Fasudil | Mll Signal

Rho‑associated kinases contribute to the regulation of tau phosphorylation and amyloid metabolism during neuronal plasticity

Hatice Saray1 · Cem Süer1 · Bilal Koşar1 · Burak Tan1 · Nurcan Dursun1

Abstract

Background Neural plasticity under physiological condition develops together with normal tau phosphorylation and amyloid precursor protein (APP) processing. Since restoration of PI3-kinase signaling has therapeutic potential in Alzheimer’s disease, we investigated plasticity-related changes in tau and APP metabolism by the selective Rho-kinase inhibitor fasudil. Methods Field potentials composed of a field excitatory post-synaptic potential (fEPSP) and a population spike (PS) were recorded from a granule cell layer of the dentate gyrus. Plasticity of synaptic strength and neuronal function was induced by strong tetanic stimulation (HFS) and low-frequency stimulation (LFS) patterns. Infusions of saline or fasudil were given for 1 h starting from the application of the induction protocols. Total and phosphorylated tau levels and soluble APPα levels were measured in the hippocampus, which was removed after at least 1 h post-induction period.
Results Fasudil infusion resulted in attenuation of fEPSP slope and PS amplitude in response to both HFS and LFS. Fasudil reduced total tau and phosphorylated tau at residue Thr181 in the HFS-stimulated hippocampus, while Thr231 phosphorylation was reduced by fasudil treatment in the LFS-stimulated hippocampus. S er416 phosphorylation was increased by fasudil treatment in both HFS- and LFS-stimulated hippocampus. Fasudil significantly increased soluble APPα in LFS-stimulated hippocampus, but not in HFS-stimulated hippocampus.
Conclusion In light of our findings, we suggest that increased activity of Rho kinase could trigger a mechanism that goes awry during synaptic plasticity which is reversed by a Rho-kinase inhibitor. Thus, Rho-kinase inhibition might be a therapeutic target in cognitive disorders.

Keywords Rho kinase · Synaptic plasticity · Protein tau · Amyloid peptide · Hippocampus

