Aberrant stress-induced phosphorylation of perikaryal neurofilaments.

The aberrant phosphorylation of the neurofilament high molecular weight subunit (NFH) in the neuronal perikaryon is a common feature of several neurological diseases. We demonstrated a strong correlation between hyperphosphorylation of the NFH carboxyl-terminal domain and activation of stress-activated protein kinase (SAPK) -γ in PC12 cells. Agents that activated SAPKγ in PC12 cells also caused the hyperphosphorylation of perikaryal NFH in cultured dorsal root ganglion neurons. The NFH carboxyl-terminal domain was phosphorylated by SAPKγ in vitro, and the use of peptide substrates indicated that this event occurred preferentially at KSPXE motifs. We propose that SAPKγ, perhaps in concert with other SAPKs, is involved in the abnormal phosphorylation of perikaryal NFH. This finding could lead to new insights into the etiology of several neurological diseases.

These KSP repeats represent the major phosphorylation sites in NFH and NFM (14,15), which account for the high phosphoserine content of the two proteins (7,16). The electrophoretic mobilities of NFH and NFM on SDS-polyacrylamide gels are retarded significantly by phosphorylation of the KSP repeats (16).
The knowledge that KSP repeats contain the major phosphorylation sites in NFM and NFH has focused efforts to identify the relevant kinases within the superfamily of proline-directed protein kinases. Several neuronal enzymes in this category have been shown to phosphorylate NFH in vitro. These include glycogen synthase kinase-3 (17), extracellular signal-regulated kinases (ERKs) (18,19), and cyclin-dependent kinase-5 (20,21). Since KSP motifs located in different sequence contexts are phosphorylated in vivo (15), several kinases may participate in tail domain phosphorylation, perhaps through a hierarchical mechanism (22).
The development of monoclonal antibodies that could distinguish between phosphorylated and unphosphorylated KSP repeats in the tail domains of NFH and NFM led to the discovery that axonal NFs are normally more highly phosphorylated than those located in the perikaryon and dendrites (23,24). This normal phosphorylation pattern is characteristically altered in a variety of neuropathologies, where perikaryal NFs become hyperphosphorylated (25,26).
We recently showed (27) that N-acetyl-Leu-Leu-norleucinal (CI), a potent calpain (28) and proteasome inhibitor (29,30), stimulated phosphorylation of the tail domain of NFH in neuron-like PC12 cells and in cell bodies of dorsal root ganglion (DRG) neurons. We have now found that agents known to activate stress response pathways have an effect similar to that of CI. These pathways involve proline-directed kinases of the mitogen-activated protein (MAP) kinase family, which include the ERKs, the stress-activated protein kinases (SAPKs), and p38. (31). The MAP kinases are related structurally and are activated by similar cascades in response to diverse external stimuli. The SAPK pathway responds to intra-and extracellular stress stimuli and may promote inhibition of cell growth (32,33). There is a degree of cross-talk between the different MAP kinase signaling cascades as evidenced by the activation of ERKs, SAPKs, and p38 by hyperosmolarity (34,35). However, the various MAP kinases also respond differently to certain stimuli, one example being the activation by arsenite of p38, but not the ERKs, in PC12 cells (36).
We report here that CI caused the prolonged activation of ERK-1/2 and SAPK␥. The stimulation of NFH tail domain phosphorylation in PC12 cells by various stress response activators correlated with the degree of SAPK␥ activation and the kinase phosphorylated recombinant NFH tail domain in vitro. These results suggest that stress-activated protein kinases may be responsible for the hyperphosphorylation of perikaryal NFs seen in various neuropathologies.
