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Regulation of Microglial Tyrosine Phosphorylation in Response to Neuronal Injury
Experimental Neurology 161, 297–305 (2000) doi:10.1006/exnr.1999.7257, available online at http://www.idealibrary.com on
Regulation of Microglial Tyrosine Phosphorylation in Response to Neuronal Injury Ronald Griffith, Jennie Soria, and John G. Wood Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322 Received May 17, 1999; accepted September 27, 1999
INTRODUCTION The regulation and substrate specificity of microglial phosphotyrosine (ptyr) increases accompanying motor neuron degeneration in the rat spinal cord induced by injection of the cytotoxic lectin, ricin, into sciatic nerve were examined using specific enzyme inhibitors, immunohistochemistry, and Western blot analyses. Optical density measurements of immunostained sections show that microglial ptyr levels are elevated at 3 days postinjection. This period coincides with initial stages of neuronal degeneration, and ptyr levels are maximal at 7 days. We next asked whether this increase is due to increased tyrosine kinase or decreased tyrosine phosphatase activities by assaying ptyr immunostaining in animals that received osmotic pump infusion of the nonreceptor tyrosine kinase inhibitor, herbimycin A, for the 7-day survival period. When compared to the control ventral horn, microglial ptyr on the experimental side was attenuated by at least 45% in the presence of herbimycin A. In order to identify microglial substrates undergoing increased tyrosine phosphorylation, Western blot analysis was performed on hemicord and punch biopsy samples from control and experimental sides following ricin injection. A subset of two proteins was identified whose increased ptyr was almost completely attenuated in the herbimycin-A-treated animals. We conclude that the data support earlier indications that upregulation of microglial tyrosine phosphorylation is a key early event in response to neuronal injury. Further, this upregulation is due to turning on tyrosine kinase activities, particularly nonreceptor kinases, and the end product is phosphorylation of a very limited number of substrates. This suggests the activation of specific tyrosine phosphorylation pathways, which may represent critical therapeutic intervention points, rather than a global response. The results are discussed in terms of recent cell culture models of microglial activation and earlier data demonstrating elevated microglial ptyr in neurodegenerative disease. r 2000 Academic Press Key Words: microglia; tyrosine phosphorylation; neurodegeneration; nonreceptor tyrosine kinases.
Microglial activation is one of the earliest cellular responses to brain injury. This activation is characterized by a cascade of process and soma enlargement, conversion to ameboid morphology, proliferation, expression of cell surface antigens such as major histocompatibility complex, immunoglobulin Fc receptors, 2 integrins, and the vitronectin receptor as well as various cytokines, proteases, and free radical intermediates (cf., 5, 7, 12, 17, 26, 27). The detailed nature of microglial activation is dependent upon the type of insult inflicted upon the brain (cf., 12), but the intracellular signaling events guiding the response are surprisingly poorly understood. Microglia harbor an interesting population of inwardly rectifying potassium channels which may be involved in the process (11, 14). Microglia also contain receptors for neurotransmitters (31), peptides such as calcitonin gene-related peptide as well as ATP (21, 30), to which they may respond by turning on transcriptional mechanisms (cf., 21). Although these various signaling pathways are candidates as potential players in the complex cascade of events driving microglial response to neuronal injury, there is little direct evidence indicating how these and other pathways might converge in vivo. This is in part due to the fact that much of the work involving signaling pathways has been done in cultures of microglia which lack neurons and other glial cells involved in neuronal degeneration in vivo. Further, cultured microglia are usually obtained from developing rodent brain tissue. In this case the resultant cultured cells are primarily ameboid and therefore already at least partially activated microglia rather than the ramified, ‘‘resting’’ form found in brain tissue prior to neuronal injury. Thus, a primary gap we examine here is the investigation of signaling pathways utilized in microglia responding to injury in intact neural tissue. One of the best models to study microglial response to injury is injection of the toxic lectin Ricinus communis (ricin) into peripheral nerves where it is retrogradely delivered to those neurons giving rise to axons in the nerve, leading to neuronal degeneration (cf., 23). The
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0014-4886/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.
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advantage of the model is that the blood–brain barrier is not disrupted as is the case with contusion or lesion models of CNS injury. Thus, interpretation of microglial response data is not complicated by contributions from infiltrating lymphocytes and monocytes. This model has been used to assess levels of tyrosine phosphorylation in microglia responding to neuronal degeneration in the mesencephalic trigeminal nucleus (10). Ameboid microglia in the degenerating nucleus exhibit a robust increase in staining using anti-phosphotyrosine antibodies, but, of even greater interest, ramified microglia adjacent to the nucleus also exhibit increased tyrosine phosphorylation prior to showing any other signs of activation (10). Since at least some of the ramified microglia are in the process of changing morphology to become ameboid, it may be that this upregulation of tyrosine phosphorylation is part of a switch that initiates this morphological change. Characterization of such an early step in microglial response to injury is essential to subsequent efforts directed toward identifying compounds capable of attenuating the response. Important questions in this regard include whether increased phosphotyrosine (ptyr) in microglia during injury is a result of increased tyrosine kinase or decreased tyrosine phosphatase activity. Further, the nature of the substrates involved in vivo remains elusive. We demonstrate that the increased ptyr is primarily due to enhanced activation of protein tyrosine kinase activity which results in the phosphorylation of a limited subset of microglial substrates. The results suggest that the complex microglial response to neuronal injury in vivo may be driven by activation of restricted tyrosine phosphorylation systems, amenable to further dissection, rather than a global upregulation of tyrosine phosphorylation. MATERIALS AND METHODS
Materials. The anti-phosphotyrosine antibody, PY69, and bovine serum albumin (BSA, Fraction V) were obtained from ICN Pharmaceuticals (Costa Mesa, CA). Protogel acrylamide stock solution was from National Diagnostics (Atlanta, GA). The Vectastain Elite kit was obtained from Vector Laboratories (Burlingame, CA). The PVDF membrane was from Millipore Corporation (Bedford, MA). Prestained molecular weight standards were from Bio-Rad laboratories, (Hercules, CA). The ECL reagents were from Amersham Life Science (Arlington Heights, IL). Alzet miniosmotic pumps were from Alza Corporation (Palo Alto, CA). All other chemicals and reagents were from Sigma Chemical (St. Louis, MO). Surgical procedures. Sprague–Dawley rats were anesthetized with ketamine (50 mg/kg body weight, im) diluted 1:1 in xylazine. The right sciatic nerve was exposed in the upper thigh by blunt dissection. The
