The amount of neurofilaments aggregated in the cell body is controlled by their increased sensitivity to trypsin-like proteases

F. Fasani, A. Bocquet, P. Robert, A. Peterson* and J. Eyer{ddagger}

Laboratoire Neurobiologie and Transgenese, UPRES-EA 3143, INSERM, 4 rue Larrey, bâtiment Montéclair, CHU 49033 Angers CEDEX, France.

{ddagger} Author for correspondence (e-mail: eyer{at}univ-angers.fr)

Accepted 14 October 2003


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurofilaments are synthesised and assembled in neuronal cell bodies, transported along axons and degraded at the synapse. However, in several pathological situations they aggregate in cell bodies or axons. To investigate their turnover when separated from their normal site of degradation, we used a previously described transgenic model characterised by perikaryal retention of neurofilaments, and compared the basic features of both neurofilament synthesis and degradation with that observed in normal mice. Despite the massive perikaryal aggregates, neurofilament transcript levels were found to be unchanged, whereas the total accumulation of neurofilament proteins was markedly reduced. Neurofilaments isolated from transgenic samples are more sensitive to both trypsin and {alpha}-chymotrypsin mediated proteolysis. Consistent with their greater in vitro sensitivity, trypsin immunolabeling of cell bodies was stronger in transgenic mice. These results show a novel mechanism to regulate the amount of neurofilaments when they abnormally aggregate.

Key words: Neurofilaments, Aggregation, Transgenic mice, Trypsin proteolysis


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurofilaments are heteropolymers of three subunits: light (NFL, 70 kDa), medium (NFM, 150 kDa) and heavy (NFH, 200 kDa), which co-assemble through their central domain. The C-terminal regions of NFM and NFH are highly phosphorylated and form perpendicular projections involved in inter-neurofilament interactions, the axonal transport of neurofilaments and the axonal calibre (Hirokawa, 1982Go; Carden et al., 1987Go; Eyer and Leterrier, 1988Go; De Waegh et al., 1992Go). Following their synthesis, each subunit assembles into filaments that are transported by the slow axonal flow (ScA).

As neurofilaments are actively produced, an active degradation process also exists. Leupeptin injection in the optic tectum of goldfish induces the synaptic accumulation of neurofilaments, arguing for their synaptic degradation by a calcium-activated protease (Roots 1983Go). Similar proteases are found in human tissues, and degrade neurofilaments from squid and rat (Paggi and Lasek 1984Go; Schlaepfer et al., 1985Go; Gallant et al., 1986Go; Vitto et al., 1986). At micromolar calcium concentrations a limited proteolysis of NFM occurs during the axonal transport. At higher concentrations (i.e. following axonal transections), a pronounced degradation of neurofilaments occurs (Nixon et al., 1986Go; Banik et al., 1997Go). Neurofilaments are also proteolysed by the lysosomial cathepsin D in rat, bovine and human tissues (Nixon and Marotta 1984Go; Banay-Schwartz et al., 1987Go; Suzuki et al., 1988Go), or by trypsin and {alpha}-chymotrypsin. Such trypsin proteolytic strategies were particularly useful to analyze the spatial architecture of neurofilaments (Chin et al., 1983Go; Chin et al., 1989Go). However, despite the recent availability of multiple transgenic lines of mice characterized by the aggregation of neurofilaments, the proteolysis of these filaments in mice has not yet been described.

Several human neuropathological situations are also characterised by abnormal aggregations of neurofilaments. In Amyotrophic Lateral Sclerosis they precipitate in cell bodies or the proximal part of axons from motor neurons (Hirano, 1991Go; Corbo and Hays, 1992Go). They accumulate in Lewy bodies of Parkinson's disease (Hill et al., 1993Go), in Neurofibrillary Tangles of Alzheimer's disease (Leigh et al., 1989Go), and following intoxication by aluminium, hexanedione, acrylamide or ß,ß'-Iminodipropionitrile (IDPN) (Eyer et al., 1989Go; Leterrier et al., 1992Go). A decreased amount of their transcripts is also observed in Amyotrophic Lateral Sclerosis where NFL mRNA is decreased by 60%, and in Alzheimer's disease by 70% (Bergeron et al., 1994Go; Perry et al., 1991Go). However, it is unclear whether this reduction reflects the degeneration of neurons, or is an active process of the disease responsible for the downregulation of neurofilament production. Moreover, in all these pathological situations the cellular and molecular mechanisms used to eliminate the neurofilamentous aggregates are still unknown, as well as the mechanism responsible for regulating the turnover of neurofilaments. Recently, it has been shown that trypsin-like proteases are expressed in neurons (Gschwend et al., 1997Go; Yamashiro et al., 1997Go; Scarisbrick et al., 2001Go), and they accumulate within pathological neurofilamentous aggregates (Chou et al., 1998Go). These data suggest a functional importance of such proteases in the degradation of neurofilaments in vivo, but such a possibility has not yet been analysed in pathological samples.