Introduction

Plasticity is the process by which synapses modulate their strength and form new connections with other neurons. In this way, neurons alter the communication ability with each other in response to specific patterns of electrical stimulation and/or neurotrophic factors. The most studied forms of long-lasting synaptic plasticity in mammals, particularly rodents, are long-term potentiation (LTP) and long-term depression (LTD), which refer to long-lasting increases or decreases, respectively, in synaptic strength. It is widely accepted that the expressed form depends on the kinetics of inward Ca2+currents from an N-methyl-D-aspartate (NMDA) receptor and/or voltage-gated calcium channels. Thus, LTD is induced by slow and low rises in intracellular Ca2+ concentrations, whereas rapid and higher rises are required to induce LTP. The increases in Ca2+ concentrations are followed by the activation of signal transduction pathways involving multiple kinases and phosphatases. Some of these proteins then control synaptic removal from or insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, a critical process for induction of early phases of LTP/LTD [1]. In the case of LTP, protein kinase A and Ca2+ / calmodulin-dependent kinase II (CaMKII) stimulate the insertion of AMPA receptors on the postsynaptic surface [2]. In the case of LTD, activation of protein phosphatases such as calcineurin and protein phosphatase 2A ultimately results in the removal of AMPA receptors from the post-synaptic surface [3]. These early processes can be followed by synthesis of new synaptic proteins. This is achieved when the signals propagate in the nucleus [4, 5] and produce a persistent late form of synaptic plasticity. For instance, a single tetanic stimulus leads only to early LTP, but it can result in the protein synthesis-dependent late phase of LTP as long as stimulation is repeated within a specific window of time [6]. On the other hand, prolonged low-frequency stimulation evokes LTD. Similar to LTP, there is also a persistent late form of LTD that requires transcription and translation [7].
Rho-associated kinases ROCK1 and ROCK2 are serine/ threonine kinases that are downstream targets of the small GTPases RhoA, RhoB, and RhoC. These kinases mediate the control of the actin cytoskeleton by Rho family GTPases that have emerged as key regulators of structural plasticity in response to extracellular signals. In addition, a previous study showed a role of the Rho family of small GTPases in activity-dependent spine enlargement in the hippocampus [8]. ROCKs phosphorylate and thereby activate LIM domain kinases (LIMK) 1 and 2 [9] which would then result in inactivation of an actin de-polymerizing factor (cofilin) [10]. Therefore, ROCK leads to an increased number of stable actin filaments and a reduced actin turnover in the cells, thus counteracting cell growth and the regeneration of cell processes like neuritis [11]. This actin polymerization by the RhoA–ROCK–cofilin pathway is needed for LTP stabilization [12]. Moreover, inhibition of actin polymerization mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD [13]. An important prediction from the aforementioned argument is that ROCK inhibition will disrupt LTP consolidation but consolidate the LTD induction. However, only a restricted number of studies have researched the in vivo effects of ROCK inhibition on LTP, and have given controversial results [14–16].
Recent studies have provided evidence for the presence of direct and indirect mechanisms by which the microtubulestabilizing protein tau and amyloid precursor protein (APP) can modulate synaptic function and plasticity. For instance, tau is transported to dendrites and spines upon phosphorylation of two to three residues together with synaptic proteins [17] that contribute to synaptic plasticity following neuronal activity [18]. Soluble APPα (sAPPα), a product of non-amyloidogenic α-cleavage of APP, enhances learning and memory [19–22] and modulates synaptic function in the neonatal hippocampus [23], and thereby is largely considered to have neurotrophic and neuroprotective properties. Pathologically, the abnormal hyper-phosphorylation of tau (it contains 5–9 mol of phosphate/mole of the protein or more) tau forms neurofibrillary tangles [24, 25], and β- and γ-cleavage of APP are involved in the pathogenesis of several neurodegenerative disorders, including Parkinson’s disease, frontotemporal dementia, and Alzheimer’s disease (AD). Interestingly, many protein kinases and phosphatases which are activated during the induction phase are able to regulate the phosphorylation/dephosphorylation balance of the protein tau [26, 27] and APP processing [28]. Therefore, it is reasonable to conclude that physiological plasticity develops with normal tau phosphorylation and APP processing which can potentially result in the formation of the major hallmarks of AD [29, 30]. Albeit with these findings, so far, conditions that may lead to physiological and pathological tau phosphorylation / APP processing overlap during the formation of memory traces by changing the activity of the signaling pathways responsible for LTP and LTD have remained elusive.
Pharmacological inhibition of the ROCKs decreased tau phosphorylation in cellular models of tauopathy [31] and inhibition of both ROCK isoforms by fasudil lowered β-secretase activity resulting in decreased production of brain Aβ in the APP/PS1 mouse model of AD [32]. In the present study, we investigated the effect of the selective Rhokinase inhibitor fasudil on LTP- and LTD-related changes in tau and APP metabolism.

Materials and methods

Animals

The experiments were carried out on adult male Wistar rats between the ages of 2 and 3 months in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) regarding the protection of animals used for experimental purposes, and with the guiding principles for the care and use of laboratory animals approved by Erciyes University. Rats were randomly assigned to two groups of infusion with saline (n = 16) and infusion with fasudil (n = 16). Eight rats of each infusion group were used in LTP experiments and the remaining rats in LTD experiments. Another set of 6 rats were used to determine the effect of fasudil on baseline synaptic strength. All rats were obtained from the Experimental Research and Application Center of Erciyes University (ERAC, Kayseri, Turkey), housed in a controlled environment (20 °C and 60% humidity with lights on at 8:00 and off at 20:00), and were fed with tap water and Purina rodent chow ad lib. On the day that the experiment was performed, the rats were anesthetized and transported from ERAC to the experimental room for recording.