nerve was ligated with a silk suture and 1 µl of a solution containing 1.5 µg of ricin lectin was injected using a 5-µl Hamilton syringe and a size 32 needle. The syringe needle (bevel up) is passed approximately 2 mm into the nerve and then withdrawn approximately 1 mm for injection. This provides space for the ricin solution and minimizes leakage from the nerve after needle withdrawal. The nerve is cut below the ligature, the ligature is removed, and the incision is closed with sutures. Immunohistochemistry. Animals under deep sodium barbiturate anesthesia were sacrificed by vascular perfusion of the PLP (paraformaldehyde, lysine, periodate) mixture of McLean and Nakane (18). Appropriate blocks of spinal cord were fixed for 3 additional h in PLP and embedded in 4% agar at 55°C. Fortymicrometer vibratome sections were cut from lumbar spinal cord segments L3 and L4 in 0.1 M phosphate buffer, pH 7.4, at 4°C. Representative sections were stained with cresyl violet to confirm motor neuron degeneration. The other sections were processed for immunohistochemistry using gentle agitation on a rotating platform. Free-floating sections were incubated with the monoclonal PY69 ptyr antibody (ICN, 1:1000) overnight at 4°C in 24-well culture dishes (Corning). The antibody was diluted in 0.01 M PBS containing 0.1% Triton X-100 and 1% normal horse serum. This was followed by incubation for 45 min at room temperature with a horse anti-mouse IgG (H&L chain) biotinylated secondary antibody which was preadsorbed against normal rat serum (Vector, at 1:250). Sections were then incubated for 45 min at room temperature with avidin–HRP complex (Vectastain elite, Standard, Vector Laboratories). The chromogenic substrate, 0.05% 3838-diaminobenzidine (Sigma), was added to 0.005% hydrogen peroxide in 0.05 M Tris buffer at pH 7.0. The reaction was stopped after 1.5–3 min by dilution with 0.01 M PBS. The sections were mounted on gelatin-coated slides, air dried overnight, and coverslipped for image analysis. A total of 137 sections from seven rats treated with ricin/herbimycin and 100 sections from six rats treated with ricin alone were analyzed. Anti-phosphotyrosine optical density (OD) was measured with the aid of a BioQuant IV image analyzer (R&M Biometrics, Nashville, TN) employing background linearity correction for the light source and optics. The spinal cord contralateral to the side of ricin injection served as the control in each tissue section measured. The average pixel transform in a uniform measurement window on the ipsilateral ricin-treated side was expressed as the percentage of change from the control side [(control ⫺ experimental/ control) ⫻ 100]. In order to minimize distribution error due to potential nonlinear relationships between transmittance and absorbance (20), transmittance was transformed to OD pixel by pixel before calculating the
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average OD, and a uniform measurement field was used for all animals. Statistical evaluation of the measurements was carried out using a two-tailed Student’s t test. Some sections were labeled with antibodies to glial fibrillary acidic protein to identify astrocytes. Other sections were labeled with fluorescent secondary antibodies in order to permit double labeling with antibodies to OX-42, CD45, or PTP SH2 to confirm the phosphotyrosine-containing cells as microglia (cf., 10). In these experiments appropriate secondary antibodies tagged with fluorescein or rhodamine were employed and visualization was by epifluorescence microscopy. Western blots. At times after ricin injection selected on the basis of the quantitative light microscopic time course of phosphotyrosine upregulation in response to ricin, unfixed tissue containing the area of degeneration as well as the corresponding area from the contralateral control side were taken for Western blot analysis of the potential substrates involved. Anesthetized rats were perfused through the left ventricle with 150 mM saline containing 1 mM sodium orthovanadate (Na3VO4) plus 15 µl of 10% PMSF and 25 KIU of aprotinin/ml to inhibit tyrosine dephosphorylation and proteolysis. Appropriate blocks of tissue were snap frozen in liquid nitrogen and sectioned into 100- to 200-µm-thick slices using a Zeiss Microm cryostat. Punch biopsies of the spinal cord ventral horn from lesioned and control sides were taken under a dissecting microscope using stainless steel punches. The tissue was homogenized in the following buffer (10 mM Hepes, pH 7.4, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Triton X, 1 mM Na3VO4, 10 µM leupeptin, and 1 mM PMSF) added 1:5 (w/v). Aliquots were taken for protein analysis using a Bradford (1) protein assay read in a Bio-Rad Model 3550-UV microplate reader. Equal amounts of total protein (20–25 µg) were loaded per lane. Sample aliquots were combined with 2⫻ sample buffer (0.05 M Tris, pH 6.8, with 30% glycerol, 2% SDS, 8% dithiothreitol, and 0.06% pyronin Y) to achieve the desired protein concentration and boiled for 5 min before being loaded onto the gel. Samples were electrophoresed using the Bio-Rad MiniProtean II cell and a 0.75-mm-thick, 10% polyacrylamide resolving gel with a 4% polyacrylamide stacking gel using the buffer system described by Laemmli (13). The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane using a Bio-Rad minitransblot electrophoretic transfer cell at 30 V for 14 h or 100 V for 1 h at 4°C in a buffer containing 25 mM Tris, 263 mM glycine, 20% methanol, and 0.1% SDS, pH 8.3. After transfer of proteins, the membranes were incubated in blocking solution (5% BSA, Fraction V), in Tris-buffered saline (TBS) for 1 h and then incubated for 1 h in the same PY69 antibody used for the
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immunohistochemical analysis diluted in TBS/0.1% Tween 20 (TTBS). After four 10-min washes in TTBS, membranes were processed essentially as described for the tissue sections and reaction product was quantified using a Bio-Rad Model GS 670 densitometer. Molecular weights of immunoblotted proteins were determined using a standard mixture of prestained proteins (BioRad) that are electrophoresed and transblotted along with the other samples. Linear regression analysis was used to determine molecular weights. Tyrosine kinase inhibition. Rats were anesthetized as described above and, under aseptic conditions, a miniosmotic pump (Alzet, Model 2001) was implanted subcutaneously for inhibitor delivery. Ricin injections were performed either at that time or between 1 and 24 h after introduction of the pump. Herbimycin A (Kamiya Biomedical) was dissolved in dimethyl sulfoxide at a concentration of 2 mg/ml and diluted 1:1 with sterile saline immediately before loading into the osmotic pumps. The pumps were incubated at 37°C for 4 h prior to implantation to assure that the drug was being delivered at the time of implantation. Each pump delivers 1 µg/h of inhibitor for up to 7 days. In all cases a bolus of inhibitor (100 µg) was introduced at the time of pump implantation by a single subcutaneous injection to establish a circulating concentration of drug. The time course data for phosphotyrosine staining after ricin injury were used to determine the period of maximal response and animals were sacrificed at this time (7 days) and prepared for immunohistochemical and Western blot analyses exactly as described above. RESULTS
The ricin-induced neuronal degeneration model employed in these studies is illustrated in Fig. 1A. Phosphotyrosine-positive microglia reside throughout the gray and white matter of the lumbar ventral horn and some of these are closely associated with motor neurons (Fig. 1B). Injection of ricin into the sciatic nerve results in motor neuron degeneration in the posterior and gastrocnemius motor columns in lumbar segments three and four beginning 3 days after ricin injection. Motor neuron loss is complete within 7 days of ricin injection (Fig. 2). Ricin-induced motor neuron degeneration initiates a microglial reaction characterized by proliferation and increased phosphotyrosine immunostaining (Fig. 2B). The increase in microglial ptyr begins between 3 and 5 days after ricin injection at the time when motor neurons begin to degenerate. A marked increase in microglial ptyr is clearly visible 7 days after ricin injection (Fig. 3). Microglia occupying the contralateral uninjured lumbar ventral horn do not exhibit degeneration or increased ptyr staining. Since the blood–brain barrier is not physically disrupted, no
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FIG. 1. The cytotoxic lectin from Ricinus communis (castor bean) is injected into the sciatic nerve in the flank and the nerve is cut distal to the injection site (A). Ricin is retrogradely transported through both motor and dorsal root ganglion (DRG) axons. The microglial reaction is elicited by motor neuron death in the ventral horn and degeneration of central processes of DRG neurons in the dorsal horn. Motor neuron death occurs approximately 72 h after ricin injection and nerve cut. (B) Light micrograph showing a phosphotyrosinepositive microglial cell in close association with a motor neuron (MN) and blood vessel (BV).