A central question we addressed in this study is to determine how the turnover of neurofilaments is adjusted when the site of their accumulation is separated from the synapse, which is their normal site of degradation. Here, using a transgenic preparation in which neurofilaments are sequestered in cell bodies (Eyer and Peterson, 1994Go), we show that the amount of neurofilaments is strongly reduced in transgenic samples whereas their transcript level is unchanged. Moreover, neurofilaments isolated from transgenic samples are degraded more efficiently by exogenous trypsin and {alpha}-chymotrypsin than those isolated from control animals, whereas their degradation profile is unchanged and comparable to other species. Finally, trypsin immunolabeling was more pronounced in perikarya of transgenic tissues. These results indicate that when neurofilaments are retained in the cell body, their amount is not controlled at the transcriptional level but is reduced via an increased susceptibility to trypsin-like proteolysis.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
RNA analysis by northern blots
Brains and spinal cords were removed from 2-day, 4-week, 6-month and 18-month-old transgenic and control mice, immediately frozen in liquid nitrogen and stored at –80°C. Total RNA extraction and northern blots were performed and evaluated as previously described (Robert et al., 2001Go). NFH and NFHLacZ mRNAs were identified using a 12 kb EcoRV-KpnI fragment from the mouse NFH gene (Julien et al., 1988Go). Probe for NFL was a 7.5 kb EcoRI fragment from the mouse NFL gene (Lewis and Cowan, 1985Go) and probe for NFM was a 0.9 kb SacI-SacII fragment from the mouse NFM cDNA (Levy et al., 1987Go).

Isolation of neurofilament proteins
Neurofilaments were purified according to the procedure described by Leterrier and Eyer (Leterrier and Eyer, 1987Go), and modified for mouse samples as follows. Brains from 3- to 6-month-old control and transgenic mice were homogenised in buffer A (MES 0.1 M, pH 6.8, EGTA 1 mM, MgCl2 1 mM). The homogenate was centrifuged at 100,000 g for 1 hour at 4°C. The supernatant (S1) was made 4 M glycerol and incubated for 2 hours at 4°C to prevent microtubule assembly, but allowing neurofilaments to form reticulated networks. This suspension was centrifuged at 100,000 g for 1 hour at 4°C. The resulting pellet (P2) was homogenised in buffer A, and a third centrifugation was performed to recover neurofilaments in the third pellet (P3). The amount of proteins present in each sample was evaluated using the BCA Protein assay kit (Pierce). Each fraction of such a preparation was stored at –20°C before analysis.

In vitro proteolysis
Kinetics of proteolysis was performed using 10 or 25 µg of neurofilaments. Proteins were degraded for various times (0 to 60 minutes) with respectively 4 ng or 20 ng of trypsin or {alpha}-chymotrypsin (Boehringer Mannheim) at 30°C in MES 50 mM, MgCl2 10 mM, pH 6.5. Proteolysis was stopped at 0, 10, 20, 40, 60 minutes by the addition of an equal volume of the SDS-PAGE sample buffer (Tris 0.125 M, pH 6.8; glycerol 20%, SDS 2%, ß-mercaptoethanol 2%, bromophenol blue) and thermic denaturation (100°C for 4 minutes).

To determine which part of each neurofilament subunit was solubilized following proteolysis, the reaction products were centrifuged at 100,000 g at 4°C for 30 minutes. To each fraction (supernatant and pellet) an equal volume of SDS-PAGE sample buffer was added. After boiling for 4 minutes at 100°C, samples were stored at –20°C before western-blot analysis.

Western-blot analysis
Proteins were separated on a 7.5% acrylamide SDS-PAGE (Mini-Protean II Cell, BioRad) according to Laemmli (Laemmli, 1970Go), and then transferred onto nitrocellulose membranes (Millipore) for immunoblotting analysis (Towbin et al., 1979Go). Primary antibodies used to reveal neurofilament proteins and dilutions employed were as follows: monoclonal mouse anti-NFH antibodies N0142, 1:2000 (Sigma), monoclonal mouse anti-NFM antibodies N5264, 1:1000 (Sigma), monoclonal mouse anti-NFL antibodies N5139, 1:1000 (Sigma). The enhanced chemiluminescence detection system (ECL, Amersham Life Science) was used to reveal immunoreactive bands on radiographic films. Following revelation, membranes were washed with TBS and stripped for 30 minutes at 50°C in Tris 62.5 mM, pH 6.7, ß-mercaptoethanol 100 mM, SDS 2%. Extensive washing with TBS preceded a new incubation overnight in the blocking solution (10% dry milk in TBS), which allows probing of the same membrane with another antibody the next day. Band intensities were determined using the ImageQuant or NIH-Image software and transferred to Microsoft Excel files. Results from at least three experiments were analysed for their means and standard deviations using Microsoft Excel. The amount of each proteolysed product was estimated by comparing its intensity value with the undigested neurofilament subunit control band (incubation time: 0 minutes; or no enzyme added), which was run on the same immunoblot.