Chemicals

Fasudil (Lot.: FCB059815) was purchased from the Flourochem Company, and 1 M stock solutions of this drug were prepared by dissolving in a minimal volume of 10 mM DMSO and diluting with saline. The drug was diluted to 10 µM (final concentration) when used. The primary antibodies used in the Western blot analysis were as follows: mouse mAb-Tau (Tau46; 1:1000, 4019, Cell Signaling), rabbit mAb-p-Ser416-Tau (1:1000, D7U2P, 15,013, Cell Signaling), pAb-p-Ser396-Tau (1:1000, 44-752G, Invitrogen, Thermo Scientific), mAb-p-Thr181-Tau (1:500, AT270, MN1050, Invitrogen, Thermo Scientific), Ab-pThr231-Tau (1:500, PHF-6, sc-32276, Santa Cruz Biotechnology), and Ab-β-Actin (1:1000, AC-15, sc-69879, Santa Cruz Biotechnology). The secondary antibodies were mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz Biotechnology) and m-IgGκ BP-HRP (sc-516102, Santa Cruz Biotechnology).

Electrophysiology

Under urethane (1.2 g/kg) anesthesia, a double-barrel glass micropipette (Borosilicate, outer diameter 1.5 mm, length 10 cm; World Precision Instruments) was inserted into the granule cell layer of the Dentate Gyrus (DG) in the right hemisphere (in mm, from bregma: anteroposterior: − 3.0; mediolateral: 2.15; dorsoventral: 2.5–3 mm below the dura). The recording barrel was filled with 3 M NaCl (tip resistance: 2–10 MΩ) for recording of field potentials. The other was filled with saline or fasudil and was connected to a Hamilton syringe (25 μL) driven by a syringe pump (Stoelting Co., Wood Dale, IL, USA). A bipolar tungsten electrode (stainless steel, Teflon-coated, 127 μm in diameter, insulated except at its tips) was used to stimulate the medial perforant path (PP, from bregma, in mm: anteroposterior: − 6.5; mediolateral: 3.8; dorsoventral: 2–2.5 below the dura) of the right hemisphere. The depth of recording and stimulating electrodes (dorsoventral coordinate) was adjusted to obtain a large positive excitatory post-synaptic potential (EPSP) followed by a negative-going population spike (PS) in response to the PP stimulation. After obtaining an input–output curve and a 15-min baseline recording, plasticity was induced by application of a tetanic stimulation consisting of four trains of high frequency stimuli (HFS, 100 Hz, 1 s duration) or by application of low-frequency stimulation (LFS, 1 Hz, 900 pulses). Infusions of %0.9 saline solution or fasudil (10 µM) were given for 1 h starting from the induction, using a Hamilton pump (a 20 μL volume, at a rate of 0.33 μL/min). The ratio of 5-min averages of the EPSP slopes and PS amplitudes at the end of recording was used as a measure of the magnitude of LTP.

Measurement of computed parameters

The slope of the EPSP was calculated as the amplitude change at 20–80% of the voltage difference between the start and the peak of the waveform. The PS amplitude was calculated as the average of the two potential differences of the negative spike peak to the preceding and following positive peaks. The mean value of the EPSP slope and the PS amplitude during baseline recording was chosen to represent 100 percent, and each EPSP slope and PS amplitude was expressed as a percentage of this value. For the analysis, the EPSP slope and PS amplitude values were averaged over 5-min data bins at 70–75 min, and the magnitude of LTP and LTD was calculated based on these averaged fEPSP slopes and PS amplitudes.

