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2 Huntington's Disease Transgenic Mice
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Molecular and Cellular Neuroscience 20, 638 – 648 (2002) doi:10.1006/mcne.2002.1152
Abnormal Phosphorylation of Synapsin I Predicts a Neuronal Transmission Impairment in the R6/2 Huntington’s Disease Transgenic Mice Jean-Charles Lie´vens, Benjamin Woodman, Amarbirpal Mahal, and Gillian P. Bates 1 Medical and Molecular Genetics, GKT School of Medicine, London SE1 9RT, United Kingdom
Motor and cognitive deficits in Huntington’s disease (HD) are likely caused by progressive neuronal dysfunction preceding neuronal cell death. Synapsin I is one of the major phosphoproteins regulating neurotransmitter release. We report here an abnormal phosphorylation state of synapsin I in the striatum and the cerebral cortex of R6/2 transgenic mice expressing the HD mutation. These changes are mostly characterized by an early overphosphorylation at sites 3–5, whereas phosphorylation at site 1 remains unchanged and at site 6 becomes reduced only close to the end stage of the disease. Such changes do not result from modification in protein expression levels. However, we show a decreased expression of the calcineurin regulatory subunit-B, which may contribute to an imbalance between kinase and phosphatase activities. Together the results suggest that an early impairment in synapsin phosphorylation– dephosphorylation may alter synaptic vesicle trafficking and lead to defective neurotransmission in HD.
INTRODUCTION Huntington’s disease (HD) 2 is an autosomal dominant neurodegenerative disorder characterized by motor and cognitive deficits (Harper, 1996). The disease is caused by an expanded CAG repeat that is translated into an abnormally long polyglutamine expansion in 1 To whom correspondence should be addressed at Division of Medical and Molecular Genetics, GKT School of Medicine, 8th Floor Guy’s Tower, Guy’s Hospital, London SE1 9RT, UK. Fax: 020 7955 4444. E-mail: [email protected]. 2 Abbreviations used: CaM, calcium/calmodulin; CaMK, calcium/ calmodulin-dependent protein kinase; cdk5, cyclin-dependent kinase 5; ERK, extracellular signal-regulated kinase; HD, Huntington’s disease; PKA, cAMP-dependent protein kinase.
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the N-terminus of the huntingtin protein (Huntington’s Disease Collaborative Research Group (HDCRG), 1993). Unaffected individuals carry repeats ranging from 6 to 35 CAGs and symptomatic patients present from 36 to ⬎200 CAG repeats. The neuronal cell death occurs primarily in the caudate nucleus and putamen and to a lesser extent in the cerebral cortex and the cerebellum of HD patients. Evidence has been provided that the neuronal death is secondary to neuronal dysfunction since motor (Myers et al., 1998; Smith et al., 2000) and cognitive impairments (Foroud et al., 1995; Lawrence et al., 1998) can be detected before the loss of neurons in HD patients. The mechanism by which the mutation leads to cellular dysfunction and selective neuronal death is still unknown. Insights into the molecular basis of HD have arisen from an R6 transgenic mouse model expressing the first exon of the huntingtin gene with an expanded CAG repeat (Mangiarini et al., 1996; Davies et al., 1997); the R6/2 mice being the most extensively characterized. Although no obvious neuronal loss is detected in R6/2 mice before their premature death between 14 and 16 weeks, they show progressive neurological disorders measurable with subtle behavioral test from 4 weeks but becoming more obvious from 8 weeks (Carter et al., 1999; Lione et al., 1999; Murphy et al., 2000). Studies indicate that an early defect in neuronal transmission could account for the motor and cognitive deficits observed in R6/2 mice. This includes alterations in expression of neurotransmitter receptors (Cha et al., 1998, 1999; Denovan-Wright and Robertson, 2000; LuthiCarter et al., 2000), proteins in signaling pathways (Bibb et al., 2000; Luthi-Carter et al., 2000), and the Met-enkephalin neuropeptide (Menalled et al., 2000). It has also 1044-7431/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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been proposed that a slow excitotoxic process (Cha et al., 1998, 1999; Lievens et al., 2001) and defects in mitochondrial function (Tabrizi et al., 2000; Perez-Severiano et al., 2000) may enhance the formation of free radicals, resulting in oxidative stress. Synapsin I is considered to be one of the major proteins involved in the regulation of neurotransmitter release and synapse formation. Synapsin I cross-links the synaptic vesicles to the cytoskeleton including actin microfilaments (Greengard et al., 1993; Benfenati et al., 1989, 1992), microtubules (Baines and Bennett, 1986), and brain spectrin (Sikorski et al., 1991; Iga et al., 1997). It can also bundle microtubules and microfilaments (Bahler and Greengard, 1987; Bennett and Baines, 1992). These interactions via synapsin I tightly depend on its phosphorylation state at six sites: site 1 (Ser-9) is phosphorylated by the cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent (CaM) protein kinases I and IV (Czernik et al., 1987); sites 2 and 3 (Ser-566 and Ser-603, respectively) are phosphorylated by CaM kinases II and IV (Czernik et al., 1987); sites 4 – 6 (Ser-62, -67, and -549 respectively) are phosphorylated by extracellular-signal-regulated kinases 1 and 2 (ERK1,2) (Jovanovic et al., 1996; Matsubara et al., 1996). Site 6 also undergoes phosphorylation by cyclin-dependent kinase 5 (cdk 5). Dephosphorylation of synapsin I is achieved at sites 4 – 6 by the calcineurin (calcium/ calmodulin-dependent protein phosphatase 2B) and at sites 1 to 3 mainly by the protein phosphatase 2A but also by calcineurin (Jovanovic et al., 2001). The present study was designed to investigate the expression and phosphorylation state of synapsin I in the striatum and cerebral cortex of R6/2 transgenic mice. We found an alteration of synapsin I phosphorylation, which is mostly characterized by an early overphosphorylation at sites 3–5. These changes may contribute to abnormal neurotransmission in the cerebral cortex and the striatum of R6/2 mice.
RESULTS The Expression of Synapsin I Remained Unchanged in R6/2 The expression of synapsin I was studied in the whole cerebral cortex and the striatum of R6/2 mice. Analysis was performed at 8 weeks, when the phenotype is first apparent by home cage observation, and 12 weeks, when mice exhibit a pronounced phenotype, close to the end stage.
Synapsin I expression was found unchanged in the striatum and the cerebral cortex of transgenic mice as compared to littermate controls at 8 and 12 weeks (Fig. 1). These data confirm and further extend the previous observations showing no significant change in synapsin I level in the striatum between 2 and 8 weeks (Bibb et al., 2000).