Immunofluorescence analysis
Mice were lethally anaesthetised with avertin (8 mg/kg), perfused transcardialy first with 20 ml of phosphate buffer to remove blood, and fixed with 20 ml of 4% paraformaldehyde in phosphate buffer. Samples were dissected, post-fixed in the same fixation buffer for one hour, and gradually transferred to 30% sucrose before being frozen and stored at –80°C. Cryostat sections (10 µm) were rinsed three times with phosphate buffer before blocking at room temperature for one hour with 5% BSA plus 5% goat serum. Sections were then rinsed (3x5 minutes) with phosphate buffer and incubated for 90 minutes with the first primary antibody in 1% BSA plus 1% goat serum. Sections were rinsed (3x5 minutes) and incubated with the second primary antibody in the same conditions. To reveal the location of the two primary antibodies, sections were rinsed (3x5 minutes) and incubated with fluorescent-labelled secondary antibodies. Each labelled antibody was incubated consecutively for 1.5 hours and rinsed (3x5 minutes). Slides were mounted with anti-fading medium and stored at 4°C in the dark before observation with a confocal microscope (Olympus BX50 with Olympus Fluoview software 3.O). Primary antibodies and dilutions employed were as follows: polyclonal rabbit anti-trypsin antibodies AB1823, 1:100 (Chemicon), monoclonal mouse anti-NFH antibodies N0142, 1:1000 (Sigma), polyclonal rabbit anti-peripherin antibodies AB1530, 1:500 (Chemicon); Alexa Fluor 488 goat anti-mouse IgG antibodies A11001, 1:200 (Interchim), Alexa Fluor 568 goat anti-rabbit IgG antibodies A11011, 1:200 (Interchim).


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NFHLacZ transgenic mice express low levels of an NFH-ß-galactosidase fusion protein in projection neurons (Eyer and Peterson, 1994Go). Electrophoretic analysis of spinal cord homogenates from NFHLacZ mice revealed a fusion protein of the predicted molecular mass (214 kDa) at a concentration approximating 10% of the endogenous NFH protein, and poorly phosphorylated (Eyer and Peterson, 1994Go; Tu et al., 1997Go). Such low levels of NFH-ß-galactosidase accumulation are sufficient to aggregate the entire neurofilament cytoskeleton in the perikaryal compartment, and to prevent their axonal export.

Analysis of neurofilament protein and transcript levels in NFHLacZ transgenic mice
To evaluate the level at which neurofilaments expression might be influenced by their perikaryal aggregation, we measured the total accumulation of neurofilament proteins in NFHLacZ transgenic mice. Homogenates from brain and spinal cord tissues of adult control and transgenic mice were prepared, and 20 µg of proteins from brain and 30 µg from spinal cord homogenates were analysed by western blots. Compared with control mice, the accumulated levels of all three neurofilament subunits, both in brain and spinal cord samples, were strongly reduced (Fig. 1A, Table 1A). Note that the majority of NFH present in transgenic samples migrates as a lower molecular weight subunit compared with non-transgenic samples, which indicates its poorly phosphorylated status (Fig. 1A).



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 1 Evaluation of the accumulated amount of neurofilament proteins and transcripts in transgenic and control samples. (A) A typical western blot of brain and spinal cord crude extracts from control (–) and transgenic (+) mice was revealed using antibodies against NFH, NFM and NFL. A much lower amount of each subunit was regularly found in transgenic samples. (B-B') The levels of NFHLacZ mRNA (B') were found to be between 12- and 80-fold lower compared with the endogenous NFH mRNA (B). No major difference in the expression of the endogenous NFH gene was observed between control and transgenic mice during ageing. (C) A 10-fold lower level of NFM mRNA was detected in brain compared with spinal cord without significant difference between control and transgenic tissues. (D-D') A similar pattern of expression of NFL was found between control and transgenic samples. In spinal cord, a 4-fold increase of the light transcript (2.5 kb) has been observed between the earlier stage of life and the other stages.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Quantification of neurofilament proteins and transcripts by western (A) and northern blot (B) experiments

 

To determine whether this reduction of accumulated neurofilaments in NFHLacZ mice resulted from pre- or post-translational events, levels of accumulated transcripts for the three neurofilament subunits were compared with control values using northern blots. In control brain samples the level of the endogenous NFH mRNA increased between post-natal day 2 (P2) and the 4th week (4w) by sevenfold and thereafter remained stable. A similar pattern was observed for spinal cord samples (Fig. 1B, Table 1B). Neither the developmental program nor the absolute levels of accumulated NFH mRNA were altered in transgenic samples. Compared with the NFH transcript, accumulated levels of NFHLacZ were between 12.4 and 80.6 times lower (Fig. 1B', Table 1B). NFM mRNA accumulation revealed a large difference between brain and spinal cord samples from control mice, but unlike NFH, no marked upregulation in transcript levels was observed during the ex utero development. Both the developmental programming and the amounts in transgenic samples were similar to non-transgenic samples (Fig. 1C, Table 1B). Finally, accumulated levels of both 2.5 and 4 kb transcripts of NFL revealed no differences between control and transgenic samples (Fig. 1D, Fig. 1D', Table 1B). Thus, the decreased levels of accumulated neurofilament proteins cannot be attributed to differences of the accumulated amount of transcripts.