Cell Culture-Embryos were obtained from Sprague-Dawley rats that were previously anesthetized with ether and sacrificed by cervical dislocation. DRGs from E15 rat embryos were dissected, dissociated with trypsin, and maintained in defined medium N1 (37) containing 30 g ml Ϫ1 bovine apotransferrin, with added 0.9% bovine serum albumin, 6 ng ml Ϫ1 2.5 S NGF, and antibiotics. Culture dishes were coated with cross-linked collagen (38) in a procedure involving overnight precipitation of 50 g ml Ϫ1 collagen followed by cross-linking for 2 h at room temperature with 130 g ml Ϫ1 of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide-p-toluenesulfonate. Dissociated DRGs were plated either dispersely or in a volume of 10 l at the center of a 35-mm dish. In the latter case, the cells were allowed to attach for 30 min before flooding with defined medium. The localization of neuronal perikarya in a small centrally located region allowed them to be separated manually from the surrounding halo of neurites. Localized and dispersed DRG cultures were maintained for 19 -20 days before being used for experiments.
The PC12 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained according to published procedures (39,40).
Immunoprecipitation Kinase Assay of ERK-1/ERK-2-The assay was similar to that for SAPK␥ except for the following changes. The lysis buffer consisted of 50 mM NaCl, 5 mM EGTA, 10 mM Tris, pH 7.6, 0.2% Nonidet-P40, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, and 10 g/ml leupeptin. The kinases were immunoprecipitated with 5 l each of anti-ERK-1 (C-16) (100 g/ml) and anti-ERK-2 (C-14) (100 g/ml) polyclonal antibodies. ERK kinase buffer consisted of 30 mM Hepes, pH 7.2, 10 mM MgCl 2 , and 1 mM dithiothreitol. Assays were initiated by adding to the sedimented beads 20 l of ERK kinase buffer containing 10 g of myelin basic protein and 50 M [␥-32 P]ATP (5 Ci/mmol). The reactions were terminated after 20 min at 30°C by boiling for 5 min in SDS sample buffer. The phosphorylation of myelin basic protein was quantified by liquid scintillation counting of Coomassie Blue R250-staining protein bands excised from SDS-PAGE gels.
Gel Electrophoresis and Western Blotting-For the separate analysis of neurite and perikaryal fractions, dissociated DRGs were plated on a small area at the center of a dish as described above. This allowed for the manual separation of a central zone rich in neuronal cell bodies from the halo of neurites by using a circular punch with a diameter slightly larger than that of the neuronal cell body mass. The DRG fractions were solubilized in SDS sample buffer. PC12 cells were harvested in 2% SDS, 50 mM Tris, pH 6.8, and protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce). Equal amounts of protein were resolved on slab gels by SDS-PAGE (41). Proteins were electrophoretically transferred to Immobilon-P membrane (Millipore Corp.) in buffer containing 48 mM Tris, 39 mM glycine, and 5% methanol. The membrane was then blocked with 1% skimmed milk powder in Tris-buffered saline/Tween (20 mM Tris, pH 7.7, 137 mM NaCl, and 0.1% Tween 20), incubated with primary antibodies, and developed using the ECL Western blotting Detection Kit (Amersham Corp.).

Effect of Different Agents on NFH Phosphorylation in PC12
Cells-The effects on NFH phosphorylation of agents that are known to activate members of the MAP kinase family were assessed by Western blotting using three different monoclonal antibodies. N52 is a NFH-specific, phosphorylation-independent antibody (42) that was used to assess increases in NFH phosphorylation by detecting species with a reduced mobility on SDS-PAGE (16). SMI  of the NFH to shift to a more slowly migrating form ( Fig. 1) and resulted in the greatest increase in NFH immunoreactivity to SMI 31 and SMI 34 among the various agents that were tested. Treatment of PC12 cells with CII (30 M) for 10 h did not cause any detectable change in NFH phosphorylation. NGF, an activator of ERKs in PC12 cells (44,45), also had no noticeable effect on NFH phosphorylation (Fig. 1). Neither did treatment with anisomycin or TNF␣, two SAPK activators (46 -48).