The Phosphorylation of Synapsin I Increased at Site 3 but Not at Site 1 in R6/2 Synapsin I can undergo phosphorylation at six specific sites thereby changing its function. Phosphorylation sites 2 and 3 are particularly involved in the regulation of the interaction of synapsin I with the synaptic vesicles and actin (Schiebler et al., 1986; Bahler and Greengard, 1987; Petrucci and Morrow, 1987; Benfenati et al., 1989; Valtorta et al., 1992). In contrast, phosphorylation at site 1 has very moderate effect on the binding of synapsin I to lipids and actin (Schiebler et al., 1986; Bahler and Greengard, 1987). To examine the phosphorylation state of these sites, immunoblots were performed using the antibodies G257, which specifically detect synapsin I/II phosphorylated by PKA and CaMK I/IV at site 1 and Ru19 antibodies which recognize synapsin I phosphorylated by CaMK II/IV at site 3. An increase in the phosphorylation of site 3 was detected in the striatum and the cerebral cortex of 8-week transgenic mice compared to their littermate controls (Fig. 1). The quantitative analysis revealed that the overphosphorylation at site 3 was more pronounced in the cerebral cortex (⫹408% versus control, P ⬍ 0.01) than in the striatum (⫹34% vs control, P ⬍ 0.05). This increase in site 3 phosphorylation persisted in the cerebral cortex of 12-week transgenic mice (⫹145% vs littermate control, P ⬍ 0.05), but not in the striatum. In contrast, the state of site 1 phosphorylation was found unchanged at any of the time points studied. Examination of frontal brain sections immunostained with Ru19 antibodies revealed no change or in some cases a slight increase in the density of synapsin I phosphorylation at site 3 in the striatum of 12-week R6/2 mice compared to littermate controls (Fig. 2A). A more pronounced increase in the site 3 phosphorylated form was observed in the superficial layers (layers I and II) of the cerebral cortex but not in the cortical deep layers. In contrast, the density of synapsin I remained unchanged in the striatum and the cerebral cortex (Fig. 2B).
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The Phosphorylation of Synapsin I Increased at Sites 4 and 5 in R6/2 In addition, three other synapsin I phosphorylation sites (sites 4 – 6) are the target of MAP kinases ERK1,2 and regulate the interaction of synapsin I with actin (Jovanovic et al., 1996; Matsubara et al., 1996). Site 6 is also phosphorylated by cdk5. G526 and G555 antibodies, used here, recognize synapsin I phosphorylated on the sites 4/5 and on site 6, respectively (Fig. 1). The R6/2 mice showed increased amounts of the phosphorylated sites 4/5 at 8 weeks in the striatum and the cerebral cortex (⫹172%, P ⬍ 0.05 and ⫹91%, P ⬍ 0.05 respectively). These changes remained persistent at 12 weeks in the two cerebral structures (⫹55%, P ⬍ 0.05 and ⫹87%, P ⬍ 0.01 in the striatum and the cerebral cortex, respectively). The only change in the level of site 6 phosphorylation was a decrease by about 69% (P ⬍ 0.05) in the striatum at 12 weeks. The Phosphorylation of Neurofilament H Remained Unchanged in R6/2 No significant change in the level of the phosphorylated form of neurofilament H was observed in the striatum and the cerebral cortex in R6/2 line at 8 and 12 weeks of age compared to their littermate controls (Fig. 1). ERK1,2 Phosphorylation State Previous studies have reported changes in mRNA or protein levels of calcium-dependent kinases in R6/2 (Luthi-Carter et al., 2000; Deckel et al., 2001) and cyclic AMP-dependent kinase (Bibb et al., 2000) but to our knowledge the activity of MAP kinases ERK1,2 has not yet been investigated in R6 transgenic mouse model. For this purpose we used specific antibodies directed against the active phosphorylated form of ERK1,2 on Western blot. A dramatic increase in the phosphorylation levels of ERK1,2 was found in the striatum (⫹720%, P ⬍ 0.01) at 12 weeks but not at 8 weeks compared to littermate controls (Fig. 3). In contrast the examination
of cerebral cortex homogenates revealed a 68% (P ⬍ 0.05) decrease in the phosphorylation of ERK1,2 at 12 but not 8 weeks compared to controls. To determine the cellular type (neurons or glia) showing the changes in the ERK1,2 phosphorylation, frontal sections from R6/2 brains at 12 weeks of age were immunostained with phospho-ERK1,2 and a neuronal (neuron-specific nuclei protein) or astroglial (glutamine synthetase) marker. As clearly shown in Fig. 4, ERK1,2 phosphorylation was induced in striatal neurons but not in astrocytes. In contrast no change in phospho-ERK1,2 immunolabeling was observed in the cerebral cortex, the levels of phospho-ERK1,2 being too low to detect a decrease. Calcineurin-B Expression Decreased in R6/2 Synapsin I is also targeted by phosphatases such as calcineurin and protein phosphatase 2A (Jovanovic et al., 2001). Previous data from Luthi-Carter et al. (2000) reported a decrease in the striatal mRNA expression of calcineurin subunit-B in R6/2. To determine whether or not this downregulation affects the protein level, immunoblots using antibodies directed against calcineurin-B have been performed from striatal and cortical homogenates at 8 and 12 weeks of age. Figure 5 shows a slight reduction in the protein level of calcineurin-B in both the striatum and cerebral cortex of R6/2 compared to their littermate controls. The change in calcineurin-B was already significant at 8 weeks of age, a decrease (by about 22%) that was maintained between 8 and 12 weeks.
DISCUSSION As a major finding, we report here a defect in the phosphorylation state of synapsin I in line R6/2 whereas its expression level remains unchanged. An overphosphorylation of synapsin I occurs in the cerebral cortex at sites 3–5 by 8 weeks. The R6/2 striatum
FIG. 1. Western blot analysis of synapsin I and neurofilament H phosphorylation states. The analysis was performed within homogenates from the whole striatum or the cerebral cortex of control and R6/2 transgenic animals at 8 and 12 weeks of age. (A) Digitized prints of Western blot showing the phosphorylation state of synapsin I at sites 1 (G257), 3 (Ru 19), 4/5 (G526), and 6 (G555); the phosphorylated form of neurofilament H; and protein levels of synapsin I and ␣-tubulin. The immunoblots for each cerebral structure and each time point were performed separately, permitting comparisons only between transgenic mice and the age-matched littermate controls for each structure and at each time point. (B) Quantitative analysis of Western blots. Optical densities for each animal were expressed as percentages of control mean. Data from four to six animals per condition were averaged and presented as means ⫾ SEM. Statistical comparisons of transgenic versus control values are performed using a Student’s t test. *P ⬍ 0.05, **P ⬍ 0.01.
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FIG. 2. Photomicrographs of brain sections showing the density of total synapsin I protein (B) or that phosphorylated at site 3 (A) in the striatum and the cerebral cortex of transgenic R6/2 animals at 12 weeks. Magnification ⫻63. *Pial surface of sections.