Proteolysis of neurofilaments by trypsin
The steady state level of neurofilaments in the cell body is the result of an equilibrium between several factors, including the rate of synthesis, the rate of export to axons and dendrites, and the rate of local degradation. In these NFHLacZ transgenic mice neurofilaments are sequestered into cell bodies, and consequently not transported into axons (Eyer and Peterson, 1994Go). Therefore, the decreased amount of neurofilaments in these transgenic mice (while their transcript level is unaffected) could be because of a local degradation. Moreover, as neurofilaments sequestered in the cell body are less phosphorylated (Fig. 1A), one possibility could be that the reduced amount of neurofilaments results from an increased susceptibility of these filaments to proteolysis (Goldstein et al., 1987Go; Pant, 1988Go). To test this hypothesis, we analysed the proteolysis pattern of each neurofilament subunit by SDS-PAGE to determine whether their spatial conformation is modified in transgenic samples. We also measured the rate of degradation of neurofilaments by exogenous proteases depending whether filaments were isolated from control or transgenic tissues.

When neurofilaments were purified from normal and transgenic tissues and incubated at 30°C alone, for up to two hours, no particular degradation process occurred. Moreover, when calpain and other calcium-activated proteases were tested, the proteolysis was very low (not shown). Therefore, we used trypsin and {alpha}-chymotrypsin enzymes, as neurofilaments isolated from other species were shown to be good substrates for such proteases (Chin et al., 1983Go; Chin et al., 1989Go). Moreover, recently it has been shown that trypsin-like proteases are expressed in the nervous system (Gschwend et al., 1997Go; Chou et al., 1998Go; Scarisbrick et al., 2001Go), and therefore could be involved in the turnover of neurofilaments.

Kinetic proteolyses were realised (0 to 60 minutes), and for each neurofilament subunit the proteolytic products were analysed by western blotting to determine the molecular weight of the resulting fragments. Fig. 2A shows typical proteolytic patterns obtained following 20 minutes of proteolysis. The intact mouse NFH protein (t=0 minutes) showed two closely migrating bands that correspond to phosphorylated and dephosphorylated forms. Following 20 minutes of proteolysis by trypsin, the intensity of these bands was strongly reduced while a breakdown product appeared with a lower molecular weight of approximately 160 kDa. Following the same proteolysis conditions, a considerable amount of intact NFM protein (single band at 150 kDa) was still observed, together with four major breakdown products (120, 100, 40 and 30 kDa). For NFL, the intact protein (single band of 70 kDa) was proteolysed into three fragments of 65, 45 and 40 kDa. As the same membrane was used to evaluate systematically the extent of proteolysis of each subunit, it appears that NFM is the most resistant to trypsin degradation as the intensity of the intact protein band was still intense after 20 minutes of proteolysis.



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 2. Trypsin proteolysis of neurofilaments isolated from control (WT) and NFHLacZ transgenic (TG) mice. (A) Neurofilaments (25 µg) were incubated during 20 minutes with 20 ng (for NFM) or 4ng (for NFH and NFL) of trypsin at 30°C. The reaction was stopped by the addition of Laemmli buffer, boiled during 5 minutes and analyzed by western blot. (B) 50 µg of neurofilaments were digested for 20 minutes at 37°C with trypsin (2 ng for NFH and NFL, and 4 ng for NFM), then sedimented for 30 minutes at 100,000 g and 4°C. Supernatant (S) and pellet (P) fractions were analysed by western blot. Similar proteolytic patterns and solubility of fragments were observed between control and transgenic mice.

 

Following trypsin proteolysis, the reaction products were centrifuged in order to sediment in the pellet the undigested subunits which are still assembled into intermediate filaments, from proteolytic fragments liberated from the filament and therefore present in the supernatant. The intact subunits were found only in the pellet (Fig. 2B). The breakdown products of NFH were found mostly in the supernatant, whereas for NFL no fragment was solubilized by centrifugation. For NFM a similar amount of fragments was found in both the pellet and supernatant. The same investigation was performed with neurofilaments isolated from NFHLacZ transgenic mice. The degradation patterns of neurofilaments isolated from transgenic samples and the solubility of their proteolytic fragments were similar to control samples, indicating that their perikaryal retention did not affect their proteolytic conformation.