infiltration of blood-borne monocytes or macrophages is seen in cresyl-violet-stained sections (Fig. 2). Continuous infusion of herbimycin A was used to test effects of protein tyrosine kinase inhibition on the microglial ptyr response to ricin-induced motor neuron degeneration. The robust increase in microglial ptyr immunostaining after motor neuron degeneration (Fig. 4B) was attenuated by herbimycin A and is clearly visible in the immunostained sections (Fig. 4D). There was no apparent change in phosphotyrosine staining on the control side of herbimycin-treated animals (Fig. 4C) compared to the control side of those animals that had been treated with ricin alone (compare Figs. 4A and
4C). Immunostaining was quantified using computerassisted image analysis. In order to compensate for interanimal variability and variability in immunostained sections, OD measurements were made from the contralateral uninjured ventral horn of each section and compared with the OD measurement from the injured side of the same section. The percentage of change in OD was expressed as the percentage of change between the two sides and was calculated using the following formula: %OD change ⫽ [(control ⫺ experimental)/control] ⫻ 100. The mean OD increase in seven rats treated with only ricin and the mean OD increase in six rats treated with herbimycin A and ricin were calculated. The result was a statistically significant (P ⬍ 0.001) difference in mean OD change between the two groups of rats with approximately 45% attenuation of the microglial ptyr response in response to herbimycin A. In order to address the issue that herbimycin might be affecting cell division, we used the microglial marker OX-42 to assess microglial numbers. There was no difference in this staining of ricin/herbimycintreated animals compared to ricin-treated animals (data not shown). Tissue section immunohistochemical results were verified and extended using SDS–PAGE. Tissue punches from uninjured and injured lumbar ventral horn were collected from a separate group of ricin and ricin ⫹ herbimycin-A-treated rats. Equal amounts of total protein were run in adjacent lanes, transferred to PVDF membrane, and immunostained with the same ptyr antibody used in tissue section immunohistochemistry. It is important to note that we have previously characterized this antibody as being microglial specific in normal and degenerating rat tissue (10). Several bands were visible on the autoradiograms with changes occurring predominately in two proteins with molecular weights of 52 and 57 kDa. Phosphotyrosine was clearly upregulated in these proteins in tissue isolated from the injured ventral horn. Continuous infusion of herbimycin A attenuated this response. In fact, the Western blot analyses consistently showed a return of ptyr labeling of these bands to levels indistinguishable from control levels (Fig. 5). DISCUSSION
We have previously demonstrated that phosphotyrosine levels in microglia were apparently increased in response to neuronal injury (10). This is not just the result of an increased microglial proliferation because individual microglia on the injured side, including some still in the ramified state, show robust elevation of phosphotyrosine compared to those on the control side (10). In this study we have systematically examined the time course, enzymatic activities, and substrates involved in generating the increased phosphotyrosine.
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FIG. 2. Light micrographs of cresyl-violet-stained sections from the lumbar spinal cord of a rat 7 days after ricin injection and nerve cut. The notch in the ventral lateral white matter indicates the control uninjured side (*). Motor neurons in the ventral horn have degenerated on the right (ricin-treated) side (A). Immunostaining for phosphotyrosine reveals an intense upregulation of tyrosine phosphorylation in the region of motor neuron degeneration. Higher magnification images of the cresyl-violet-stained sections show motor neurons in the control ventral horn (C) and the ricin-treated ventral horn (D). The apparent increase in small nuclei in D is correlated with the increased staining of microglial phosphotyrosine in B. Bar in A, 1 mm. Bar in D, 50 µm.
Phosphotyrosine comprises less than one-tenth of a percent of all normal cellular phosphoamino acids (22). The first tyrosine kinase identified, pp60v-src (8), is a 60-kDa phosphoprotein product of the transforming gene, v-src, of the Rous sarcoma virus which infects chickens. As the ability of the virus to transform its host cell depends on tyrosine phosphorylation (22), it was postulated that viral tyrosine kinases either directly or indirectly modify key regulators of cellular growth. Interestingly, the retroviral oncogenes encoding tyrosine kinases originated from cellular genes that were coopted by viruses and subsequently accumulated minor alterations (cf., 24). There are both receptor- and nonreceptor-associated tyrosine kinases and recent work from a number of laboratories implicates tyrosine kinase systems as playing key roles in signal transduction mechanisms as well as regulation of critical stages
in the cell cycle (cf., 3, 4, 9). It has also recently been recognized that protein tyrosine phosphatases are at least as important as the kinase limb of these pathways (cf., 19, 25). Despite this progress in assigning functional roles for tyrosine phosphorylation in a variety of cell types, there is very little understanding of the functional role of tyrosine phosphorylation in neuronal response to injury as well as which enzymes and substrates are critical to the process. Since there are many interactions between glial cells in the normal and injured CNS, the choice of injury model used to study a given glial cell type is critical. This is particularly true of microglia since the immunohistochemical markers used to label them also label blood-borne cells such as macrophages and monocytes. For this reason, we chose an injury model which does not cause direct or invasive trauma to the CNS with
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FIG. 3. Time course of phosphotyrosine upregulation in spinal cord ventral horn after ricin injection into sciatic nerve. Optical density (OD) measurements are made through the control and ricin-treated sides of immunostained multiple sections from at least two animals for each survival time. The mean change in OD between sides is expressed as a percentage of increase in OD. Bars represent mean ⫾SEM. The upregulation of ptyr immunostaining reaches maximal levels at 7 days after ricin injection.