Arsenite and osmotic shock, which have been shown to activate stress response pathways in PC12 cells (36, 49), each caused a partial shift in the mobility of NFH (Fig. 1). Increased osmolarity rendered NFH immunoreactive with both SMI 31 and SMI 34, whereas arsenite treatment produced NFH that was more immunoreactive with SMI 31. PC12 cells were treated with 0.4 M NaCl or 0.5 mM arsenite for no longer than 4 h, as they began detaching from the substratum beyond this time point. Treatment with 0.05 mM arsenite for 10 h yielded results similar to those with 0.5 mM arsenite for 4 h. The band slightly above NFH that was weakly reactive with SMI 31 antibody did not appear to be a slower migrating form of NFH, since it was not detected by N52 antibody.
SAPK␥ Activation Parallels the Increase in NFH Phosphorylation-Since only certain of the agents known to activate SAPKs caused an increase in NFH phosphorylation, we determined whether the discrepancies were due to variation in the extent of enzyme activation. Hyperosmolarity produced a relatively strong activation of SAPK␥ in PC12 cells, and the high level of activity was maintained for approximately 1 h (Fig. 2A). Arsenite produced approximately the same level of activation as hyperosmolarity, although the activation was more prolonged (Fig. 2B). The increase in NFH phosphorylation was nevertheless comparable in the two cases (Fig. 1), suggesting that toxicity may have dampened the effect of more prolonged activation by arsenite. Anisomycin produced a very modest level of SAPK␥ activation which peaked at 0.5 h (Fig. 3A). TNF␣ also caused only a modest and transient activation of SAPK␥ (Fig. 3B), similar to that reported in HeLa cells (48). CI produced a very strong and prolonged activation of SAPK␥ which lasted for at least 10 h (Fig. 3C). CII also caused a prolonged activation of SAPK␥ but at a lower level than obtained with CI (Fig. 3D).
Activation of ERK-1/2 by NGF, CI, and CII-Since ERK-2 has been implicated in the phosphorylation of NFH in vitro (18,19), we compared the effects of CI and CII on ERK-1/2 activity with that of NGF, a known ERK activator. NGF caused a prolonged activation of ERK-1/2 (Fig. 4) similar to what has already been reported (44,45). Prolonged ERK activation was also seen following CI treatment, but at a lower level than was obtained with NGF. The lowest level of ERK activation was observed with CII treatment.
Increased Phosphorylation of Perikaryal NFH in DRG Neurons-As shown in Fig. 5, CI treatment of DRG cultures produced a complete shift in the mobility of perikaryal NFH, to a level normally seen only in axonal NFH. CII, which did not cause an observable increase in NFH phosphorylation in PC12 cells, effected a partial decrease in the mobility of perikaryal NFH in DRGs. Hyperosmotic shock caused a more extensive shift in the mobility of perikaryal NFH. Arsenite treatment caused a broadening of the NFH band, whereas anisomycin treatment had no effect.
CI treatment and hyperosmotic shock promoted the staining of neuronal perikarya with SMI 34 (Fig. 6, B and D, respectively). Treatment with CII (Fig. 6C), anisomycin, or arsenite (data not shown) did not enhance the staining of neuronal perikarya with SMI 34.
Since hyperphosphorylation of perikaryal NFH and NFM has been observed following axonal damage (50), we tested whether this effect could be reproduced in vitro. The neurites of DRG neurons in localized cultures were severed from their cell bodies, and the latter were harvested at different times after injury. The mobility of NFH on SDS-PAGE was unchanged at 2 and 5 h following neurite disruption (Fig. 7). A partial decrease in mobility was seen at 12 and 24 h, which was reversed by 48 h. It is likely that the elevated phosphorylation state of perikaryal NFH that occurs in both mechanically damaged cultures and in cultures exposed to other stressing agents is due to the activation of the same or related kinase(s).