Proteolysis of neurofilaments by {alpha}-chymotrypsin
The proteolysis of mouse neurofilaments was also tested using {alpha}-chymotrypsin, another enzyme known to degrade neurofilaments. The action of this enzyme resulted in the appearance of one main fragment of 175 kDa for NFH, and one of 106 kDa for NFM (Fig. 3A). For NFL, three breakdown products were produced: one migrated very close to the intact protein (60 kDa), and two with lower molecular weights (45 kDa and 40 kDa).



View larger version (65K):
[in this window]
[in a new window]
 
Fig. 3. Immunoblot analysis of neurofilaments from control (WT) and NFHLacZ transgenic (TG) mice treated with {alpha}-chymotrypsin. (A) Isolated neurofilaments (10 µg) were treated during 20 minutes with 4 ng of {alpha}-chymotrypsin at 30°C, and then analysed by western blotting. (B) Similarly, neurofilaments (50 µg) were digested for 20 minutes at 37°C with {alpha}-chymotrypsin (2.5 ng for NFH and NFL, or 10 ng for NFM) and centrifuged for 30 minutes at 100,000 g and 4°C. Supernatants (S) and pellets (P) were separated and analysed by western blot.

 

Proteolytic fragments were also separated from the filament by centrifugation (Fig. 3B). The breakdown products of NFH were found almost only in the supernatant, whereas the fragments of NFM (106 kDa) were distributed both in the supernatant and the pellet. Finally, for NFL the intact subunit and the three breakdown products were present only in the pellet. As for trypsin, the amount of {alpha}-chymotrypsin necessary to degrade NFM was higher than for NFH and NFL (20 ng versus 4 ng), indicating an increased resistance of this subunit towards proteolysis. The proteolytic products obtained from neurofilaments isolated from NFHLacZ transgenic mice (TG) were similar to non-transgenic samples, as well as their solubility.

Proteolysis rate of neurofilaments: comparison between control and transgenic mice
As no difference was observed for the proteolytic pattern and the solubility of neurofilament fragments between normal and transgenic samples, we evaluated the rate of neurofilament proteolysis by measuring the amount of undegraded subunits following a proteolysis reaction. Following 40 minutes of proteolysis with trypsin, 84.66±15% of intact NFH was present in normal samples, whereas only 60±24% was present in transgenic samples. Following 60 minutes of {alpha}-chymotrypsin proteolysis, 41±2% of intact NFH was detected in normal samples, whereas only 19±8% was present in transgenic samples. Therefore, NFH degradation with both enzymes is more efficient in transgenic samples compared with control.

For NFM subunits, 56±8% of intact NFM was detected in control samples following a 40-minute proteolysis reaction with trypsin and only 11±1% in transgenic samples (Fig. 4A). However, using {alpha}-chymotrypsin the degradation rates of NFM were similar between transgenic and control samples (Fig. 4B).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4. Comparison of the extent of neurofilament proteolysis between control and NFHLacZ transgenic samples. Neurofilaments purified from brain were digested at 30°C during increasing times (0-60 minutes) with 4 ng trypsin (A) or {alpha}-chymotrypsin (B), and the intact subunits were revealed by western blot. Their amount was a proportion of the initial time (t=0). Means and standard deviations of a typical triplicate experiment are shown. Results for control mice are represented by black lanes and those for transgenic NFHLacZ mice by white lanes.

 

For NFL subunits, only half of the proteins present in control samples were degraded by trypsin after 40 minutes of incubation, whereas in the same conditions more than 90% was proteolyzed in transgenic samples (Fig. 4A). Similarly, a stronger degradation of NFL isolated from transgenic samples was observed using {alpha}-chymotrypsin. Together, these results indicate that neurofilaments isolated from transgenic samples are degraded more efficiently than those isolated from control samples.

Immunohistochemical detection of trypsin in control and transgenic mice
As neurofilaments isolated from transgenic mice were more susceptible to proteolysis compared with normal animals, we analysed by immunocytochemistry the subcellular distribution of trypsin using an antibody that has been previously shown to detect trypsin aggregated with neurofilament conglomerates in Amyotrophic Lateral Sclerosis (Chou et al., 1998Go; Drapkin et al., 2002Go). In control mice, trypsin was present mostly in the grey matter of spinal cord, particularly in cell bodies and axonal extensions, and a weaker immunolabeling was observed in the white matter (Fig. 5). Compared with the distribution of phosphorylated NFH subunits present massively in axons (white matter), there was a mild co-localisation between these two proteins. By contrast, in NFHLacZ transgenic mice, the majority of neurofilaments are aggregated in perikarya as previously described (Eyer and Peterson, 1994Go), together with a strong co-localisation of trypsin within these aggregates.