subsequent infiltration of blood-borne elements. Injection of the dimeric protein ricin into the peripheral sciatic nerve results in a lesion caused by motor neuron degeneration in the lumbar spinal cord. This injury model leaves the blood–brain barrier intact and allows a clear assessment of the microglial reaction without the complication of distinguishing resident microglia from macrophages or monocytes. We were therefore unambiguously able to show that the elevated microglial phosphotyrosine response in the spinal cord ventral horn is staged approximately 3 days after the ricin injection. This time point coincides with the earliest signs of motor neuron degeneration. These results in spinal cord, together with those of our previous studies in cranial nerve nuclei (10), strongly support a role for tyrosine phosphorylation in the activation of microglia in response to CNS injury in vivo. We next asked whether specific microglial substrates exhibiting elevated levels of phosphotyrosine could be identified in punch biopsy samples of spinal cord ventral horn from ricin or control animals. Somewhat surprisingly, only two bands of approximately 52 and 57 kDa consistently showed elevated phosphotyrosine levels although the degree of this increase was striking. This result suggests that the complicated microglial response to neuronal injury may be driven, at least in part, by activation of a limited number of tyrosine
phosphorylation pathways which should be amenable to further dissection using the ricin injury model. The increased microglial phosphotyrosine following neuronal injury could be due either to increased tyrosine kinase activity or to decreased tyrosine phosphatase activity. Therefore, we tested the hypothesis that continuous infusion of the tyrosine kinase inhibitor herbimycin A attenuates increased microglial phosphotyrosine in response to CNS injury. The results indicate significant attenuation of the microglial phosphotyrosine signal globally, as demonstrated by immunohistochemical analysis, and specifically in a subset of tyrosine kinase substrates demonstrated by Western blot analysis in which the elevated phosphotyrosine returns essentially to control values in herbimycin-treated rats. In our hands Western blot analysis has consistently yielded more reliable quantitative results than immunohistochemical analysis. Further, the immunohistochemical analysis was performed to include the entire ventral horn, whereas the punch biopsy procedure was limited to the area of maximal neuronal degeneration. Thus, while we cannot rule out the possibility that the portion of immunohistochemical levels of phosphotyrosine not attenuated globally in ricin-treated animals represents additional tyrosine kinase or phosphatase pathways, we believe that the Western blot results may be most relevant to the actual in vivo events accompanying ricin-induced elevations of microglial phosphotyrosine. Herbimycin A is a benzenoid ansamycin antibiotic protein tyrosine kinase inhibitor isolated from the broth of Streptomyces cultures. Although first isolated for its herbicidal activity, it was subsequently found to reduce phosphotyrosine content in RSV-transformed cell types. Herbimycin A irreversibly inhibits the nonreceptor or cytosolic protein tyrosine kinases. The mechanism of action of herbimycin A is through binding of reactive sulfhydryl groups on or near the active site of the tyrosine kinase. Support for this mechanism comes from studies using sulfhydryl compounds to block the effects of herbimycin A (28, 29). Herbimycin A does not inhibit the action of PKA or PKC (6). Based on this specificity we conclude that the bulk of ricin-induced increases in microglial phosphotyrosine is due to the action of nonreceptor tyrosine kinase activities. This is particularly true for the 52- and 57-kDa bands identified by Western blot analysis. In terms of downstream events which may be relevant to the response of microglia to injury are the recent observations that herbimycin A is effective in inhibiting lipopolysaccharide interferon-␥-induced nitric oxide expression in a cloned microglial cell line (15). In two interesting and related papers signal transduction pathways in cultured microglia responding to amyloid fibrils and prion proteins were examined (2, 16). In both cases tyrosine-kinase-dependent pathways
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FIG. 4. Light micrographs of ptyr immunostaining in control and ricin-treated ventral horns of non-herbimycin (A, B) and herbimycin (C, D) treated rats. The increase in ptyr immunostaining in response to ricin (B) is significantly attenuated in the rats receiving herbimycin infusions (D). Bar in D, 50 µm.
were rapidly activated resulting in the phosphorylation of specific substrates. In addition it was shown the Lyn, Syk, FAK, and the recently described calcium-sensitive tyrosine kinase PYK2 were activated in response to amyloid fibrils or prion proteins. It is difficult to directly compare our results to the results of McDonald et al. (16) and Combs et al. (2) for several reasons: (1) with the possible exception of PYK2, the tyrosine kinases involved in their studies are primarily receptorassociated whereas the increased microglial tyrosine phosphorylation we observe is primarily the result of nonreceptor tyrosine phosphorylation, at least over a sustained time course; (2) with the exception of one 48-h time course employing immunohistochemical analysis, their results were examining rapid induction of tyrosine phosphorylation, and the substrates identified at these early time points were apparently of higher apparent molecular weight than those identified in our studies; (3) their studies employed cultured
microglia which are primarily ameboid and already activated, whereas we examined the response of resting ramified microglia in the presence of a full complement of other neural cells. We believe that the results shown here are reflective of the response of microglia to neuronal injury in their native environment, and the sustained elevation of phosphotyrosine levels in a limited number of substrates may be directly relevant to sustained levels of phosphotyrosine in microglia associated with neuritic plaques in Alzheimer’s disease (unpublished observations; 32). This is not to say that the pathogenesis is identical, only that sustained elevated levels of microglial phosphotyrosine are characteristic of both processes. Future studies of these pathways will include use of the ricin injury model together with infusion of inhibitors or activators of specific tyrosine kinases or components either up- or downstream of the tyrosine phosphorylation events, together with immunoprecipitation of punch biopsies of ricin-
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FIG. 5. (Left) Cresyl-violet-stained section from the lumbar segment of a rat spinal cord illustrating the location of tissue punches which are used for immunoblotting. A 1-mm-diameter tissue punch is used to collect biopsies from fresh spinal cord ventral horn. Thirty micrograms of protein from punch biopsies is loaded into each lane and subjected to SDS–PAGE and Western blotting for ptyr immunostaining. Increased staining of two bands at 52 and 57 kDa (arrows) is apparent in the ventral horns (two left lanes) of ricin-treated rats (VHE) compared to the control side (VHC). In the presence of herbimycin (two right lanes), this increase is essentially completely attenuated (VHE) compared to the control side (VHC).
treated animals and preparative gel electrophoresis to obtain sufficient quantities of the 52- and 57-kDa substrates for sequence analysis.