Phosphorylation of NFH and Peptides by SAPK␥-GST-NFH is a recombinant fusion protein that contains the entire tail domain of mouse NFH (amino acids 412-1087; Ref. 12) in a completely unphosphorylated state. 2 GST-NFH was phosphorylated by SAPK␥ in an immunoprecipitation kinase assay (Fig. 8A), whereas GST alone was not phosphorylated under the same conditions (data not shown). GST-NFH was not as good a SAPK␥ substrate as GST-c-Jun; 30 -35 times more 32 P was incorporated into GST-c-Jun than GST-NFH when the immunoprecipitation kinase assays were done in parallel.

DISCUSSION
In this study we have shown that treatment of PC12 cells and DRG neurons with agents that activate stress response pathways can promote the phosphorylation of KSP repeats in the tail domain of NFH. The extent to which NFH phosphorylation in PC12 cells was increased correlated with the degree of SAPK␥ activation by various agents. The strongest activator, CI, as well as hyperosmotic shock, also increased the phosphorylation of perikaryal NFH in DRG neurons. Although other stress-activated kinases, in addition to SAPK␥, may also participate in this process, the ERKs did not appear to play a 2 M. G. Sacher, unpublished results. Localized DRG cultures were prepared as described under "Experimental Procedures." The cultures were maintained for 19 -20 days and then treated as indicated above each lane. The neuronal cell bodies were manually separated from the neurites as described under "Experimental Procedures" and subjected to Western blot analysis using the anti-NFH monoclonal antibody, N52. pNFH and dpNFH refer to hyper-and hypophosphorylated NFH, respectively. The neurites were manually severed from the cell bodies using a punch with a diameter slightly larger that the cell body mass, and the cell bodies were harvested at different times indicated above each lane. The samples were subjected to Western blot analysis using the anti-NFH monoclonal antibody, N52. pNFH and dpNFH refer to hyper-and hypophosphorylated NFH, respectively. significant role in view of the failure of NGF treatment to cause a detectable increase in the phosphorylation state of NFH ( Figs. 1 and 4). In addition, treatment of PC12 cells with arsenite, which activates SAPK␥ (Fig. 2), but not ERKs (36), caused an increase in NFH phosphorylation.
The evidence linking SAPK␥ to the hyperphosphorylation of NFH is compelling. There was a strong correlation between the extent of KSP repeat phosphorylation and the degree of SAPK␥ activation in PC12 cells. Only when SAPK␥ was strongly activated, as in the case of treatment with 30 M CI, 0.4 M NaCl, or 0.5 mM arsenite, was there a detectable increase in phosphorylation of the tail domain of NFH. More modest increases in SAPK␥ activity caused by TNF␣, anisomycin, or 30 M CII were without apparent effect. However, lower levels of phosphorylation in the latter cases could have gone undetected since multiple phosphorylation events are required to cause the extensive shifts in gel electrophoretic mobility (16), and concurrent appearance of phosphoepitopes (14), that were monitored in the present study.
In addition to these suggestive correlations, we have shown that SAPK␥ phosphorylates recombinant NFH tail domain in vitro, as well as a peptide with KSPXE sequence motifs that occur in NFH. Recombinant NFH was not as good an in vitro substrate for SAPK␥ as c-Jun. This could explain the need for strong activation of SAPK␥ in PC12 cells to obtain detectable shifts in NFH mobility and the concurrent appearance of phosphoepitopes. The SAPK phosphorylation sites in the aminoterminal domain of c-Jun consist of a Ser followed by a Pro and an acidic residue (SPD or SPE) (51). Similar although not identical sequences (SPXE) in the tail domain of murine and rat NFH are relatively frequent (12,13), and SAPK␥ phosphorylated the Ser in these sequences in peptide-(601-615) (Fig.  8B). Peptide-(854 -867) was a poorer SAPK␥ substrate; perhaps the Lys residue that immediately follows Glu in the KSPEK motif has a neutralizing effect that renders the site an unsuitable substrate.