also shows an increase in the phosphorylation state of synapsin I at site 3 but only at 8 weeks and at sites 4 and 5 by 8 weeks. In contrast, phosphorylation at site 1 remains unaffected at 8 and 12 weeks and the only significant change in phosphorylation state at site 6 is a decrease at 12 weeks in the striatum. Synapsin I dynamically maintains a “reserve pool” of synaptic vesicles in the axonal terminals (Greengard et al., 1993; Hilfiker et al., 1999) by interacting with actin and synaptic vesicles. Phosphorylation of synapsin I profoundly modifies its conformation and then changes its function. For instance, synapsin I phosphorylated at sites 2 and 3 by CaM kinases showed a lower affinity for synaptic vesicles (Schiebler et al., 1986) and almost no affinity for F- and G-actin (Bahler and Greengard, 1987; Petrucci and Morrow, 1987; Valtorta et al., 1992). In contrast, phosphorylation at site 1 has minor effects since its phosphorylation leads to only subtle conformational changes (Benfenati et al., 1990). More recently in vitro studies have reported that phosphorylation at sites 4 – 6 by MAP kinase results in a decrease in the
ability of synapsin I to interact with G- and F-actin but does not affect the binding to synaptic vesicles (Jovanovic et al., 1996). Then, in response to a nerve terminal stimulation, the phosphorylation of synapsin I sites 2 and 3 leads to a dissociation of synapsin I from synaptic vesicles (Sihra et al., 1989; Torri Tarelli et al., 1992) and may result in a dispersion of synapsin I (Chi et al., 2001) and a translocation of synaptic vesicles from the “reserve” pool to the so-called “releasable” pool docked at the plasma membrane (Hilfiker et al., 1999). However, it has also been proposed that a concomitant calcineurin-dependent dephosphorylation of sites 4 – 6 may act as a constraint on processes promoting neurotransmitter release (Jovanovic et al., 2001). In addition, synapsins may also act downstream from vesicle docking, in either priming or fusion reactions (for review see Hilfiker et al., 1999). Therefore, the abnormal overphosphorylation of synapsin I at sites 3–5 in R6/2 line may affect the neurotransmission at a presynaptic level in the cerebral cortex and the striatum of these transgenic mice. It is probably premature to speculate in which
Synapsin I Phosphorylation in Huntington’s Disease
FIG. 3. Western blot analysis of activated ERK1,2 and ␣-tubulin within homogenates from the whole striatum of control and R6/2 transgenic animals. The analysis was performed at 8 and 12 weeks. (A) Digitized prints of Western blot. (B) Quantitative analysis of Western blots. Optical densities for each animal were expressed as percentages of control mean. Data from four to six animals per condition were averaged and presented as means ⫾ SEM. Statistical comparisons of transgenic versus control values are performed using a Student’s t test. *P ⬍ 0.05, **P ⬍ 0.01.
way the overphosphorylation of synapsin I will change the neurotransmitter release. However, it is possible that sustained synapsin I overphosphorylation might have two effects: (1) increase “releasable” pool of synaptic vesicles docked at the plasma membrane and (2)
643 disrupt the response to a repeated stimulation by interfering with the recycling of synaptic vesicles back to the reserve pool. Few data are currently available about the neurotransmitter release efficiency in R6/2. A study from Nicniocaill et al. (2001) using microdialysis has reported an enhanced increase in excitatory amino acids release subsequently to a KCl exposure in the striatum of another transgenic mouse model for Huntington’s disease (R6/1). More recently, an alteration in efferent striatal neuron responses to the repeated activation of striatal inputs has been described in R6/2, indicating a loss of glutamatergic synaptic inputs from the cerebral cortex and probably the thalamus (Klapstein et al., 2001). The loss of dentritic spines on striatal and cortical neurons (Klapstein et al., 2001) together with the decrease in glutamate uptake (Lievens et al., 2001) and the presynaptic changes as shown here might contribute to altered glutamatergic synaptic transmission in these both cerebral structures. Whether or not the synapsin I phosphorylation changes occur at different types of synapses and in other cerebral structures such as hippocampus where abnormal synaptic plasticity occurs in R6/2 (Murphy et al., 2000) should be investigated. Presynaptic changes in glutamatergic terminals may account for the relative resistance of R6/2 neurons to excitotoxins (Hansson et al., 1999; Morton and Leavens, 2000), the excitotoxic processes in vivo requiring the integrity of the glutamatergic corticostriatal inputs (Biziere and Coyle, 1979; McGeer et al., 1978; Galarraga et al., 1990; Orlando et al., 2001). Moreover, changes in other presynaptic proteins implicated in the synaptic vesicle fusion machinery such as complexin II has also been described early in R6/2 (Morton and Edwardson, 2001), but the effects remain unknown. The changes in the phosphorylated state of synapsin I could be due to multiple processes. Our results, together with the data from Bibb et al. (2000) show that the expression of synapsin I is not altered between wild-type and transgenic mice. Changes in phosphorylation are more likely caused by an imbalance between protein kinase and phosphatase activities. Reduced protein levels of PKA (Bibb et al., 2000) and CaM kinases II/IV (Deckel et al., 2001) have been measured in R6/2. We report here opposing changes in active levels of ERK1,2 at a late stage in the striatum and the cerebral cortex of R6/2, which alone cannot account for the phosphorylation state of sites 4 – 6. The increase in ERK1,2 phosphorylation in the striatal neurons could result from accumulated reactive oxygen species or/ and excitotoxic process (Guyton et al., 1996; Vanhoutte et al., 1999; Schwarzschild et al., 1999; Fuller et al., 2001)
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FIG. 4. Photomicrographs of brain sections showing the increase in activated ERK1,2 (red) in neurons but not in astrocytes (green) in the striatum and the cerebral cortex of transgenic R6/2 animals at 12 weeks. (A) Astrocytes are detected by glutamine synthetase immunocytochemistry. (B) Neurons are visualized by neuronal nuclei immunocytochemistry. Magnification ⫻16.
and may have deleterious long-term effects (Stanciu et al., 2000; Stanciu and DeFranco, 2002). More important, the mRNA level of the calcineurin subunit-B is decreased in the R6/2 striatum (Luthi-Carter et al., 2000) and we show here that it translates into an alteration of the protein expression at an early stage in the striatum and the cerebral cortex. Increases in the mRNA levels of immunophilins (cyclophilin A and immunophilin P59), which could inhibit calcineurin activity (Liu et al., 1992; Sabatini et al., 1997), also occur as early as 6 weeks of age in R6/2 (Luthi-Carter et al., 2000). Inhibition of calcineurin activity has been previously shown to increase the phosphorylation of synapsin I mainly at sites 4 and 5 (Jovanovic et al., 2001). It is then tempting to propose that a continuous alteration of calcineurin activity may play a role in the overphosphorylated state of synapsin I at sites 4 and 5 but also at site 3. It would be of interest to assess the activity of protein phosphatase 2A, which seems to be responsible for the dephosphorylation at sites 1–3 (Jovanovic et al., 2001). In conclusion, we here suggest that an imbalance between kinase and phosphatase activities occurs in Huntington’s disease, resulting in an altered phosphorylation of synapsin I. Such a change may be responsible for an abnormal cycling of synaptic vesicles, which
profoundly modifies the presynaptic response to excitation and synaptic plasticity in this disease.