Total protein extraction and western blot analysis

The entire hippocampus was dissected out from the brain immediately after recording and lysed in RIPA buffer (sc24948; Santa Cruz Biotechnology, Santa Cruz, California, USA) with thorough homogenization. This buffer was supplemented with a protease inhibitor cocktail, PMSF, and sodium orthovanadate (sc-24948; Santa Cruz Biotechnology) by adding 10 μl of each to 1 mL of RIPA on ice immediately before use. The homogenates were centrifuged at 15,000 g for 20 min at 4 °C. Forty micrograms of total protein from each sample were separated on a 10% SDSpolyacrylamide gel and transblotted onto a PVDF membrane (GE Healthcare 10,600,021 Amersham Hybond).
The membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween20 (PBS-T) for 1 h at room temperature before being incubated with the primary antibodies overnight at 4 °C. The membranes were then washed briefly with TBST and incubated for 1 h at room temperature in a 1:4000 dilution of goat anti-rabbit IgG (sc2004; Santa Cruz Biotechnology) or m-IgGκ BP-HRP (sc-516102, Santa Cruz Biotechnology) secondary antibodies conjugated to horseradish peroxidase label. The bound antibodies were visualized by a Syngene G:Box XR5System (Beacon House, Cambridge, UK) using an enhanced chemiluminescence substrate (Pierce™ ECL Western Blotting Substrate, Catalog number: 32106) according to the manufacturer’s instructions. The blots were subsequently stripped and reprobed with a β-actin mouse monoclonal antibody (AC-15, sc-69879–Santa Cruz Biotechnology) to confirm equal loading of protein samples in the gel. Optical density (OD) of the blots was measured with Image J software (National Institutes of Health, USA). OD of each band was normalized to the corresponding β-actin band. Relative OD (ROD) was calculated by dividing the optical density of the analyzed sample by the first band of each blot.

ELISA

In the present study, we measured hippocampus APP levels, which can be expected to change during or after induction of plasticity because Ca2+ influx is essential to establish longterm plastic changes, using the sAPPα ELISA (Invitrogen KHB0051) kit according to the instruction manual. Briefly, 100 µL of each sample/standard was added to the pre-coated wells and the wells for chromogen blanks were left empty. All wells were incubated for 2 h at room temperature, then washed, and 100 µL of biotin conjugate solution was added into each well except the chromogen blanks and was again incubated at room temperature for 1 h. Afterwards, 100 µL Streptavidin-HRP solution was added into each well except the chromogen blanks, incubated at room temperature for 30 min, washed, 100 µL of stabilized chromogen to each well was added and incubated at room temperature in dark for 30 min. Lastly, 100 µL of stop solution was added and the samples were measured in the Multiskan FC (at 450 nm, Thermo Scientific). The sAPPα values were calculated according to the standard curve. The analytical sensitivity of this assay was < 0.4 ng/mL sAPPα.

Statistical analysis

The EPSP slope and PS amplitude in the I/O curves were analyzed for significance using repeated-measures ANOVA with stimulus intensity (8 levels of intensity) as a withinsubjects factor. LTP and LTD magnitudes were analyzed for significance using the Student’s t test. Statistical analysis of western blot and ELISA results was performed by univariate ANOVA with two factors (protocol: HFS or LFS; treatment: saline or fasudil). Post hoc independent t test of means was performed when appropriate. Significance was set at p < 0.05 (two-tailed).SPSS 15.0 software was used for the statistical analysis.

Results

Input/output curves

I/O curves taken before delivering the HFS (Fig. 1A) and LFS (Fig. 1B) are shown in Fig. 1.As expected, the EPSP slope and the PS amplitude increased systematically as a function of perforant pathway stimulus intensity in all experiments (Intensity effect, all p < 0.001). There was no overall significant difference between pre-LTP I/O curves and between pre-LFS I/O curves, as attested by the absence of a significant Infusion Group effect and no interaction between the two factors. Figure 1C shows the I/O curves taken before and after a 15-min infusion of fasudil. Fasudil infusion has no effect on the increases in fEPSP slope and PS amplitude produced by increasing stimulus intensity as attested by the absence of a significant interaction effect between intensity and fasudil infusion (p > 0.05). These results show that baseline synaptic transmission of dentate gyrus synapses was not different before induction of synaptic plasticity and was not dependent on Rho kinase activity.