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ACKNOWLEDGMENTS
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Supported by NIH grant AG11123 and the University Research Committee. We thank Dr. Henry Tomasiewicz and Denise Flaherty for helpful discussions.
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Regulation of Microglial Tyrosine Phosphorylation in Response to Neuronal Injury Ronald Griffith, Jennie Soria, and John G. Wood Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322 Received May 17, 1999; accepted September 27, 1999
INTRODUCTION The regulation and substrate specificity of microglial phosphotyrosine (ptyr) increases accompanying motor neuron degeneration in the rat spinal cord induced by injection of the cytotoxic lectin, ricin, into sciatic nerve were examined using specific enzyme inhibitors, immunohistochemistry, and Western blot analyses. Optical density measurements of immunostained sections show that microglial ptyr levels are elevated at 3 days postinjection. This period coincides with initial stages of neuronal degeneration, and ptyr levels are maximal at 7 days. We next asked whether this increase is due to increased tyrosine kinase or decreased tyrosine phosphatase activities by assaying ptyr immunostaining in animals that received osmotic pump infusion of the nonreceptor tyrosine kinase inhibitor, herbimycin A, for the 7-day survival period. When compared to the control ventral horn, microglial ptyr on the experimental side was attenuated by at least 45% in the presence of herbimycin A. In order to identify microglial substrates undergoing increased tyrosine phosphorylation, Western blot analysis was performed on hemicord and punch biopsy samples from control and experimental sides following ricin injection. A subset of two proteins was identified whose increased ptyr was almost completely attenuated in the herbimycin-A-treated animals. We conclude that the data support earlier indications that upregulation of microglial tyrosine phosphorylation is a key early event in response to neuronal injury. Further, this upregulation is due to turning on tyrosine kinase activities, particularly nonreceptor kinases, and the end product is phosphorylation of a very limited number of substrates. This suggests the activation of specific tyrosine phosphorylation pathways, which may represent critical therapeutic intervention points, rather than a global response. The results are discussed in terms of recent cell culture models of microglial activation and earlier data demonstrating elevated microglial ptyr in neurodegenerative disease. r 2000 Academic Press Key Words: microglia; tyrosine phosphorylation; neurodegeneration; nonreceptor tyrosine kinases.
Microglial activation is one of the earliest cellular responses to brain injury. This activation is characterized by a cascade of process and soma enlargement, conversion to ameboid morphology, proliferation, expression of cell surface antigens such as major histocompatibility complex, immunoglobulin Fc receptors, 2 integrins, and the vitronectin receptor as well as various cytokines, proteases, and free radical intermediates (cf., 5, 7, 12, 17, 26, 27). The detailed nature of microglial activation is dependent upon the type of insult inflicted upon the brain (cf., 12), but the intracellular signaling events guiding the response are surprisingly poorly understood. Microglia harbor an interesting population of inwardly rectifying potassium channels which may be involved in the process (11, 14). Microglia also contain receptors for neurotransmitters (31), peptides such as calcitonin gene-related peptide as well as ATP (21, 30), to which they may respond by turning on transcriptional mechanisms (cf., 21). Although these various signaling pathways are candidates as potential players in the complex cascade of events driving microglial response to neuronal injury, there is little direct evidence indicating how these and other pathways might converge in vivo. This is in part due to the fact that much of the work involving signaling pathways has been done in cultures of microglia which lack neurons and other glial cells involved in neuronal degeneration in vivo. Further, cultured microglia are usually obtained from developing rodent brain tissue. In this case the resultant cultured cells are primarily ameboid and therefore already at least partially activated microglia rather than the ramified, ‘‘resting’’ form found in brain tissue prior to neuronal injury. Thus, a primary gap we examine here is the investigation of signaling pathways utilized in microglia responding to injury in intact neural tissue. One of the best models to study microglial response to injury is injection of the toxic lectin Ricinus communis (ricin) into peripheral nerves where it is retrogradely delivered to those neurons giving rise to axons in the nerve, leading to neuronal degeneration (cf., 23). The
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advantage of the model is that the blood–brain barrier is not disrupted as is the case with contusion or lesion models of CNS injury. Thus, interpretation of microglial response data is not complicated by contributions from infiltrating lymphocytes and monocytes. This model has been used to assess levels of tyrosine phosphorylation in microglia responding to neuronal degeneration in the mesencephalic trigeminal nucleus (10). Ameboid microglia in the degenerating nucleus exhibit a robust increase in staining using anti-phosphotyrosine antibodies, but, of even greater interest, ramified microglia adjacent to the nucleus also exhibit increased tyrosine phosphorylation prior to showing any other signs of activation (10). Since at least some of the ramified microglia are in the process of changing morphology to become ameboid, it may be that this upregulation of tyrosine phosphorylation is part of a switch that initiates this morphological change. Characterization of such an early step in microglial response to injury is essential to subsequent efforts directed toward identifying compounds capable of attenuating the response. Important questions in this regard include whether increased phosphotyrosine (ptyr) in microglia during injury is a result of increased tyrosine kinase or decreased tyrosine phosphatase activity. Further, the nature of the substrates involved in vivo remains elusive. We demonstrate that the increased ptyr is primarily due to enhanced activation of protein tyrosine kinase activity which results in the phosphorylation of a limited subset of microglial substrates. The results suggest that the complex microglial response to neuronal injury in vivo may be driven by activation of restricted tyrosine phosphorylation systems, amenable to further dissection, rather than a global upregulation of tyrosine phosphorylation. MATERIALS AND METHODS
Materials. The anti-phosphotyrosine antibody, PY69, and bovine serum albumin (BSA, Fraction V) were obtained from ICN Pharmaceuticals (Costa Mesa, CA). Protogel acrylamide stock solution was from National Diagnostics (Atlanta, GA). The Vectastain Elite kit was obtained from Vector Laboratories (Burlingame, CA). The PVDF membrane was from Millipore Corporation (Bedford, MA). Prestained molecular weight standards were from Bio-Rad laboratories, (Hercules, CA). The ECL reagents were from Amersham Life Science (Arlington Heights, IL). Alzet miniosmotic pumps were from Alza Corporation (Palo Alto, CA). All other chemicals and reagents were from Sigma Chemical (St. Louis, MO). Surgical procedures. Sprague–Dawley rats were anesthetized with ketamine (50 mg/kg body weight, im) diluted 1:1 in xylazine. The right sciatic nerve was exposed in the upper thigh by blunt dissection. The