EXPERIMENTAL METHODS Mice R6/2 mice are transgenic for exon 1 of the HD gene carrying an expanded CAG repeat (Mangiarini et al., 1996). The R6/2 line was maintained by backcrossing to (C57BL/6 ⫻ CBA)F 1 mice and the genotype and CAG repeat size was determined as previously described (Mangiarini et al., 1997). The CAG repeat size of the R6/2 mice used in this study was 191.83 ⫾ 1.56 (SEM). Nontransgenic littermates were used in the present study as controls. R6/2 mice (Jackson code B6CBATgN(HDexon1)62) can be obtained from the Induced Mutant Resource (Jackson Laboratory, Bar Harbor, ME). Antibodies The monoclonal mouse antibody raised against ␣-tubulin was from Sigma Chemical Co. (St. Louis, MO). Polyclonal rabbit antibodies raised against the phosphorylated form of ERK1,2 (p42 and p44) were from
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New England Biolabs (Beverly, MA). Synapsin 1 was detected using rabbit polyclonal antibodies from Chemicon (Temecula, CA). Rabbit polyclonal antibodies to detect synapsin I specifically phosphorylated at residues Ser-9 (referred to as P-site 1), Ser-603 (P-site 3), Ser-62 (P-sites 4), Ser-67 (P-site 5), or at residue 549 (P-site 6) were a generous gift from P. Greengard (Rockefeller University, New York, NY) and were produced as previously described (Czernik et al., 1991; Jovanovic et al., 1996); they had the same specificity as the original antibodies G257 (P-site 1 Ab), Ru19 (P-site 3 Ab) G526 (P-sites 4 and 5 Ab), and G555 (P-site 6 Ab). Glutamine synthetase, neuron-specific nuclear protein, and the phosphorylated form of neurofilaments H were detected using monoclonal mouse antibodies from Chemicon (Temecula, CA). Rabbit polyclonal antibodies raised against subunit-B of calcineurin were a generous gift from M. Moriya (Hokkaido University, Sapporo, Japan) and were generated as previously described (Moriya et al., 1995). A Texas red goat anti-rabbit and anti-mouse IgG were both from Molecular Probes (Eugene, OR). Immunohistochemistry
FIG. 5. Western blot analysis of calcineurin subunit-B and ␣-tubulin within homogenates from the whole striatum of control and R6/2 transgenic animals. The analysis was performed at 8 and 12 weeks. (A) Digitized prints of Western blot. (B) Quantitative analysis of Western blots. Optical densities for each animal were expressed as percentages of control mean. Data from four to six animals per condition were averaged and presented as means ⫾ SEM. Statistical comparisons of transgenic versus control values are performed using a Student’s t test. *P ⬍ 0.05, **P ⬍ 0.01.
Immunostaining of total synapsin I or that phosphorylated at site 3 (Ru19 antibodies) and dual immunolabeling of activated ERK1,2 (phospho-ERK1,2 antibodies) with either astroglial (glutamine synthetase antibody) or neuronal (neuron-specific nuclei protein antibody) markers were performed using fluorescent secondary antibodies. R6/2 and littermate control mice at 12 weeks (n ⫽ 6 each) were anesthetized with an intraperitoneal injection of sodium pentobarbitone (Sagatal, Rhone Merieux) at the dose of 6 mg/30 g body wt and transcardially perfused with 4% paraformaldehyde in 0.1 M PBS. The brains were then quickly removed, postfixed overnight in the same fixative at 4°C, cryoprotected with 30% sucrose in 0.1 M PBS overnight at 4°C, frozen in isopentane on dry ice, and stored at ⫺70°C. Frontal sections 20 m were cut at striatal level with a cryostat (Bright Instrument Co. Ltd. Huntingdon, Cambs, UK), mounted on Superfrost Plus slides, and kept frozen at ⫺70°C. Slide-mounted sections were fixed for 30 min in a 0.1 M PBS, pH 7.4, containing 4% paraformaldehyde. Reagents were prepared in TBS (50 mM Tris, 150 mM NaCl, pH 7.5) and each step was followed by rinses in TBS solution. Sera were prepared in 2% BSA. Sections were successively incubated in serum (15 min) followed by the same solution containing the first antibodies (dilutions: Ru 19 1:200, synapsin I 1:200, phosphoERK1,2 1:350, neuron-specific nuclei protein 1:500, and glutamine synthetase 1:1000) for 16 h at 4°C. The sections were then incubated with the appropriate secondary an-
646 tibodies (Alexa 488 anti-mouse IgG 1:100, Texas red antirabbit IgG 1:100). Coverslips were mounted with Mowiol (Calbiochem). Microscopic examination of synapsin I immunostaining was performed using a LCM confocal microscope (Zeiss) and Zeiss LSM 5 image browser software. The same fluorescence calibration was used between the different experimental conditions (laser, rhodamine 543 nm; amplification offset, ⫺0.085; gain detector, 827; pinhole, 1.00 airy units). Double labeling of activated ERK1,2 with astroglial or neuronal markers was examined on a fluorescence microscope (Zeiss axioPlan II) and image processing using the Smart Capture VP and IP lab software. Identical calibrations of the microscope were used between the different experimental conditions. Western Blotting Immunodetection on blots was performed from four to six R6/2 and control animals at 8 and 12 weeks. The striata and the whole cerebral cortex were rapidly dissected, frozen in isopentane on dry ice, and stored at ⫺70°C. Tissue samples were thawed, homogenized in 20 mM NaPi (15.5 mM Na 2HPO, 4.5 mM NaH 2PO 4, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Boehringer Mannheim, Germany) and sonicated for 30 s. Protein concentrations were determined by the Bradford assay, using BSA as standard, and 1/6 (v/v) sample buffer was added (300 mM Tris–HCl, pH 6.8, 10% SDS, 50% glycerol, 0.05% bromphenol blue, and 300 mM DTT). Total protein (5–15 g protein per lane as determined in a preliminary experiment) was electrophoresed through a stacking gel and a 8 –12% resolving gel, and electroblotted (250 mA, 1 h) onto Hybond-P membrane (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). Before immunodetection, blots were stained with Ponceau-S to assess if the same quantity of protein had been loaded in each well and to visualize the transfer efficiency. The blots were then successively incubated in blocking buffer: 1⫻ TBS (50 mM Tris, 150 mM NaCl pH 7.6) containing 3% dry milk (Marvel) for 1 h and blocking buffer containing the primary antibody overnight at 4°C. The dilutions of the primary antibodies used here were dilution 1:750 for anti-phospho ERK1,2; dilution 1:1000 for G257, Ru19, G526, and G555; dilution 1:1500 for anti-calcineurin-B and anti-phosphoneurofilaments; and dilution 1:2500 for anti-synapsin 1. Blots were washed in TBST (1⫻ TBS containing 0.1% Tween 20) for 3 ⫻ 5 min and incubated with affinity-purified peroxidase-labeled secondary antibody (anti-rabbit secondary antibodies, 1/3000; anti-mouse secondary antibodies, 1/5000) for 1 h. The signal was visualized on film with
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enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). Digitized images of immunoreactive proteins were quantified using a KS300 image analysis system (Carl Zeiss Vision GmbH, Hallbergmoos, Germany) from autoradiographic films. The blots were then washed in a stripping buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 100 mM -mercaptoethanol) for 30 min at 50°C and reprobed using anti-␣-tubulin antibodies (1:1000). The immunoblots were performed separately for each cerebral structure and each time point, permitting comparisons for each structure only between transgenic mice and the age-matched littermate controls. Optical densities for each animal were therefore expressed as percentage of control mean. Data from n animals per condition were averaged and presented as means ⫾ SEM. Statistical analysis of data was performed using a Student’s t test. A significance of P ⬍ 0.05 was required for rejection of the null hypothesis.
ACKNOWLEDGMENTS We thank Professor Paul Greengard and Dr. Hung-Teh Kao for providing us antibodies against the phosphorylated form of synapsin I and Dr. Megumi Moriya for calcineurin-B antibodies. We also thank Dr. Patrick Brundin and Roman Gonitel for helpful discussions. This work was supported by grants from the Wellcome Trust (051897) and the Human Frontiers Science Program (RG0132).