Neuronal plasticity

Figure 2 shows the time course of the fEPSP slope (A and B) and the PS amplitude (C and D) before and after a strong tetanus protocol (A and C) and before and after prolonged low-frequency stimulation (B and D). The averages of the fEPSP slopes and the PS amplitudes, between 70 to 75 min, were used as a measure of the synaptic and somatic components of neuronal plasticity, respectively. The fEPSP slope potentiated by 120.6% ± 4.4% of baseline 1 h after HFS, but did not change 1 h after LFS (98.0% ± 2.3%), showing failed expression of a significant LTD in the saline infusion group. However, in the fasudil infusion group, a significant LTD of fEPSP slope (72.0% ± 4.8%) was obtained, while HFS did not evoke any potentiation of fEPSP slope (96.2% ± 5.41%). On the other hand, PS amplitude potentiated after both HFS (218.4% ± 7.6%) and LFS (150.6% ± 8.2%) in the saline group, while less potentiated PS amplitude was obtained in the fasudil infusion group (HFS: 116.2% ± 19.2%, LFS: 119.2% ± 11.2%). The statistical analysis of fEPSP slopes by a 2 × 2 ANOVA showed that intra-hippocampal administration of fasudil attenuates synaptic plasticity in both protocols (Protocol effect: F1,28 = 25.188, p < 0.001), fasudil effect: F1,28 = 32.739, p < 0.001, interaction: F1,28 = 0.36, p > 0.05), causing impaired LTP and facilitated LTD. According to the analysis of the PS amplitude, the effect by the fasudil was found to be slightly different. There was again a significant effect of the protocol (F1,28 = 6.760, p = 0.015) and a significant effect of the fasudil (F1,28 = 28.739, p < 0.001), but interaction between the two factors was found to be significant (F1,28 = 8.061, p < 0.001). These data indicate protocol dependency of the impairment of somatic plasticity by fasudil. Pair-wise comparisons revealed that the PS amplitude was significantly lower in the fasudil-infused rats compared to the saline-infused rats after stimulation with HFS (t14 = 4.82, p < 0.001), but not with LFS (p > 0.05).