View larger version (76K):
[in this window]
[in a new window]
 
Fig. 5. Distribution of trypsin in the spinal cord of control and NFHLacZ transgenic mice. Frozen spinal cord sections of normal and transgenic mice were immunolabeled with anti-NFH and anti-trypsin antibodies. In transgenic samples a strong immunolocalisation of both epitopes was present in the cell bodies of motor neurons. Merged images are shown on the right.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The interval between synthesis and assembly of each neurofilament subunit is very short, and no free subunits can be detected in the cell (Black et al., 1986Go). Then, filaments are anterogradely transported to the synapse where they are degraded by calcium activated proteases (Roots, 1983Go). In NFHLacZ transgenic mice neurofilaments are sequestered in cell bodies, and therefore cannot be degraded by this pathway. This study clearly indicates that in such a situation, the amount of accumulated transcript for each subunit is unaltered, while a strong decrease of their protein level was observed (Fig. 1). Moreover, neurofilaments isolated from transgenic mice were systematically more efficiently degraded by exogenous trypsin and {alpha}-chymotrypsin than those isolated from normal animals, despite similar proteolytic patterns and solubility of their fragments (Figs 2, 3, 4).

Noticeably, similar incubation protocols but without the addition of exogenous proteases revealed no detectable proteolytic activities associated with purified neurofilaments both in transgenic and control samples, and up to two hours of incubation. This suggests that the local concentration of trypsin-like proteases in such purified neurofilaments is too low to be active. Moreover, western-blotting analysis of the crude extracts using the anti-trypsin antibody failed to detect significant difference between normal and transgenic samples (not shown). We previously showed that such neurofilaments retained in cell bodies are less phosphorylated (Fig. 1A) (Eyer and Peterson, 1994Go; Tu et al., 1997Go). Therefore, the lower phosphorylation level of neurofilaments isolated from transgenic mice could account for their increased susceptibility to degradation as previously described by Goldstein et al. (Goldstein et al., 1987Go) and Pant (Pant, 1988Go). Alternatively, the assembly of neurofilaments present in NFHLacZ transgenic mice could be looser, as indicated by their increased solubility (Riederer et al., 2003Go), and such a structural fragility of the filament could also contribute to an increased susceptibility of their degradation.

Following the degradation of neurofilaments by the addition of exogenous trypsin and {alpha}-chymotrypsin, the proteolytic profile of neurofilaments isolated from mouse tissues was similar to that reported for other species (Chin et al., 1983Go; Chin et al., 1989Go; Carden et al., 1987Go). However, it was interesting to observe a differential solubility of the proteolytic fragments. Although all degradation products from NFH were soluble following centrifugation, those from NFL were all present in the pellet with the filaments. An intermediate situation occurs for NFM (Figs 2, 3). As suggested by several studies (Carden et al., 1987Go; Chin et al., 1983Go), degradation products of NFH would correspond to the detachment of the carboxy-tail side-arms of NFH. However, the detachment of the C-terminal tail of NFM from the axis is incomplete, as similar amounts of fragments were present both in the pellet and the supernatant. This result suggests that some portion of the C-terminal domain of NFM interacts directly with the axis. Moreover, the increased susceptibility of NFL to degradation compared with NFM suggests that some parts of this subunit are particularly accessible to proteases, and not hidden by the fixation of NFM and NFH. Interestingly, no fragment of NFL was solubilized, indicating their strong interaction to form the filament.

Several human pathological situations characterised by abnormal neurofilament accumulations were found associated with strong reductions of their mRNAs levels (Bergeron et al., 1994Go; Griffin and Watson, 1988Go; Eyer et al., 1998Go; Hirano, 1991Go; Perry et al., 1991Go). Although the synthesis of neurofilaments could be seen as central to cell maintenance, growth and development, it is not clear whether such a reduction of neurofilaments is directly involved in the pathogenesis, or reflects the neuronal loss because of the disease. This study shows that in NFHLacZ transgenic mice most neurons survive during their lifetime (Eyer and Peterson, 1994Go; Tu et al., 1997Go; Eyer et al., 1998Go) despite a strong reduction of neurofilament proteins (Fig. 1). This indicates that a reduced production of neurofilaments is not detrimental for the survival of neurons. Moreover, in the light of these data the reduced levels of neurofilament transcripts observed in human pathological situations may rather indicate a loss of neurons occurring at the end stages of the pathogenesis than an active process involved in the pathogenesis.

The results of this study suggest that trypsin-like proteases could be functionally important for the degradation of neurofilaments when they are abnormally accumulated in the cell body. As a similar immunostaining of trypsin-like enzymes was observed into neurofilamentous conglomerates in Amyotrophic Lateral Sclerosis (Chou et al., 1998Go), it will be particularly crucial to determine whether such a mechanism takes place in pathological situations like Alzheimer's and Parkinson's diseases or Amyotrophic Lateral Sclerosis to regulate the turnover of neurofilaments. Moreover, it will be interesting to determine whether, and how far, this conclusion can be generalised to other transgenic models and pathological situations characterised by abnormal aggregation of intermediate filaments.