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Molecular and Cellular Neuroscience 20, 638 – 648 (2002) doi:10.1006/mcne.2002.1152
Abnormal Phosphorylation of Synapsin I Predicts a Neuronal Transmission Impairment in the R6/2 Huntington’s Disease Transgenic Mice Jean-Charles Lie´vens, Benjamin Woodman, Amarbirpal Mahal, and Gillian P. Bates 1 Medical and Molecular Genetics, GKT School of Medicine, London SE1 9RT, United Kingdom
Motor and cognitive deficits in Huntington’s disease (HD) are likely caused by progressive neuronal dysfunction preceding neuronal cell death. Synapsin I is one of the major phosphoproteins regulating neurotransmitter release. We report here an abnormal phosphorylation state of synapsin I in the striatum and the cerebral cortex of R6/2 transgenic mice expressing the HD mutation. These changes are mostly characterized by an early overphosphorylation at sites 3–5, whereas phosphorylation at site 1 remains unchanged and at site 6 becomes reduced only close to the end stage of the disease. Such changes do not result from modification in protein expression levels. However, we show a decreased expression of the calcineurin regulatory subunit-B, which may contribute to an imbalance between kinase and phosphatase activities. Together the results suggest that an early impairment in synapsin phosphorylation– dephosphorylation may alter synaptic vesicle trafficking and lead to defective neurotransmission in HD.
INTRODUCTION Huntington’s disease (HD) 2 is an autosomal dominant neurodegenerative disorder characterized by motor and cognitive deficits (Harper, 1996). The disease is caused by an expanded CAG repeat that is translated into an abnormally long polyglutamine expansion in 1 To whom correspondence should be addressed at Division of Medical and Molecular Genetics, GKT School of Medicine, 8th Floor Guy’s Tower, Guy’s Hospital, London SE1 9RT, UK. Fax: 020 7955 4444. E-mail: [email protected]. 2 Abbreviations used: CaM, calcium/calmodulin; CaMK, calcium/ calmodulin-dependent protein kinase; cdk5, cyclin-dependent kinase 5; ERK, extracellular signal-regulated kinase; HD, Huntington’s disease; PKA, cAMP-dependent protein kinase.
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the N-terminus of the huntingtin protein (Huntington’s Disease Collaborative Research Group (HDCRG), 1993). Unaffected individuals carry repeats ranging from 6 to 35 CAGs and symptomatic patients present from 36 to ⬎200 CAG repeats. The neuronal cell death occurs primarily in the caudate nucleus and putamen and to a lesser extent in the cerebral cortex and the cerebellum of HD patients. Evidence has been provided that the neuronal death is secondary to neuronal dysfunction since motor (Myers et al., 1998; Smith et al., 2000) and cognitive impairments (Foroud et al., 1995; Lawrence et al., 1998) can be detected before the loss of neurons in HD patients. The mechanism by which the mutation leads to cellular dysfunction and selective neuronal death is still unknown. Insights into the molecular basis of HD have arisen from an R6 transgenic mouse model expressing the first exon of the huntingtin gene with an expanded CAG repeat (Mangiarini et al., 1996; Davies et al., 1997); the R6/2 mice being the most extensively characterized. Although no obvious neuronal loss is detected in R6/2 mice before their premature death between 14 and 16 weeks, they show progressive neurological disorders measurable with subtle behavioral test from 4 weeks but becoming more obvious from 8 weeks (Carter et al., 1999; Lione et al., 1999; Murphy et al., 2000). Studies indicate that an early defect in neuronal transmission could account for the motor and cognitive deficits observed in R6/2 mice. This includes alterations in expression of neurotransmitter receptors (Cha et al., 1998, 1999; Denovan-Wright and Robertson, 2000; LuthiCarter et al., 2000), proteins in signaling pathways (Bibb et al., 2000; Luthi-Carter et al., 2000), and the Met-enkephalin neuropeptide (Menalled et al., 2000). It has also 1044-7431/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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been proposed that a slow excitotoxic process (Cha et al., 1998, 1999; Lievens et al., 2001) and defects in mitochondrial function (Tabrizi et al., 2000; Perez-Severiano et al., 2000) may enhance the formation of free radicals, resulting in oxidative stress. Synapsin I is considered to be one of the major proteins involved in the regulation of neurotransmitter release and synapse formation. Synapsin I cross-links the synaptic vesicles to the cytoskeleton including actin microfilaments (Greengard et al., 1993; Benfenati et al., 1989, 1992), microtubules (Baines and Bennett, 1986), and brain spectrin (Sikorski et al., 1991; Iga et al., 1997). It can also bundle microtubules and microfilaments (Bahler and Greengard, 1987; Bennett and Baines, 1992). These interactions via synapsin I tightly depend on its phosphorylation state at six sites: site 1 (Ser-9) is phosphorylated by the cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent (CaM) protein kinases I and IV (Czernik et al., 1987); sites 2 and 3 (Ser-566 and Ser-603, respectively) are phosphorylated by CaM kinases II and IV (Czernik et al., 1987); sites 4 – 6 (Ser-62, -67, and -549 respectively) are phosphorylated by extracellular-signal-regulated kinases 1 and 2 (ERK1,2) (Jovanovic et al., 1996; Matsubara et al., 1996). Site 6 also undergoes phosphorylation by cyclin-dependent kinase 5 (cdk 5). Dephosphorylation of synapsin I is achieved at sites 4 – 6 by the calcineurin (calcium/ calmodulin-dependent protein phosphatase 2B) and at sites 1 to 3 mainly by the protein phosphatase 2A but also by calcineurin (Jovanovic et al., 2001). The present study was designed to investigate the expression and phosphorylation state of synapsin I in the striatum and cerebral cortex of R6/2 transgenic mice. We found an alteration of synapsin I phosphorylation, which is mostly characterized by an early overphosphorylation at sites 3–5. These changes may contribute to abnormal neurotransmission in the cerebral cortex and the striatum of R6/2 mice.
RESULTS The Expression of Synapsin I Remained Unchanged in R6/2 The expression of synapsin I was studied in the whole cerebral cortex and the striatum of R6/2 mice. Analysis was performed at 8 weeks, when the phenotype is first apparent by home cage observation, and 12 weeks, when mice exhibit a pronounced phenotype, close to the end stage.
Synapsin I expression was found unchanged in the striatum and the cerebral cortex of transgenic mice as compared to littermate controls at 8 and 12 weeks (Fig. 1). These data confirm and further extend the previous observations showing no significant change in synapsin I level in the striatum between 2 and 8 weeks (Bibb et al., 2000).