Tau phosphorylation

Figure 3 shows the optical densities of blot bands normalized in front of actin for total and phosphorylated tau in the Thr181, Ser202–Thr205, Thr231, and S er416 epitopes of Fig. 2 Effect of fasudil, a Rho-kinase (ROCK) inhibitor, on HFSinduced and LFS-induced neuronal plasticity. The time course of the fEPSP slope as a function of synaptic strength (A and B) and the PS amplitude as a function of neuronal output (C and D) before and after a strong tetanus protocol (Left panel; 100 Hz, 4 times, 5-min intervals; arrows) and before and after prolonged low-frequency stimulation (Right panel; LFS, 1 Hz, 900 pulses; 0–15 min) are presented. Saline or fasudil (10 μM) was infused starting from time point zero at a rate of 0.33 μL/min (long black line). Each fEPSP and PS is expressed as a percentage of the average from the baseline period (between − 15 and 0 min). Note that the averages of the fEPSP slopes and PS amplitudes between 70 to 75 min are lower in the fasudil infusion group (n = 8/group, Student’s t test, *p < 0.01). E Representative traces of field potential recordings made immediately before the application of HFS/LFS (black traces) and at the end of the recording (gray traces). E and F Representative traces of field potential recordings made immediately before the application of LFS (black traces) and at the end of the recording (gray traces). Note that representative trace of a rat in the fasudil infusion group shows decreased LTP by HFS (E, as indicated by decreased ratio of the fEPSP slope and PS amplitude in the gray trace to those in the black trace) and increased LTD by LFS (F, as indicated by decreased ratio of the fEPSP slope in the gray trace to that in the black trace)
Fig. 3 Effect of fasudil, a Rho-kinase (ROCK) inhibitor, on HFSinduced and LFS-induced tau phosphorylation in the hippocampus. Optical densities of blot bands normalized in front of actin for total (A) and phosphorylated tau at T hr181 (B), Ser202–Thr205 (C), Thr231 (D), and Ser416 (E) epitopes of the saline- and fasudil-infused group at least 1 h after application of HFS/LFS. Bars represent the arithmetic mean of six samples per group ± standard error of the mean. Univariate ANOVA with two factors followed by independent t test when appropriate, *p < 0.05, **p < 0.01, ***p < 0.001. F Representatives membrane indicating that fasudil decreased phosphorylation of tau at Thr181 (in the HFS-stimulated hippocampus) and Thr231 epitopes (in the LFS-stimulated hippocampus), but increased the phosphorylation at Ser416 in the HFS/LFS-stimulated hippocampus. f fasudil; s saline the saline- and fasudil-infused groups at least 1 h after application of HFS/LFS. Figure 3A shows that plasticityrelated total tau levels were decreased by the fasudil. An univariate ANOVA yielded a significant main effect of treatment (F1,23 = 4.965, p = 0.038), significant main effect of induction protocol (F1,23 = 5.034, p = 0.036) but no significant effect of interaction (F1,23 = 2.079, p = 0.165). A post hoc independent t test of means indicated that fasudil significantly decreased total tau levels in HFS-stimulated hippocampus (t10 = 2.290, p = 0.045). This effect of the fasudil was at the limit of significance for LFS-stimulated hippocampus (t10 = 1.863, p = 0.090).
Figure 3B shows that fasudil increases the plasticityrelated phosphorylation of Tau at Ser416 regardless of the induction protocol. This was confirmed by a significant main effect of treatment (F1,23 = 16.092, p = 0.001) together with non-significant main effect of the protocol (F1,23 = 2.072, p = 0.165) and non-significant interaction effect (F1,23 = 0.107, p = 0.747). Post hoc independent t test of means indicated that levels of pSer416-tau were significantly higher in HFS-stimulated hippocampus (t10 = 2.315, p = 0.043) and LFS-stimulated hippocampus (t10 = 5.267, p < 0.001) under fasudil infusion. In saline infusion experiments, but not in fasudil infusion experiments, the HFS resulted in increased tau phosphorylation at this residue compared with the LFS (t10 = 3.179, p = 0.01).
Figure 3C shows that fasudil reduced tau phosphorylation at T hr181 epitope in HFS-stimulated hippocampus. This was confirmed by a significant main effect of the treatment (F1,23 = 11.199, p = 0.003) and a significant interaction effect between treatment and induction protocol (F1,23 = 5.167, p = 0.034) together with no significant effect of the induction protocol (F1,23 = 2.089, p > 0.05). Post hoc independent t test of means indicated that levels of p Thr181-tau in HFS-stimulated hippocampus was significantly decreased by fasudil (t10 = 4.482, p = 0.001). In addition, HFS resulted in increased tau phosphorylation at this residue in saline infusion experiments compared with LFS given under saline (t10 = 3.276, p = 0.008) and fasudil (t10 = 2.669, p = 0.024).
Figure 3D shows that fasudil modulates plasticityrelated tau phosphorylation at Thr231 epitope. This was confirmed by a significant main effect of induction protocol (F1,23 = 23.438, p < 0.001) and significant interaction effect between induction protocol and treatment (F1,23 = 6.217, p = 0.027) with no significant main effect of treatment (F1,23 = 0.010, p > 0.10). Post hoc independent t test of means indicated that fasudil significantly decreased the levels of pThr231-tau in LFS-stimulated hippocampus (t10 = 3.051, p = 0.012). In addition, HFS resulted in increased tau phosphorylation at this residue compared with LFS in both saline infusion (t10 = 2.425, p = 0.036) and fasudil infusion experiments (t10 = 4.191, p = 0.002). In contrast to these findings, phosphorylation in Ser202–Thr205 was not affected by treatment or protocol (Fig. 3E, all ps > 0.1).

