatible with our Western blot result (Fig. 3 D) and with a report indicating that some unmyelinated sensory axons express peripherin in the absence of NFs (Goldstein et al. 1996).
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16% of motor axons at both 6 and 20 mo of age. 6-mo-old mice overexpressing peripherin in NF-L null background exhibited declines of 45–50% in the number of their motor axons. The progressive nature of the overt phenotypes observed in TPer;L–/– and Per;L–/– mice prompted us to monitor the loss of motor axons in mice of different age. Analysis of ventral root axons in the TPer;L–/– mice at 2, 6, and 14 mo of age revealed a parallel increase of motor dysfunction and loss of motor axons culminating in the loss of 59% of motor axons in paralyzed mice at 14 mo of age (Table ). To examine whether the massive loss of motor axons was accompanied by a corresponding death of neurons, we counted the number of motor neurons in a spinal cord region spanning a 3-mm rostral to caudal region at the level of L5 ventral roots. These counts were performed on Nissl-stained sections (Fig. 7). Two mice from each genotype were used for the analysis. Since similar results were obtained for the two mice of each genotype, the data were pooled and the mean number of large motor neuron per ventral horn was calculated (Table ). This analysis revealed a dramatic reduction in the mean number of large spinal motor neurons (64% neuronal loss) present in the ventral horns of 14-mo-old TPer;L–/– mice as compared with normal, TPer, and L–/– mice (Fig. 7 and Table ). Moreover, the loss of motor neurons during aging of the TPer;L–/– mice was progressive since 5-mo-old mice showed a lower degree of neuronal loss (Table ).
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Early Detection of Peripherin Inclusions in Mice Bearing SOD1G37R
Taken together, our results and the previous reports that peripherin is present in the majority of IF inclusions in sporadic ALS cases (Corbo and Hays 1992; Migheli et al. 1993; Tu et al. 1996) suggest that peripherin may play a contributory role in ALS pathogenesis. Further support for this view came from our immunodetection of peripherin inclusions in spinal cord sections of paralyzed and presymptomatic mice expressing a Glycine 37 to Arginine mutant form of SOD1 (SOD1G37R) associated with familial human ALS (Wong et al. 1995). This analysis was carried out in two different lines of transgenic mice, lines 42 and 29, which are characterized by disease onset at 5 and 11 mo, respectively. Staining of spinal cord sections from normal mice did not show any peripherin inclusions but allowed the detection of peripherin in axons from the dorsal column and in some motor axons of the ventral horns (Fig. 9A and Fig. E). In contrast, peripherin inclusion bodies were observed in paralyzed mice from both SOD1G37R lines (Fig. 9B and Fig. D). Staining of sections from presymptomatic mice of line 29 revealed the presence of peripherin inclusions in 5-mo-old mice (Fig. 9 C). Similar inclusions were also observed in one out of three mice studied at 3 mo of age, indicating that the formation of peripherin inclusions in motor axons occurs at an early stage of the disease. It is noteworthy that peripherin inclusions were also detected in the dorsal column in paralyzed but not in presymptomatic mice from both SOD1G37R lines (Fig. 9, F–H).