The Phosphorylation of Synapsin I Increased at Site 3 but Not at Site 1 in R6/2 Synapsin I can undergo phosphorylation at six specific sites thereby changing its function. Phosphorylation sites 2 and 3 are particularly involved in the regulation of the interaction of synapsin I with the synaptic vesicles and actin (Schiebler et al., 1986; Bahler and Greengard, 1987; Petrucci and Morrow, 1987; Benfenati et al., 1989; Valtorta et al., 1992). In contrast, phosphorylation at site 1 has very moderate effect on the binding of synapsin I to lipids and actin (Schiebler et al., 1986; Bahler and Greengard, 1987). To examine the phosphorylation state of these sites, immunoblots were performed using the antibodies G257, which specifically detect synapsin I/II phosphorylated by PKA and CaMK I/IV at site 1 and Ru19 antibodies which recognize synapsin I phosphorylated by CaMK II/IV at site 3. An increase in the phosphorylation of site 3 was detected in the striatum and the cerebral cortex of 8-week transgenic mice compared to their littermate controls (Fig. 1). The quantitative analysis revealed that the overphosphorylation at site 3 was more pronounced in the cerebral cortex (⫹408% versus control, P ⬍ 0.01) than in the striatum (⫹34% vs control, P ⬍ 0.05). This increase in site 3 phosphorylation persisted in the cerebral cortex of 12-week transgenic mice (⫹145% vs littermate control, P ⬍ 0.05), but not in the striatum. In contrast, the state of site 1 phosphorylation was found unchanged at any of the time points studied. Examination of frontal brain sections immunostained with Ru19 antibodies revealed no change or in some cases a slight increase in the density of synapsin I phosphorylation at site 3 in the striatum of 12-week R6/2 mice compared to littermate controls (Fig. 2A). A more pronounced increase in the site 3 phosphorylated form was observed in the superficial layers (layers I and II) of the cerebral cortex but not in the cortical deep layers. In contrast, the density of synapsin I remained unchanged in the striatum and the cerebral cortex (Fig. 2B).
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The Phosphorylation of Synapsin I Increased at Sites 4 and 5 in R6/2 In addition, three other synapsin I phosphorylation sites (sites 4 – 6) are the target of MAP kinases ERK1,2 and regulate the interaction of synapsin I with actin (Jovanovic et al., 1996; Matsubara et al., 1996). Site 6 is also phosphorylated by cdk5. G526 and G555 antibodies, used here, recognize synapsin I phosphorylated on the sites 4/5 and on site 6, respectively (Fig. 1). The R6/2 mice showed increased amounts of the phosphorylated sites 4/5 at 8 weeks in the striatum and the cerebral cortex (⫹172%, P ⬍ 0.05 and ⫹91%, P ⬍ 0.05 respectively). These changes remained persistent at 12 weeks in the two cerebral structures (⫹55%, P ⬍ 0.05 and ⫹87%, P ⬍ 0.01 in the striatum and the cerebral cortex, respectively). The only change in the level of site 6 phosphorylation was a decrease by about 69% (P ⬍ 0.05) in the striatum at 12 weeks. The Phosphorylation of Neurofilament H Remained Unchanged in R6/2 No significant change in the level of the phosphorylated form of neurofilament H was observed in the striatum and the cerebral cortex in R6/2 line at 8 and 12 weeks of age compared to their littermate controls (Fig. 1). ERK1,2 Phosphorylation State Previous studies have reported changes in mRNA or protein levels of calcium-dependent kinases in R6/2 (Luthi-Carter et al., 2000; Deckel et al., 2001) and cyclic AMP-dependent kinase (Bibb et al., 2000) but to our knowledge the activity of MAP kinases ERK1,2 has not yet been investigated in R6 transgenic mouse model. For this purpose we used specific antibodies directed against the active phosphorylated form of ERK1,2 on Western blot. A dramatic increase in the phosphorylation levels of ERK1,2 was found in the striatum (⫹720%, P ⬍ 0.01) at 12 weeks but not at 8 weeks compared to littermate controls (Fig. 3). In contrast the examination
of cerebral cortex homogenates revealed a 68% (P ⬍ 0.05) decrease in the phosphorylation of ERK1,2 at 12 but not 8 weeks compared to controls. To determine the cellular type (neurons or glia) showing the changes in the ERK1,2 phosphorylation, frontal sections from R6/2 brains at 12 weeks of age were immunostained with phospho-ERK1,2 and a neuronal (neuron-specific nuclei protein) or astroglial (glutamine synthetase) marker. As clearly shown in Fig. 4, ERK1,2 phosphorylation was induced in striatal neurons but not in astrocytes. In contrast no change in phospho-ERK1,2 immunolabeling was observed in the cerebral cortex, the levels of phospho-ERK1,2 being too low to detect a decrease. Calcineurin-B Expression Decreased in R6/2 Synapsin I is also targeted by phosphatases such as calcineurin and protein phosphatase 2A (Jovanovic et al., 2001). Previous data from Luthi-Carter et al. (2000) reported a decrease in the striatal mRNA expression of calcineurin subunit-B in R6/2. To determine whether or not this downregulation affects the protein level, immunoblots using antibodies directed against calcineurin-B have been performed from striatal and cortical homogenates at 8 and 12 weeks of age. Figure 5 shows a slight reduction in the protein level of calcineurin-B in both the striatum and cerebral cortex of R6/2 compared to their littermate controls. The change in calcineurin-B was already significant at 8 weeks of age, a decrease (by about 22%) that was maintained between 8 and 12 weeks.
DISCUSSION As a major finding, we report here a defect in the phosphorylation state of synapsin I in line R6/2 whereas its expression level remains unchanged. An overphosphorylation of synapsin I occurs in the cerebral cortex at sites 3–5 by 8 weeks. The R6/2 striatum
FIG. 1. Western blot analysis of synapsin I and neurofilament H phosphorylation states. The analysis was performed within homogenates from the whole striatum or the cerebral cortex of control and R6/2 transgenic animals at 8 and 12 weeks of age. (A) Digitized prints of Western blot showing the phosphorylation state of synapsin I at sites 1 (G257), 3 (Ru 19), 4/5 (G526), and 6 (G555); the phosphorylated form of neurofilament H; and protein levels of synapsin I and ␣-tubulin. The immunoblots for each cerebral structure and each time point were performed separately, permitting comparisons only between transgenic mice and the age-matched littermate controls for each structure and at each time point. (B) Quantitative analysis of Western blots. Optical densities for each animal were expressed as percentages of control mean. Data from four to six animals per condition were averaged and presented as means ⫾ SEM. Statistical comparisons of transgenic versus control values are performed using a Student’s t test. *P ⬍ 0.05, **P ⬍ 0.01.
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FIG. 2. Photomicrographs of brain sections showing the density of total synapsin I protein (B) or that phosphorylated at site 3 (A) in the striatum and the cerebral cortex of transgenic R6/2 animals at 12 weeks. Magnification ⫻63. *Pial surface of sections.