Amyloid precursor protein

Non-detectable sAPPα levels were found in the non-stimulated hippocampus, while the HFS-stimulated hippocampus had higher sAPPα levels compared the LFS-stimulated hippocampus (18.26 ± 1.70 ng/mL vs. 10.42 ± 0.71 ng/ mL; Z: 2.72; p = 0.006). Moreover, 10 μM fasudil significantly increased sAPPα in the LFS-stimulated hippocampus (20.44 ± 1.16 ng/mL, Z: 2.88; p = 0.004), but not in the HFS-stimulated hippocampus (17.39 ± 0.86 ng/mL). These results indicate that HFS and to lesser extent LFS provokes non-amyloidogenic cleavage of APP, resulting in an increase in sAPPα levels in the hippocampus (Fig. 4).

Discussion

This study investigated the effect of the selective the Rhokinase inhibitor fasudil on LTP- and LTD-related changes in tau and APP metabolism. Rho/Rho kinases pathway plays a prominent role in the regulation of actin dynamics and has been implicated in post-synaptic morphological changes such as remodeling, addition, and elimination of synapses [33, 34]. However, relatively little is known about the consequence of Rho kinase inhibition on LTP and LTD induction. Herein, we show that pharmacological inhibition of ROCK by fasudil results in a depression of synaptic and somatic response in the dentate gyrus of hippocampal formation. This result agrees with a previous study in mice lacking LIM kinase-1, a downstream kinase of ROCK, in which both impaired long-term memory and late-phase LTP were observed [35]. Additionally, we have shown that fasudil down-regulates HFS-induced Thr181 phosphorylation and LFS-induced Thr231 phosphorylation, and up-regulates sAPPαlevels, suggesting its neuroprotective effect in models of Alzheimer’s disease [36], amyotrophic lateral sclerosis [37], and Parkinson’s disease [38].
A hippocampal slice study demonstrated that Y-27632, another specific inhibitor of Rho Kinase, increased the magnitude of LTP in the CA1 region [39]. However, we found that LTP was impaired by fasudil in the dentate gyrus. Fasudil and Y-27632 inhibit both ROCK I isoform, which is mainly expressed in the lung, liver, spleen, kidney and testis, and ROCK II isoform, which is distributed mostly in the brain and heart [40] at similar concentrations. Therefore, we thought that the observed effect is mainly due to the inhibition of ROCK1. However, these two ROCK inhibitors showed differential effects on axonal regeneration after injuring the optic nerve [41]. Moreover, Y-27632 enhanced the size of the post-synaptic density and maintains the dynamic process of spine shaping in rat hippocampal organotypic cultures [42, 43], suggesting an effect that promotes structural LTP. On the other hand, no study has investigated the role of Rho kinases in LTD. Herein, we show that pharmacological inhibition of ROCK by fasudil increases LFS-induced depression of synaptic strength without change of neuronal output, suggesting a role of the small GTPases in up-regulation of synaptic strength.
The findings of attenuated response to both HFS and LFS suggest that Rho kinases may be involved in hippocampal learning and memory abilities. Because fasudil impaired LTP and facilitated LTD, it can be thought that fasudil could be beneficial in disease models showing unbalanced synaptic plasticity. However, one can speculate only about the relationship between memory abilities and the results of our study as no behavioral test was conducted in the present study. Previous studies suggested a role for the Rho–ROCK pathway in hippocampus-dependent spatial memory. For instance, infusion of Rho kinase inhibitor Y27632into the lateral ventricle for 7 days enhanced spatial memory, but neither anxiety nor object recognition was changed in 8–9-week-old male C57BL/6 mice [44]. A similar result with spatial memory was reported for intra-hippocampal infusion of an inhibitor of the downstream effector kinase p160 ROCK [33]. Fasudil had beneficial effects on learning and memory dysfunction of rats impaired by Aβ1–42 [45].