nerve was ligated with a silk suture and 1 µl of a solution containing 1.5 µg of ricin lectin was injected using a 5-µl Hamilton syringe and a size 32 needle. The syringe needle (bevel up) is passed approximately 2 mm into the nerve and then withdrawn approximately 1 mm for injection. This provides space for the ricin solution and minimizes leakage from the nerve after needle withdrawal. The nerve is cut below the ligature, the ligature is removed, and the incision is closed with sutures. Immunohistochemistry. Animals under deep sodium barbiturate anesthesia were sacrificed by vascular perfusion of the PLP (paraformaldehyde, lysine, periodate) mixture of McLean and Nakane (18). Appropriate blocks of spinal cord were fixed for 3 additional h in PLP and embedded in 4% agar at 55°C. Fortymicrometer vibratome sections were cut from lumbar spinal cord segments L3 and L4 in 0.1 M phosphate buffer, pH 7.4, at 4°C. Representative sections were stained with cresyl violet to confirm motor neuron degeneration. The other sections were processed for immunohistochemistry using gentle agitation on a rotating platform. Free-floating sections were incubated with the monoclonal PY69 ptyr antibody (ICN, 1:1000) overnight at 4°C in 24-well culture dishes (Corning). The antibody was diluted in 0.01 M PBS containing 0.1% Triton X-100 and 1% normal horse serum. This was followed by incubation for 45 min at room temperature with a horse anti-mouse IgG (H&L chain) biotinylated secondary antibody which was preadsorbed against normal rat serum (Vector, at 1:250). Sections were then incubated for 45 min at room temperature with avidin–HRP complex (Vectastain elite, Standard, Vector Laboratories). The chromogenic substrate, 0.05% 3838-diaminobenzidine (Sigma), was added to 0.005% hydrogen peroxide in 0.05 M Tris buffer at pH 7.0. The reaction was stopped after 1.5–3 min by dilution with 0.01 M PBS. The sections were mounted on gelatin-coated slides, air dried overnight, and coverslipped for image analysis. A total of 137 sections from seven rats treated with ricin/herbimycin and 100 sections from six rats treated with ricin alone were analyzed. Anti-phosphotyrosine optical density (OD) was measured with the aid of a BioQuant IV image analyzer (R&M Biometrics, Nashville, TN) employing background linearity correction for the light source and optics. The spinal cord contralateral to the side of ricin injection served as the control in each tissue section measured. The average pixel transform in a uniform measurement window on the ipsilateral ricin-treated side was expressed as the percentage of change from the control side [(control ⫺ experimental/ control) ⫻ 100]. In order to minimize distribution error due to potential nonlinear relationships between transmittance and absorbance (20), transmittance was transformed to OD pixel by pixel before calculating the
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average OD, and a uniform measurement field was used for all animals. Statistical evaluation of the measurements was carried out using a two-tailed Student’s t test. Some sections were labeled with antibodies to glial fibrillary acidic protein to identify astrocytes. Other sections were labeled with fluorescent secondary antibodies in order to permit double labeling with antibodies to OX-42, CD45, or PTP SH2 to confirm the phosphotyrosine-containing cells as microglia (cf., 10). In these experiments appropriate secondary antibodies tagged with fluorescein or rhodamine were employed and visualization was by epifluorescence microscopy. Western blots. At times after ricin injection selected on the basis of the quantitative light microscopic time course of phosphotyrosine upregulation in response to ricin, unfixed tissue containing the area of degeneration as well as the corresponding area from the contralateral control side were taken for Western blot analysis of the potential substrates involved. Anesthetized rats were perfused through the left ventricle with 150 mM saline containing 1 mM sodium orthovanadate (Na3VO4) plus 15 µl of 10% PMSF and 25 KIU of aprotinin/ml to inhibit tyrosine dephosphorylation and proteolysis. Appropriate blocks of tissue were snap frozen in liquid nitrogen and sectioned into 100- to 200-µm-thick slices using a Zeiss Microm cryostat. Punch biopsies of the spinal cord ventral horn from lesioned and control sides were taken under a dissecting microscope using stainless steel punches. The tissue was homogenized in the following buffer (10 mM Hepes, pH 7.4, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Triton X, 1 mM Na3VO4, 10 µM leupeptin, and 1 mM PMSF) added 1:5 (w/v). Aliquots were taken for protein analysis using a Bradford (1) protein assay read in a Bio-Rad Model 3550-UV microplate reader. Equal amounts of total protein (20–25 µg) were loaded per lane. Sample aliquots were combined with 2⫻ sample buffer (0.05 M Tris, pH 6.8, with 30% glycerol, 2% SDS, 8% dithiothreitol, and 0.06% pyronin Y) to achieve the desired protein concentration and boiled for 5 min before being loaded onto the gel. Samples were electrophoresed using the Bio-Rad MiniProtean II cell and a 0.75-mm-thick, 10% polyacrylamide resolving gel with a 4% polyacrylamide stacking gel using the buffer system described by Laemmli (13). The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane using a Bio-Rad minitransblot electrophoretic transfer cell at 30 V for 14 h or 100 V for 1 h at 4°C in a buffer containing 25 mM Tris, 263 mM glycine, 20% methanol, and 0.1% SDS, pH 8.3. After transfer of proteins, the membranes were incubated in blocking solution (5% BSA, Fraction V), in Tris-buffered saline (TBS) for 1 h and then incubated for 1 h in the same PY69 antibody used for the
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immunohistochemical analysis diluted in TBS/0.1% Tween 20 (TTBS). After four 10-min washes in TTBS, membranes were processed essentially as described for the tissue sections and reaction product was quantified using a Bio-Rad Model GS 670 densitometer. Molecular weights of immunoblotted proteins were determined using a standard mixture of prestained proteins (BioRad) that are electrophoresed and transblotted along with the other samples. Linear regression analysis was used to determine molecular weights. Tyrosine kinase inhibition. Rats were anesthetized as described above and, under aseptic conditions, a miniosmotic pump (Alzet, Model 2001) was implanted subcutaneously for inhibitor delivery. Ricin injections were performed either at that time or between 1 and 24 h after introduction of the pump. Herbimycin A (Kamiya Biomedical) was dissolved in dimethyl sulfoxide at a concentration of 2 mg/ml and diluted 1:1 with sterile saline immediately before loading into the osmotic pumps. The pumps were incubated at 37°C for 4 h prior to implantation to assure that the drug was being delivered at the time of implantation. Each pump delivers 1 µg/h of inhibitor for up to 7 days. In all cases a bolus of inhibitor (100 µg) was introduced at the time of pump implantation by a single subcutaneous injection to establish a circulating concentration of drug. The time course data for phosphotyrosine staining after ricin injury were used to determine the period of maximal response and animals were sacrificed at this time (7 days) and prepared for immunohistochemical and Western blot analyses exactly as described above. RESULTS
The ricin-induced neuronal degeneration model employed in these studies is illustrated in Fig. 1A. Phosphotyrosine-positive microglia reside throughout the gray and white matter of the lumbar ventral horn and some of these are closely associated with motor neurons (Fig. 1B). Injection of ricin into the sciatic nerve results in motor neuron degeneration in the posterior and gastrocnemius motor columns in lumbar segments three and four beginning 3 days after ricin injection. Motor neuron loss is complete within 7 days of ricin injection (Fig. 2). Ricin-induced motor neuron degeneration initiates a microglial reaction characterized by proliferation and increased phosphotyrosine immunostaining (Fig. 2B). The increase in microglial ptyr begins between 3 and 5 days after ricin injection at the time when motor neurons begin to degenerate. A marked increase in microglial ptyr is clearly visible 7 days after ricin injection (Fig. 3). Microglia occupying the contralateral uninjured lumbar ventral horn do not exhibit degeneration or increased ptyr staining. Since the blood–brain barrier is not physically disrupted, no
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FIG. 1. The cytotoxic lectin from Ricinus communis (castor bean) is injected into the sciatic nerve in the flank and the nerve is cut distal to the injection site (A). Ricin is retrogradely transported through both motor and dorsal root ganglion (DRG) axons. The microglial reaction is elicited by motor neuron death in the ventral horn and degeneration of central processes of DRG neurons in the dorsal horn. Motor neuron death occurs approximately 72 h after ricin injection and nerve cut. (B) Light micrograph showing a phosphotyrosinepositive microglial cell in close association with a motor neuron (MN) and blood vessel (BV).