also shows an increase in the phosphorylation state of synapsin I at site 3 but only at 8 weeks and at sites 4 and 5 by 8 weeks. In contrast, phosphorylation at site 1 remains unaffected at 8 and 12 weeks and the only significant change in phosphorylation state at site 6 is a decrease at 12 weeks in the striatum. Synapsin I dynamically maintains a “reserve pool” of synaptic vesicles in the axonal terminals (Greengard et al., 1993; Hilfiker et al., 1999) by interacting with actin and synaptic vesicles. Phosphorylation of synapsin I profoundly modifies its conformation and then changes its function. For instance, synapsin I phosphorylated at sites 2 and 3 by CaM kinases showed a lower affinity for synaptic vesicles (Schiebler et al., 1986) and almost no affinity for F- and G-actin (Bahler and Greengard, 1987; Petrucci and Morrow, 1987; Valtorta et al., 1992). In contrast, phosphorylation at site 1 has minor effects since its phosphorylation leads to only subtle conformational changes (Benfenati et al., 1990). More recently in vitro studies have reported that phosphorylation at sites 4 – 6 by MAP kinase results in a decrease in the
ability of synapsin I to interact with G- and F-actin but does not affect the binding to synaptic vesicles (Jovanovic et al., 1996). Then, in response to a nerve terminal stimulation, the phosphorylation of synapsin I sites 2 and 3 leads to a dissociation of synapsin I from synaptic vesicles (Sihra et al., 1989; Torri Tarelli et al., 1992) and may result in a dispersion of synapsin I (Chi et al., 2001) and a translocation of synaptic vesicles from the “reserve” pool to the so-called “releasable” pool docked at the plasma membrane (Hilfiker et al., 1999). However, it has also been proposed that a concomitant calcineurin-dependent dephosphorylation of sites 4 – 6 may act as a constraint on processes promoting neurotransmitter release (Jovanovic et al., 2001). In addition, synapsins may also act downstream from vesicle docking, in either priming or fusion reactions (for review see Hilfiker et al., 1999). Therefore, the abnormal overphosphorylation of synapsin I at sites 3–5 in R6/2 line may affect the neurotransmission at a presynaptic level in the cerebral cortex and the striatum of these transgenic mice. It is probably premature to speculate in which
Synapsin I Phosphorylation in Huntington’s Disease
FIG. 3. Western blot analysis of activated ERK1,2 and ␣-tubulin within homogenates from the whole striatum of control and R6/2 transgenic animals. The analysis was performed at 8 and 12 weeks. (A) Digitized prints of Western blot. (B) Quantitative analysis of Western blots. Optical densities for each animal were expressed as percentages of control mean. Data from four to six animals per condition were averaged and presented as means ⫾ SEM. Statistical comparisons of transgenic versus control values are performed using a Student’s t test. *P ⬍ 0.05, **P ⬍ 0.01.
way the overphosphorylation of synapsin I will change the neurotransmitter release. However, it is possible that sustained synapsin I overphosphorylation might have two effects: (1) increase “releasable” pool of synaptic vesicles docked at the plasma membrane and (2)
643 disrupt the response to a repeated stimulation by interfering with the recycling of synaptic vesicles back to the reserve pool. Few data are currently available about the neurotransmitter release efficiency in R6/2. A study from Nicniocaill et al. (2001) using microdialysis has reported an enhanced increase in excitatory amino acids release subsequently to a KCl exposure in the striatum of another transgenic mouse model for Huntington’s disease (R6/1). More recently, an alteration in efferent striatal neuron responses to the repeated activation of striatal inputs has been described in R6/2, indicating a loss of glutamatergic synaptic inputs from the cerebral cortex and probably the thalamus (Klapstein et al., 2001). The loss of dentritic spines on striatal and cortical neurons (Klapstein et al., 2001) together with the decrease in glutamate uptake (Lievens et al., 2001) and the presynaptic changes as shown here might contribute to altered glutamatergic synaptic transmission in these both cerebral structures. Whether or not the synapsin I phosphorylation changes occur at different types of synapses and in other cerebral structures such as hippocampus where abnormal synaptic plasticity occurs in R6/2 (Murphy et al., 2000) should be investigated. Presynaptic changes in glutamatergic terminals may account for the relative resistance of R6/2 neurons to excitotoxins (Hansson et al., 1999; Morton and Leavens, 2000), the excitotoxic processes in vivo requiring the integrity of the glutamatergic corticostriatal inputs (Biziere and Coyle, 1979; McGeer et al., 1978; Galarraga et al., 1990; Orlando et al., 2001). Moreover, changes in other presynaptic proteins implicated in the synaptic vesicle fusion machinery such as complexin II has also been described early in R6/2 (Morton and Edwardson, 2001), but the effects remain unknown. The changes in the phosphorylated state of synapsin I could be due to multiple processes. Our results, together with the data from Bibb et al. (2000) show that the expression of synapsin I is not altered between wild-type and transgenic mice. Changes in phosphorylation are more likely caused by an imbalance between protein kinase and phosphatase activities. Reduced protein levels of PKA (Bibb et al., 2000) and CaM kinases II/IV (Deckel et al., 2001) have been measured in R6/2. We report here opposing changes in active levels of ERK1,2 at a late stage in the striatum and the cerebral cortex of R6/2, which alone cannot account for the phosphorylation state of sites 4 – 6. The increase in ERK1,2 phosphorylation in the striatal neurons could result from accumulated reactive oxygen species or/ and excitotoxic process (Guyton et al., 1996; Vanhoutte et al., 1999; Schwarzschild et al., 1999; Fuller et al., 2001)
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FIG. 4. Photomicrographs of brain sections showing the increase in activated ERK1,2 (red) in neurons but not in astrocytes (green) in the striatum and the cerebral cortex of transgenic R6/2 animals at 12 weeks. (A) Astrocytes are detected by glutamine synthetase immunocytochemistry. (B) Neurons are visualized by neuronal nuclei immunocytochemistry. Magnification ⫻16.
and may have deleterious long-term effects (Stanciu et al., 2000; Stanciu and DeFranco, 2002). More important, the mRNA level of the calcineurin subunit-B is decreased in the R6/2 striatum (Luthi-Carter et al., 2000) and we show here that it translates into an alteration of the protein expression at an early stage in the striatum and the cerebral cortex. Increases in the mRNA levels of immunophilins (cyclophilin A and immunophilin P59), which could inhibit calcineurin activity (Liu et al., 1992; Sabatini et al., 1997), also occur as early as 6 weeks of age in R6/2 (Luthi-Carter et al., 2000). Inhibition of calcineurin activity has been previously shown to increase the phosphorylation of synapsin I mainly at sites 4 and 5 (Jovanovic et al., 2001). It is then tempting to propose that a continuous alteration of calcineurin activity may play a role in the overphosphorylated state of synapsin I at sites 4 and 5 but also at site 3. It would be of interest to assess the activity of protein phosphatase 2A, which seems to be responsible for the dephosphorylation at sites 1–3 (Jovanovic et al., 2001). In conclusion, we here suggest that an imbalance between kinase and phosphatase activities occurs in Huntington’s disease, resulting in an altered phosphorylation of synapsin I. Such a change may be responsible for an abnormal cycling of synaptic vesicles, which
profoundly modifies the presynaptic response to excitation and synaptic plasticity in this disease.