ROCK2 is a downstream target of the small GTPases RhoA and RhoB at the concentration used in the present study. RhoA and RhoB of the Rho GTPase family members were activated by LFS, while the induction of LTP by HFS was associated with specific activation of RhoB [46]. This illustrates that these GTPases are potential mediators of synaptic transmission in the hippocampus, and raises the possibility that RhoB may play a role in plasticity in hippocampal synapses during LTP [46]. Knockout mice specifically lacking ROCK2 were normal in gross brain anatomy, but were impaired in both basal synaptic transmission and hippocampal LTP [47]. Peripheral administration of the ROCK inhibitor hydroxylfasudil improves spatial learning and working memory in the rodent model [48]. Thus, fasudil may prevent the development of abnormal behavior and spine loss induced by chronic stress, by blocking ROCK activity [49]. Despite HFS- and LFS-stimulated α-secretase processing of APP, the vast majority of studies have shown that increased calcium influx, and elevated cytosolic calcium levels, lead to a net increase in Aβ production [50–52]. Herein, we showed by ELISA that sAPPα increased 1 h after HFS and to a lesser extent after LFS, compared to a non-stimulated hippocampus. This finding suggests an increase in α‐secretases activity during synaptic plasticity because the sAPPα is the N-terminal fragment generates upon proteolytic processing of APP by this enzyme. NMDA receptors are key molecular complexes involved in synaptic plasticity induced by stimulation at different frequencies. In primary cortical neurons, conflicting findings have been reported. Activation of synaptic NMDA receptors lasting 15 min stimulated α-secretase-mediated APP cleavage [53], while sAPPα release was decreased by chronic stimulation of NMDA receptors for 24 h before harvesting neurons for analysis of APP fragments [54]. Thus, it is possible that prolonged NMDA receptor stimulation was not necessarily physiologically relevant but instead reflected a more pathophysiological situation. It was recently demonstrated that sAPPα promotes synthesis of activity-related cytoskeletalassociated protein Arc, which is an immediate early gene capable of modulating LTP [55] and LTD [56] through regulation of AMPA receptor localization in hippocampal neurons [57]. Therefore, it is likely that α-secretases activity could result in transcriptional changes in protein Arc expression, contributing to synaptic plasticity. Rapid activation of the genetic machinery can be a key mechanism underlying the enduring modification of neural networks required for the stability of memories [58–61].
Previous studies have shown that ROCK inhibition might serve as a brake to prevent Aβ production [62, 63]. We found that inhibition of ROCK by fasudil increases sAPPα levels in the dentate gyrus. Absence of such an increase after HFS points out that protein phosphatases may mediate this effect because PP2A plays an essential role in the molecular events that underlie LTD at glutamatergic synapses in vivo [64]. Regulation of PP2A has also been connected to APP. In addition PP2A inhibits the generation of toxic peptide Aβ by influencing the ability of β-secretase to cleave APP [65, 66]. Previous studies correlated the decreased sAPPα levels with the cognitive deficits observed in Alzheimer’s disease [67–70]. On the other hand, our results are consistent with a functional role for Rho kinases in tau phosphorylation during the physiological activation of excitatory synapses.
Although Rho kinases are able to phosphorylate Tau at Thr245, Thr 377, Ser409, and Ser262 to some extent in vitro [71], the present study indicates that epitopes phosphorylated by Rho kinases could be different during a physiologically relevant context of synaptic plasticity. These effects by fasudil seem to be epitope specific because the phosphorylation at Ser202–Thr205 epitopes is not affected by the fasudil.
In conclusion, we report the first results showing that fasudil favors the induction of LTD over LTP and that it induces plasticity-associated tau hypo-phosphorylation and increases expression of soluble APPα. The modification of synaptic strength produced by the LTP and LTD is widely thought to underlie cellular mechanism of learning and memory. In light of our findings, we suggest that increased activity of Rho kinase could trigger a mechanism that goes awry during synaptic plasticity which is reversed by the Rho-kinase inhibitor. Thus, Rho-kinase inhibition might be a therapeutic target in many neurodegenerative disorders.

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