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Three independent studies have shown that peripherin is present in a majority of IF inclusions in ALS patients (Corbo and Hays 1992; Migheli et al. 1993; Tu et al. 1996). We have also detected similar inclusions in presymptomatic mice expressing a mutant SOD1G37R linked to familial ALS (Fig. 9). Such IF inclusions in ALS may occur as a consequence of axonal transport defects (Zhang et al. 1997; Williamson and Cleveland 1999). Alternatively, the results presented here and the intense immunoreactivity of IF inclusions for peripherin in ALS make it plausible that such inclusion bodies could originate from an upregulation of peripherin occurring at the level of gene transcription. Peripherin is normally expressed at low levels in motor neurons (Troy et al. 1990a), but enhanced levels, and perhaps IF inclusion formation, could be part of a regenerative response observed in ALS. It is well established that after nerve injury the levels of peripherin mRNA are induced by three- to fourfold in motor neurons (Troy et al. 1990a). Other genes associated with the regenerative response after neuronal injury, such as GAP43 and 1-tubulin, have been reported to be upregulated in ALS (Parhad et al. 1992). Moreover, peripherin is upregulated by cytokines such as IL-6 and LIF (Djabali et al. 1993; Sterneck et al. 1996). There is growing evidence for an involvement of inflammatory reactions in ALS that could account for enhanced levels of peripherin. A recent study has indicated an increase of IL-6 in the cerebrospinal fluid of ALS patient (Sekizawa et al. 1998). Moreover, an upregulation of LIF has been reported in an in vivo model of glutamate excitotoxicity (Kurek et al. 1998), which is another hypothesized mechanism of toxicity associated with ALS (Lin et al. 1998). Finally, inflammatory cytokines are associated with astrogliosis, which is a common phenomenon in ALS and in other neurodegenerative diseases (Klein et al. 1997).
Our previous cell culture studies with peripherin and NF cDNA clones in SW13 cells demonstrated that peripherin can self-assemble to form an IF network and that it assembles with the NF network in the presence of NF-L (Beaulieu et al. 1999). However, in absence of NF-L, the NF-M and NF-H proteins interact with peripherin to form disorganized IF structures (Beaulieu et al. 1999). Perhaps, this could explain why the peripherin-mediated disease was exacerbated in the NF-L null background. The increased number of IF inclusion bodies in mice deficient for NF-L is probably due to abnormal interactions of peripherin and the high molecular mass NF subunits. In this regard, it is noteworthy that rare motor neurons in TPer;L–/– mice that were devoid of NF-M protein did not develop IF inclusions bodies even though peripherin was detected at elevated levels (Fig. 8 L). Similarly, small sensory neurons that express peripherin but are devoid of NFs (Ferri et al. 1990; Goldstein et al. 1996) maintained a normal IF network in the NF-L null background (Fig. 4).
The NF-L null background mimics in part the depletion in NF-L mRNA levels occurring in motor neurons of ALS patients. In situ hybridization studies revealed a striking 60% reduction of NF-L mRNA levels in the -motor neurons of ALS patients as compared with age-matched controls (Bergeron et al. 1994). Moreover, greater declines in NF-L mRNA levels (87%) were detected in motor neurons having spheroids or perikaryal filamentous accumulations (Bergeron et al. 1994). Remarkably, our data demonstrate that a partial depletion in NF-L levels is sufficient to increase the abundance of peripherin inclusions. A 50% reduction of NF-L mRNA in the TPer;L+/– and Per;L+/– mice resulted in the formation of peripherin inclusions in proximal axons of motor neurons (Fig. 8 C)
The formation of IF inclusions in motor neurons has been associated with motor dysfunction in three other types of transgenic mice (Côté et al. 1993; Xu et al. 1993; Lee et al. 1994). However, the properties of IF inclusions described here differ in several aspects from those of IF inclusions observed in mice overexpressing NF transgenes. Whereas large perikaryal NF accumulations excluding other cytoplasmic material were observed in the NF-H and NF-L transgenic mice (Côté et al. 1993; Xu et al. 1993), the inclusion bodies found in peripherin transgenic mice are relatively small, they contain membranous material including mitochondria and they occur in both cell bodies and axons. Because of their size and axonal localization, the inclusions observed in peripherin transgenic mice are more similar to the IF inclusions found in human ALS (reviewed in Chou 1995) and in mutant SOD1 transgenic mice than are the NF swellings observed in the NF transgenic mice. More importantly, unlike mice overexpressing wild-type NF proteins (Côté et al. 1993; Xu et al. 1993; our unpublished observation), the mice with peripherin inclusions exhibited axonal degeneration and massive death of motor neurons.