    Acknowledgments
 
We are grateful to Catherine Ferrier (ISTIA, University of Angers, Angers, France) for critical reading of the manuscript and thoughtful suggestions. We thank the Service Commun d'Imagerie et de Microscopie of the University of Angers for their expertise and advice in confocal microscopy and also the staff in the animal division. This work was supported by grants from AFM (Association Française contre les Myopathies) and ARC (Association de Recherche sur la Cancer) to J. Eyer.


    Footnotes
 
* Molecular Oncology Group, McGill University, Royal Victoria Hospital, 687 Pine Av. W, H3A1A1 Montreal, Canada. Back


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Banay-Schwartz, M., Dahl, D., Hui, K. S. and Lajtha, A. (1987). The breakdown of the individual neurofilament proteins by cathepsin D. Neurochem. Res. 12, 361-367.[Medline]

Banik, N. L., Matzelle, D. C., Gantt-Wilford, G., Osborne, A. and Hogan, E. L. (1997). Increased calpain content and progressive degradation of neurofilament protein in spinal cord injury. Brain Res. 752, 301-306.[CrossRef][Medline]

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.[Medline]

Black, M. M., Keyser, P. and Sobel, E. (1986). Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons. J. Neurosci. 6, 1004-1012.[Abstract]

Carden, M. J., Trojanowski, J. Q., Schlaepfer, W. W. and Lee, V. M. (1987). Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns. J. Neurosci. 7, 3489-3504.[Abstract]

Chin, T. K., Eagles, P. A. and Maggs, A. (1983). The proteolytic digestion of ox neurofilaments with trypsin and alpha-chymotrypsin. Biochem. J. 215, 239-252.[Medline]

Chin, T. K., Harding, S. E. and Eagles, P. A. (1989). Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH. Biochem. J. 264, 53-60.[Medline]

Chou, S., Taniguchi, A., Wang, H. S. and Festoff, B. W. (1998). Serpin-serine protease-like complexes within neurofilament conglomerates of motoneurons in amyotrophic lateral sclerosis. J. Neurol. Sci. 160, S73-S79.[CrossRef][Medline]

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.[Medline]

de Waegh, S. M., Lee, V. M. and Brady, S. T. (1992). Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells. Cell 68, 451-463.[Medline]

Drapkin, P. T., Monard, D. and Silverman, A.-J. (2002). The role of serine proteases and serine protease inhibitors in the migration of gonadotropin-releasing hormone neurons. BMC Dev. Biol. 2, 1-11.[CrossRef][Medline]

Eyer, J. and Leterrier, J. F. (1988). Influence of the phosphorylation state of neurofilament proteins on the interactions between purified filaments in vitro. Biochem. J. 252, 655-660.[Medline]

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.[Medline]

Eyer, J., Mclean, W. G. and Leterrier, J. F. (1989). Effect of a single dose of beta,beta'-iminodipropionitrile in vivo on the properties of neurofilaments in vitro: comparison with the effect of iminodipropionitrile added directly to neurofilaments in vitro. J. Neurochem. 52, 1759-1765.[Medline]

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]

Gallant, P. E., Pant, H. C., Pruss, R. M. and Gainer, H. (1986). Calcium-activated proteolysis of neurofilament proteins in the squid giant neuron. J. Neurochem. 46, 1573-1581.[Medline]

Goldstein, M. E., Sternberger, N. H. and Sternberger, L. A. (1987). Phosphorylation protects neurofilaments against proteolysis. J. Neuroimmunol. 14, 149-160.[CrossRef][Medline]

Griffin, J. W. and Watson, D. F. (1988). Axonal transport in neurological disease. Ann. Neurol. 23, 3-13.[Medline]

Gschwend, T. P., Krueger, S. R., Kozlov, S. V., Wolfer, D. P. and Sonderegger, P. (1997). Neurotrypsin, a novel multidomain serine protease expressed in the nervous system. Mol. Cell. Neurosci. 9, 207-219.[CrossRef][Medline]

Hill, W. D., Arai, M., Cohen, J. A. and Trojanowski, J. Q. (1993). Neurofilament mRNA is reduced in Parkinson's disease substantia nigra pars compacta neurons. J. Comp. Neurol. 329, 328-336.[Medline]

Hirano, A. (1991). Cytopathology of amyotrophic lateral sclerosis. Adv. Neurol. 56, 91-101.[Medline]

Hirokawa, N. (1982). Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J. Cell Biol. 94, 129-142.[Abstract/Free Full Text]

Julien, J. P., Cote, F., Beaudet, L., Sidky, M., Flavell, D., Grosveld, F. and Mushynski, W. (1988). Sequence and structure of the mouse gene coding for the largest neurofilament subunit. Gene 68, 307-314.[CrossRef][Medline]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]