infiltration of blood-borne monocytes or macrophages is seen in cresyl-violet-stained sections (Fig. 2). Continuous infusion of herbimycin A was used to test effects of protein tyrosine kinase inhibition on the microglial ptyr response to ricin-induced motor neuron degeneration. The robust increase in microglial ptyr immunostaining after motor neuron degeneration (Fig. 4B) was attenuated by herbimycin A and is clearly visible in the immunostained sections (Fig. 4D). There was no apparent change in phosphotyrosine staining on the control side of herbimycin-treated animals (Fig. 4C) compared to the control side of those animals that had been treated with ricin alone (compare Figs. 4A and
4C). Immunostaining was quantified using computerassisted image analysis. In order to compensate for interanimal variability and variability in immunostained sections, OD measurements were made from the contralateral uninjured ventral horn of each section and compared with the OD measurement from the injured side of the same section. The percentage of change in OD was expressed as the percentage of change between the two sides and was calculated using the following formula: %OD change ⫽ [(control ⫺ experimental)/control] ⫻ 100. The mean OD increase in seven rats treated with only ricin and the mean OD increase in six rats treated with herbimycin A and ricin were calculated. The result was a statistically significant (P ⬍ 0.001) difference in mean OD change between the two groups of rats with approximately 45% attenuation of the microglial ptyr response in response to herbimycin A. In order to address the issue that herbimycin might be affecting cell division, we used the microglial marker OX-42 to assess microglial numbers. There was no difference in this staining of ricin/herbimycintreated animals compared to ricin-treated animals (data not shown). Tissue section immunohistochemical results were verified and extended using SDS–PAGE. Tissue punches from uninjured and injured lumbar ventral horn were collected from a separate group of ricin and ricin ⫹ herbimycin-A-treated rats. Equal amounts of total protein were run in adjacent lanes, transferred to PVDF membrane, and immunostained with the same ptyr antibody used in tissue section immunohistochemistry. It is important to note that we have previously characterized this antibody as being microglial specific in normal and degenerating rat tissue (10). Several bands were visible on the autoradiograms with changes occurring predominately in two proteins with molecular weights of 52 and 57 kDa. Phosphotyrosine was clearly upregulated in these proteins in tissue isolated from the injured ventral horn. Continuous infusion of herbimycin A attenuated this response. In fact, the Western blot analyses consistently showed a return of ptyr labeling of these bands to levels indistinguishable from control levels (Fig. 5). DISCUSSION
We have previously demonstrated that phosphotyrosine levels in microglia were apparently increased in response to neuronal injury (10). This is not just the result of an increased microglial proliferation because individual microglia on the injured side, including some still in the ramified state, show robust elevation of phosphotyrosine compared to those on the control side (10). In this study we have systematically examined the time course, enzymatic activities, and substrates involved in generating the increased phosphotyrosine.
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FIG. 2. Light micrographs of cresyl-violet-stained sections from the lumbar spinal cord of a rat 7 days after ricin injection and nerve cut. The notch in the ventral lateral white matter indicates the control uninjured side (*). Motor neurons in the ventral horn have degenerated on the right (ricin-treated) side (A). Immunostaining for phosphotyrosine reveals an intense upregulation of tyrosine phosphorylation in the region of motor neuron degeneration. Higher magnification images of the cresyl-violet-stained sections show motor neurons in the control ventral horn (C) and the ricin-treated ventral horn (D). The apparent increase in small nuclei in D is correlated with the increased staining of microglial phosphotyrosine in B. Bar in A, 1 mm. Bar in D, 50 µm.
Phosphotyrosine comprises less than one-tenth of a percent of all normal cellular phosphoamino acids (22). The first tyrosine kinase identified, pp60v-src (8), is a 60-kDa phosphoprotein product of the transforming gene, v-src, of the Rous sarcoma virus which infects chickens. As the ability of the virus to transform its host cell depends on tyrosine phosphorylation (22), it was postulated that viral tyrosine kinases either directly or indirectly modify key regulators of cellular growth. Interestingly, the retroviral oncogenes encoding tyrosine kinases originated from cellular genes that were coopted by viruses and subsequently accumulated minor alterations (cf., 24). There are both receptor- and nonreceptor-associated tyrosine kinases and recent work from a number of laboratories implicates tyrosine kinase systems as playing key roles in signal transduction mechanisms as well as regulation of critical stages
in the cell cycle (cf., 3, 4, 9). It has also recently been recognized that protein tyrosine phosphatases are at least as important as the kinase limb of these pathways (cf., 19, 25). Despite this progress in assigning functional roles for tyrosine phosphorylation in a variety of cell types, there is very little understanding of the functional role of tyrosine phosphorylation in neuronal response to injury as well as which enzymes and substrates are critical to the process. Since there are many interactions between glial cells in the normal and injured CNS, the choice of injury model used to study a given glial cell type is critical. This is particularly true of microglia since the immunohistochemical markers used to label them also label blood-borne cells such as macrophages and monocytes. For this reason, we chose an injury model which does not cause direct or invasive trauma to the CNS with
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FIG. 3. Time course of phosphotyrosine upregulation in spinal cord ventral horn after ricin injection into sciatic nerve. Optical density (OD) measurements are made through the control and ricin-treated sides of immunostained multiple sections from at least two animals for each survival time. The mean change in OD between sides is expressed as a percentage of increase in OD. Bars represent mean ⫾SEM. The upregulation of ptyr immunostaining reaches maximal levels at 7 days after ricin injection.