The widespread distribution of SAPKs and ERKs in the nervous system (52) provides further support for our proposal that the hyperphosphorylation of perikaryal NFs is due to the activation of stress response pathways. Other members of the MAP kinase family in addition to SAPK␥ may be similarly involved. Differential responses to a given stressing agent might occur in different types of neurons, depending on the prominence of appropriate sensing mechanisms and on relative levels of the various MAP kinases. This may be why CII stimulated NFH phosphorylation in DRG neurons but not in PC12 cells. CII also inhibits calpains and proteasomes, although the IC 50 value is approximately 5-10-fold higher than for CI (28,30). If MAP kinases other than SAPK␥ are also involved in the neuronal stress response, they would augment the action of SAPK␥, the end result being aberrant NF phosphorylation.
ERKs and SAPKs have also been implicated in the neuronal differentiation of PC12 cells (44,53). The activation by CI of both ERK-1/2 and SAPK␥ (Figs. 3C and 4) could explain its ability to induce neurite outgrowth in PC12 cells (54).
The finding that stress-activated kinases can phosphorylate perikaryal NFs has obvious clinical implications. Abnormal phosphorylation and accumulation of perikaryal NFs occur together in several neuropathologies, suggesting that the two are somehow linked (25,26). These two characteristics are seen in neurodegenerative diseases such as Alzheimer's (55), Parkinson's (56), and amyotrophic lateral sclerosis (57)(58)(59). It is possible that a stress response activator, such as oxidative stress (60), causes the premature phosphorylation of perikaryal NFs leading to their accumulation. Other forms of stress could produce similar effects, which would be consistent with the multifactorial nature of amyotrophic lateral sclerosis (61). NF accumulations in the perikaryon or proximal axon of motor neurons have been shown to block axonal transport of NFs, tubulin, actin, and mitochondria and could eventually cause axonal degeneration (62).
NF subunits are synthesized in the perikaryon and move down the axon in the slow axonal transport compartment (5) until they reach the nerve terminal, where they are degraded (63). Phosphorylation of the KSP repeat domains in NFM and NFH normally commences in the initial axon segment and continues during transport (64 -66). The notion that aberrant tail domain phosphorylation may cause NFs to accumulate in the perikaryon (25,26) is supported by axonal transport studies. There are several reports of an apparent correlation between extensive tail domain phosphorylation and a reduction in the rate of NF transport (67)(68)(69). The premature phosphorylation of KSP repeats in the perikaryon might interfere with the association between NFs and components involved in their axonal transport. Since the latter may include microtubules, the observation that NFH tail domain phosphorylation favors the dissociation of NFH from microtubules (70) could explain how aberrant NF phosphorylation might promote perikaryal accumulation.
Several recent transgenic mouse studies indicate that perturbations in NF homeostasis brought about by mutation, or overexpression of individual NF subunits, can cause pathological neurofilamentous accumulations in neuronal cell bodies (71)(72)(73)(74). Whether there is a similar causal relationship between hyperphosphorylation and accumulation of perikaryal NFs remains to be determined. The finding that some sporadic amyotrophic lateral sclerosis patients have NFH alleles with deletions in the KSP repeat domain suggests that altered phosphorylation may indeed be a cause of neurofilamentous accumulations (75,76). Our demonstration that mechanical disruption of neurites in DRG cultures caused the hyperphosphorylation of perikaryal NFs reproduced the effects of axonal injury seen in animal studies (50). Again this finding implicates stress-activated pathways, this time in response to mechanical injury of axons.
The demonstration that a SAPK(s) is involved in the phosphorylation of perikaryal NFs provides the basis for studies to determine whether aberrant phosphorylation has deleterious effects on neuronal integrity. Our findings suggest that the  1 and 2) and peptide-(854 -867) (VKSPAKEKAKSPEK) (lanes 3 and 4) at a concentration of 0.4 mM were used as substrates in the kinase assays. The peptides were resolved on a 16.5% acrylamide, 6% bisacrylamide gel using Tricine-polyacrylamide gel electrophoresis (77) and were visualized by autoradiography.
presence of hyperphosphorylated NFs in the neuronal perikaryon can serve as a marker for SAPK activation. This type of basic information may lead to a better understanding of the etiology of several neurological diseases.