Leigh, P. N., Probst, A., Dale, G. E., Power, D. P., Brion, J. P., Dodson, A. and Anderton, B. H. (1989). New aspects of the pathology of neurodegenerative disorders as revealed by ubiquitin antibodies. Acta Neuropathol. (Berl.) 79, 61-72.[Medline]

Leterrier, J. F. and Eyer, J. (1987). Properties of highly viscous gels formed by neurofilaments in vitro. A possible consequence of a specific interfilament cross-bridging. Biochem. J. 245, 93-101.[Medline]

Leterrier, J. F., Langui, D., Probst, A. and Ulrich, J. (1992). A molecular mechanism for the induction of neurofilament bundling by aluminum ions. J. Neurochem. 58, 2060-2070.[Medline]

Levy, E., Liem, R. K., D'Eustachio, P. and Cowan, N. J. (1987). Structure and evolutionary origin of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein. Eur. J. Biochem. 166, 71-77.[Abstract]

Lewis, S. A. and Cowan, N. J. (1985). Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe. J. Cell Biol. 100, 843-850.[Abstract]

Nixon, R. A. and Marotta, C. A. (1984). Degradation of neurofilament proteins by purified human brain cathepsin D. J. Neurochem. 43, 507-516.[Medline]

Nixon, R. A., Quackenbush, R. and Vitto, A. (1986). Multiple calcium-activated neutral proteinases (CANP) in mouse retinal ganglion cell neurons: specificities for endogenous neuronal substrates and comparison to purified brain CANP. J. Neurosci. 6, 1252-1263.[Abstract]

Paggi, P. and Lasek, R. J. (1984). Degradation of purified neurofilament subunits by calcium-activated neutral protease: characterization of the cleavage products. Neurochem. Int. 6, 589-597.[CrossRef]

Pant, H. C. (1988). Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain. Biochem. J. 256, 665-668.[Medline]

Perry, M. J., Lawson, S. N. and Robertson, J. (1991). Neurofilament immunoreactivity in populations of rat primary afferent neurons: a quantitative study of phosphorylated and non-phosphorylated subunits. J. Neurocytol. 20, 746-758.[Medline]

Riederer, I. M., Robert, P., Porchet, R., Eyer, J. and Riederer, B. M. (2003). Selective changes in the neurofilament and microtubule cytoskeleton of NFHLacZ mice. J. Neurosci. Res. 71, 196-207.[CrossRef][Medline]

Robert, P., Peterson, A. C. and Eyer, J. (2001). Neurofilament cytoskeleton disruption does not modify accumulation of trophic factor mRNA. J. Neurosci. Res. 64, 487-492.[CrossRef][Medline]

Roots, B. I. (1983). Neurofilament accumulation induced in synapses by leupeptin. Science 221, 971-972.[Medline]

Schlaepfer, W. W., Lee, C., Lee, V. M. and Zimmerman, U. J. (1985). An immunoblot study of neurofilament degradation in situ and during calcium-activated proteolysis. J. Neurochem. 44, 502-509.[Medline]

Scarisbrick, I. A., Isackson, P. J., Ciric, B., Windebank, A. J. and Rodriguez, M. (2001). MSP, a trypsin-like serine protease, is abundantly expressed in the human nervous system. J. Comp. Neurol. 431, 347-361.[CrossRef][Medline]

Suzuki, H., Takeda, M., Nakamura, Y., Kato, Y., Tada, K., Hariguchi, S. and Nishimura, T. (1988). Neurofilament degradation by bovine brain cathepsin D. Neurosci. Lett. 89, 240-245.[CrossRef][Medline]

Towbin, H., Staehlin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from acrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354.[Abstract]

Tu, P. H., Robinson, K. A., de Snoo, F., Eyer, J., Peterson, A., Lee, V. M. and Trojanowski, J. Q. (1997). Selective degeneration of Purkinje cells with Lewy body-like inclusions in aged NFHLacZ transgenic mice. J. Neurosci. 17, 1064-1074.[Abstract/Free Full Text]

Vitto, A. and Nixon, R. A. (1986). Calcium-activated neutral proteinase of human brain: subunit structure and enzymatic properties of multiple molecular forms. J. Neurochem. 47, 1039-1051.[Medline]

Yamashiro, K., Tsuruoika, N., Kodama, S., Tsujimoto, M., Yamamura, Y., Tanaka, T., Nakazato, H. and Yamaguchi, N. (1997). Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain. Biochim. Biophys. Acta 1350, 11-14.[Medline]





This Article
Summary
Figures Only
Full Text (PDF)
OA All Versions of this Article:
jcs.00940v1
117/6/861    most recent
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Fasani, F.
Articles by Eyer, J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Fasani, F.
Articles by Eyer, J.