subsequent infiltration of blood-borne elements. Injection of the dimeric protein ricin into the peripheral sciatic nerve results in a lesion caused by motor neuron degeneration in the lumbar spinal cord. This injury model leaves the blood–brain barrier intact and allows a clear assessment of the microglial reaction without the complication of distinguishing resident microglia from macrophages or monocytes. We were therefore unambiguously able to show that the elevated microglial phosphotyrosine response in the spinal cord ventral horn is staged approximately 3 days after the ricin injection. This time point coincides with the earliest signs of motor neuron degeneration. These results in spinal cord, together with those of our previous studies in cranial nerve nuclei (10), strongly support a role for tyrosine phosphorylation in the activation of microglia in response to CNS injury in vivo. We next asked whether specific microglial substrates exhibiting elevated levels of phosphotyrosine could be identified in punch biopsy samples of spinal cord ventral horn from ricin or control animals. Somewhat surprisingly, only two bands of approximately 52 and 57 kDa consistently showed elevated phosphotyrosine levels although the degree of this increase was striking. This result suggests that the complicated microglial response to neuronal injury may be driven, at least in part, by activation of a limited number of tyrosine
phosphorylation pathways which should be amenable to further dissection using the ricin injury model. The increased microglial phosphotyrosine following neuronal injury could be due either to increased tyrosine kinase activity or to decreased tyrosine phosphatase activity. Therefore, we tested the hypothesis that continuous infusion of the tyrosine kinase inhibitor herbimycin A attenuates increased microglial phosphotyrosine in response to CNS injury. The results indicate significant attenuation of the microglial phosphotyrosine signal globally, as demonstrated by immunohistochemical analysis, and specifically in a subset of tyrosine kinase substrates demonstrated by Western blot analysis in which the elevated phosphotyrosine returns essentially to control values in herbimycin-treated rats. In our hands Western blot analysis has consistently yielded more reliable quantitative results than immunohistochemical analysis. Further, the immunohistochemical analysis was performed to include the entire ventral horn, whereas the punch biopsy procedure was limited to the area of maximal neuronal degeneration. Thus, while we cannot rule out the possibility that the portion of immunohistochemical levels of phosphotyrosine not attenuated globally in ricin-treated animals represents additional tyrosine kinase or phosphatase pathways, we believe that the Western blot results may be most relevant to the actual in vivo events accompanying ricin-induced elevations of microglial phosphotyrosine. Herbimycin A is a benzenoid ansamycin antibiotic protein tyrosine kinase inhibitor isolated from the broth of Streptomyces cultures. Although first isolated for its herbicidal activity, it was subsequently found to reduce phosphotyrosine content in RSV-transformed cell types. Herbimycin A irreversibly inhibits the nonreceptor or cytosolic protein tyrosine kinases. The mechanism of action of herbimycin A is through binding of reactive sulfhydryl groups on or near the active site of the tyrosine kinase. Support for this mechanism comes from studies using sulfhydryl compounds to block the effects of herbimycin A (28, 29). Herbimycin A does not inhibit the action of PKA or PKC (6). Based on this specificity we conclude that the bulk of ricin-induced increases in microglial phosphotyrosine is due to the action of nonreceptor tyrosine kinase activities. This is particularly true for the 52- and 57-kDa bands identified by Western blot analysis. In terms of downstream events which may be relevant to the response of microglia to injury are the recent observations that herbimycin A is effective in inhibiting lipopolysaccharide interferon-␥-induced nitric oxide expression in a cloned microglial cell line (15). In two interesting and related papers signal transduction pathways in cultured microglia responding to amyloid fibrils and prion proteins were examined (2, 16). In both cases tyrosine-kinase-dependent pathways
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FIG. 4. Light micrographs of ptyr immunostaining in control and ricin-treated ventral horns of non-herbimycin (A, B) and herbimycin (C, D) treated rats. The increase in ptyr immunostaining in response to ricin (B) is significantly attenuated in the rats receiving herbimycin infusions (D). Bar in D, 50 µm.
were rapidly activated resulting in the phosphorylation of specific substrates. In addition it was shown the Lyn, Syk, FAK, and the recently described calcium-sensitive tyrosine kinase PYK2 were activated in response to amyloid fibrils or prion proteins. It is difficult to directly compare our results to the results of McDonald et al. (16) and Combs et al. (2) for several reasons: (1) with the possible exception of PYK2, the tyrosine kinases involved in their studies are primarily receptorassociated whereas the increased microglial tyrosine phosphorylation we observe is primarily the result of nonreceptor tyrosine phosphorylation, at least over a sustained time course; (2) with the exception of one 48-h time course employing immunohistochemical analysis, their results were examining rapid induction of tyrosine phosphorylation, and the substrates identified at these early time points were apparently of higher apparent molecular weight than those identified in our studies; (3) their studies employed cultured
microglia which are primarily ameboid and already activated, whereas we examined the response of resting ramified microglia in the presence of a full complement of other neural cells. We believe that the results shown here are reflective of the response of microglia to neuronal injury in their native environment, and the sustained elevation of phosphotyrosine levels in a limited number of substrates may be directly relevant to sustained levels of phosphotyrosine in microglia associated with neuritic plaques in Alzheimer’s disease (unpublished observations; 32). This is not to say that the pathogenesis is identical, only that sustained elevated levels of microglial phosphotyrosine are characteristic of both processes. Future studies of these pathways will include use of the ricin injury model together with infusion of inhibitors or activators of specific tyrosine kinases or components either up- or downstream of the tyrosine phosphorylation events, together with immunoprecipitation of punch biopsies of ricin-
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FIG. 5. (Left) Cresyl-violet-stained section from the lumbar segment of a rat spinal cord illustrating the location of tissue punches which are used for immunoblotting. A 1-mm-diameter tissue punch is used to collect biopsies from fresh spinal cord ventral horn. Thirty micrograms of protein from punch biopsies is loaded into each lane and subjected to SDS–PAGE and Western blotting for ptyr immunostaining. Increased staining of two bands at 52 and 57 kDa (arrows) is apparent in the ventral horns (two left lanes) of ricin-treated rats (VHE) compared to the control side (VHC). In the presence of herbimycin (two right lanes), this increase is essentially completely attenuated (VHE) compared to the control side (VHC).
treated animals and preparative gel electrophoresis to obtain sufficient quantities of the 52- and 57-kDa substrates for sequence analysis.
8.
ACKNOWLEDGMENTS
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Supported by NIH grant AG11123 and the University Research Committee. We thank Dr. Henry Tomasiewicz and Denise Flaherty for helpful discussions.
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