EXPERIMENTAL METHODS Mice R6/2 mice are transgenic for exon 1 of the HD gene carrying an expanded CAG repeat (Mangiarini et al., 1996). The R6/2 line was maintained by backcrossing to (C57BL/6 ⫻ CBA)F 1 mice and the genotype and CAG repeat size was determined as previously described (Mangiarini et al., 1997). The CAG repeat size of the R6/2 mice used in this study was 191.83 ⫾ 1.56 (SEM). Nontransgenic littermates were used in the present study as controls. R6/2 mice (Jackson code B6CBATgN(HDexon1)62) can be obtained from the Induced Mutant Resource (Jackson Laboratory, Bar Harbor, ME). Antibodies The monoclonal mouse antibody raised against ␣-tubulin was from Sigma Chemical Co. (St. Louis, MO). Polyclonal rabbit antibodies raised against the phosphorylated form of ERK1,2 (p42 and p44) were from
645
Synapsin I Phosphorylation in Huntington’s Disease
New England Biolabs (Beverly, MA). Synapsin 1 was detected using rabbit polyclonal antibodies from Chemicon (Temecula, CA). Rabbit polyclonal antibodies to detect synapsin I specifically phosphorylated at residues Ser-9 (referred to as P-site 1), Ser-603 (P-site 3), Ser-62 (P-sites 4), Ser-67 (P-site 5), or at residue 549 (P-site 6) were a generous gift from P. Greengard (Rockefeller University, New York, NY) and were produced as previously described (Czernik et al., 1991; Jovanovic et al., 1996); they had the same specificity as the original antibodies G257 (P-site 1 Ab), Ru19 (P-site 3 Ab) G526 (P-sites 4 and 5 Ab), and G555 (P-site 6 Ab). Glutamine synthetase, neuron-specific nuclear protein, and the phosphorylated form of neurofilaments H were detected using monoclonal mouse antibodies from Chemicon (Temecula, CA). Rabbit polyclonal antibodies raised against subunit-B of calcineurin were a generous gift from M. Moriya (Hokkaido University, Sapporo, Japan) and were generated as previously described (Moriya et al., 1995). A Texas red goat anti-rabbit and anti-mouse IgG were both from Molecular Probes (Eugene, OR). Immunohistochemistry
FIG. 5. Western blot analysis of calcineurin subunit-B and ␣-tubulin within homogenates from the whole striatum of control and R6/2 transgenic animals. The analysis was performed at 8 and 12 weeks. (A) Digitized prints of Western blot. (B) Quantitative analysis of Western blots. Optical densities for each animal were expressed as percentages of control mean. Data from four to six animals per condition were averaged and presented as means ⫾ SEM. Statistical comparisons of transgenic versus control values are performed using a Student’s t test. *P ⬍ 0.05, **P ⬍ 0.01.
Immunostaining of total synapsin I or that phosphorylated at site 3 (Ru19 antibodies) and dual immunolabeling of activated ERK1,2 (phospho-ERK1,2 antibodies) with either astroglial (glutamine synthetase antibody) or neuronal (neuron-specific nuclei protein antibody) markers were performed using fluorescent secondary antibodies. R6/2 and littermate control mice at 12 weeks (n ⫽ 6 each) were anesthetized with an intraperitoneal injection of sodium pentobarbitone (Sagatal, Rhone Merieux) at the dose of 6 mg/30 g body wt and transcardially perfused with 4% paraformaldehyde in 0.1 M PBS. The brains were then quickly removed, postfixed overnight in the same fixative at 4°C, cryoprotected with 30% sucrose in 0.1 M PBS overnight at 4°C, frozen in isopentane on dry ice, and stored at ⫺70°C. Frontal sections 20 m were cut at striatal level with a cryostat (Bright Instrument Co. Ltd. Huntingdon, Cambs, UK), mounted on Superfrost Plus slides, and kept frozen at ⫺70°C. Slide-mounted sections were fixed for 30 min in a 0.1 M PBS, pH 7.4, containing 4% paraformaldehyde. Reagents were prepared in TBS (50 mM Tris, 150 mM NaCl, pH 7.5) and each step was followed by rinses in TBS solution. Sera were prepared in 2% BSA. Sections were successively incubated in serum (15 min) followed by the same solution containing the first antibodies (dilutions: Ru 19 1:200, synapsin I 1:200, phosphoERK1,2 1:350, neuron-specific nuclei protein 1:500, and glutamine synthetase 1:1000) for 16 h at 4°C. The sections were then incubated with the appropriate secondary an-
646 tibodies (Alexa 488 anti-mouse IgG 1:100, Texas red antirabbit IgG 1:100). Coverslips were mounted with Mowiol (Calbiochem). Microscopic examination of synapsin I immunostaining was performed using a LCM confocal microscope (Zeiss) and Zeiss LSM 5 image browser software. The same fluorescence calibration was used between the different experimental conditions (laser, rhodamine 543 nm; amplification offset, ⫺0.085; gain detector, 827; pinhole, 1.00 airy units). Double labeling of activated ERK1,2 with astroglial or neuronal markers was examined on a fluorescence microscope (Zeiss axioPlan II) and image processing using the Smart Capture VP and IP lab software. Identical calibrations of the microscope were used between the different experimental conditions. Western Blotting Immunodetection on blots was performed from four to six R6/2 and control animals at 8 and 12 weeks. The striata and the whole cerebral cortex were rapidly dissected, frozen in isopentane on dry ice, and stored at ⫺70°C. Tissue samples were thawed, homogenized in 20 mM NaPi (15.5 mM Na 2HPO, 4.5 mM NaH 2PO 4, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Boehringer Mannheim, Germany) and sonicated for 30 s. Protein concentrations were determined by the Bradford assay, using BSA as standard, and 1/6 (v/v) sample buffer was added (300 mM Tris–HCl, pH 6.8, 10% SDS, 50% glycerol, 0.05% bromphenol blue, and 300 mM DTT). Total protein (5–15 g protein per lane as determined in a preliminary experiment) was electrophoresed through a stacking gel and a 8 –12% resolving gel, and electroblotted (250 mA, 1 h) onto Hybond-P membrane (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). Before immunodetection, blots were stained with Ponceau-S to assess if the same quantity of protein had been loaded in each well and to visualize the transfer efficiency. The blots were then successively incubated in blocking buffer: 1⫻ TBS (50 mM Tris, 150 mM NaCl pH 7.6) containing 3% dry milk (Marvel) for 1 h and blocking buffer containing the primary antibody overnight at 4°C. The dilutions of the primary antibodies used here were dilution 1:750 for anti-phospho ERK1,2; dilution 1:1000 for G257, Ru19, G526, and G555; dilution 1:1500 for anti-calcineurin-B and anti-phosphoneurofilaments; and dilution 1:2500 for anti-synapsin 1. Blots were washed in TBST (1⫻ TBS containing 0.1% Tween 20) for 3 ⫻ 5 min and incubated with affinity-purified peroxidase-labeled secondary antibody (anti-rabbit secondary antibodies, 1/3000; anti-mouse secondary antibodies, 1/5000) for 1 h. The signal was visualized on film with
Lie´ vens et al.
enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). Digitized images of immunoreactive proteins were quantified using a KS300 image analysis system (Carl Zeiss Vision GmbH, Hallbergmoos, Germany) from autoradiographic films. The blots were then washed in a stripping buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 100 mM -mercaptoethanol) for 30 min at 50°C and reprobed using anti-␣-tubulin antibodies (1:1000). The immunoblots were performed separately for each cerebral structure and each time point, permitting comparisons for each structure only between transgenic mice and the age-matched littermate controls. Optical densities for each animal were therefore expressed as percentage of control mean. Data from n animals per condition were averaged and presented as means ⫾ SEM. Statistical analysis of data was performed using a Student’s t test. A significance of P ⬍ 0.05 was required for rejection of the null hypothesis.
ACKNOWLEDGMENTS We thank Professor Paul Greengard and Dr. Hung-Teh Kao for providing us antibodies against the phosphorylated form of synapsin I and Dr. Megumi Moriya for calcineurin-B antibodies. We also thank Dr. Patrick Brundin and Roman Gonitel for helpful discussions. This work was supported by grants from the Wellcome Trust (051897) and the Human Frontiers Science Program (RG0132).
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