Unexpectedly, the results shown here demonstrate that the presence of NF-L is not required for the formation of IF inclusions in motor neurons. This is an important finding because an involvement for NF proteins in ALS has been recently challenged by studies with L–/– mice and NF-H/lacZ mice showing that the absence of intact NFs in axons did not prevent SOD1-mediated disease (Williamson et al. 1998; Eyer et al. 1998). This led to the belief that IF proteins play a minor role in ALS pathogenesis, a notion that must be reexamined at the light of our results showing that disorganized IF structures containing high molecular weight NF proteins together with peripherin can provoke motor neuron death in absence of NF-L.
The exact mechanism underlying cell death by such IF inclusion bodies remains to be elucidated. A block of intracellular transport by multiple IF inclusions in the axon is a possible mechanism that may contribute to neurodegeneration and perhaps cell death in the peripherin transgenic mice described here. Alternatively, the peripherin-containing inclusions could provoke neurodegeneration by the sequestration of organelles such as mitochondria (Fig. 8) or other components essential for cell survival. Such a mechanism has been postulated to explain the toxicity of SOD1 aggregates detected in SOD1 mutant mice (Bruijn et al. 1998). In fact, there is growing evidence for protein aggregation as a common etiology in neurodegenerative disorders including ALS, Alzheimer's disease, prion disease, Parkinson's disease, and a class of disorders caused by expanded CAG repeats encoding polyglutamines (for review see Kakizuka 1997). The general working hypothesis is that mutations in proteins may result in misfolding and decreased protein degradation, leading to disturbance in homeostatic mechanisms. For instance, an upregulation of proteins with chaperoning activity was found to protect against aggregation of mutant ataxin-1 (Cummings et al. 1998) and also from the toxicity of SOD1 mutants (Bruening et al. 1998). Recently, polyglutamine domain proteins derived from expanded CAG repeats were found to bind specifically to NF-L and to alter NF network (Nagai et al. 1999). The data presented here introduces a novel concept in that no mutant proteins are required for the development of inclusion bodies. In future studies it would be of interest to investigate whether peripherin inclusions can also be derived by posttranslational modifications of IF proteins. It is well documented that changes in phosphorylation can alter the assembly properties of NF proteins (for review see Julien and Mushynski 1998). In addition, the nitration of tyrosine residues in the NF-L protein, a phenomenon that may occur in ALS (Crow et al. 1997), could impair its assembly and perhaps would favor interactions of peripherin with the high-molecular mass NF subunits to form noxious IF inclusions.
In conclusion, our study demonstrates for the first time that an upregulation of wild-type peripherin can cause the selective death of motor neurons during aging and that a NF-L deficiency is a factor that precipitates the disease. These results combined with the specific detection of peripherin inclusions in motor neurons of sporadic ALS cases (Corbo and Hays 1992; Migheli et al. 1993; Tu et al. 1996) and in motor neuron neurons of presymptomatic SOD1G37R mice (Fig. 9) provide compelling evidence for a potential role of peripherin in ALS pathogenesis.
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Acknowledgments |
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This research was supported by the Medical Research Council of Canada (MRC), the ALS association (USA), and the ALS society of Canada. J.-M. Beaulieu is a recipient of studentships from the Natural Science and Engineering Research Council of Canada (NSERC), from Le Fond de Recherche en Santé du Québec (FRSQ) and from the McDonald Stewart Foundation. M.D. Nguyen was a recipient of a MRC studentship. J.-P. Julien has a MRC Senior Scholarship.
Submitted: 4 June 1999
Revised: 23 September 1999
Accepted: 24 September 1999
1.used in this paper: ALS, amyotrophic lateral sclerosis; DRG, dorsal root ganglion; IF, intermediate filament; IL-6, interleukin 6; LIF, leukemia inhibitory factor; NF, neurofilament; NF-H, neurofilament heavy subunit; NF-L, neurofilament light subunit; NF-M, neurofilament medium subunit; SOD1, copper/zinc superoxide dismutase; WT, wild-type
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