Systemic Administration of Substance P Recovers Beta Amyloid-Induced Cognitive Deficits in Rat: Involvement of Kv Potassium Channels

Reduced levels of Substance P (SP), an endogenous neuropeptide endowed with neuroprotective and anti-apoptotic properties, have been found in brain and spinal fluid of Alzheimer's disease (AD) patients. Potassium (K+) channel dysfunction is implicated in AD development and the amyloid-β (Aβ)-induced up-regulation of voltage-gated potassium channel subunits could be considered a significant step in Aβ brain toxicity. The aim of this study was to evaluate whether SP could reduce, in vivo, Aβ-induced overexpression of Kv subunits. Rats were intracerebroventricularly infused with amyloid-β 25–35 (Aβ25–35, 20 µg) peptide. SP (50 µg/Kg, i.p.) was daily administered, for 7 days starting from the day of the surgery. Here we demonstrate that the Aβ infused rats showed impairment in cognitive performances in the Morris water maze task 4 weeks after Aβ25–35 infusion and that this impairing effect was prevented by SP administration. Kv1.4, Kv2.1 and Kv4.2 subunit levels were quantified in hippocampus and in cerebral cortex by Western blot analysis and immunofluorescence. Interestingly, SP reduced Kv1.4 levels overexpressed by Aβ, both in hippocampus and cerebral cortex. Our findings provide in vivo evidence for a neuroprotective activity of systemic administration of SP in a rat model of AD and suggest a possible mechanism underlying this effect.

Literature data show that Ab 25-35 -treated rodents develop behavioral impairments reminiscent of AD physiopathology [17], particularly spontaneous alternation, passive avoidance and watermaze learning deficits [16,18,19]. At CNS level, SP-immunoreactive cells are distributed in several brain regions implicated in the control of cognition and emotionality [20].
Evidences from literature suggest that SP facilitates cognitive functions when locally administered into particular brain regions or after systemic administration in rats [21][22][23]. Interestingly, consistent data indicate that SP plays a crucial role not only in memory formation and reinforcement but also in preventing brain aging-related memory decline [24,25]. Kowall and co-workers [26] demonstrated that SP, co-administered together with betaamyloid (Ab) peptide into the rat cerebral cortex, prevented the amyloid-induced neuronal loss, which is considered one of the most important histopathological hallmark of AD.
Research on the mechanisms by which Ab mediates neurotoxicity has made great strides over the last decade. Extensive growing evidence suggest that Ab alters cellular homeostasis and neuronal signalling through several mechanisms crucially involving the potassium (K + ) channels modulation [27][28][29][30]. An increased activity of plasma-membrane voltage-gated potassium (Kv) channels can induce cell death, suggesting that these channels are involved in the aetiology of Ab-induced toxicity and neuronal death [31][32][33].
In cerebellar granule neurons the increase in I kA current density induced by Ab 1-40 is due to the selective up-regulation of Kv4.2 and Kv4.3 a-subunits expression [34], while exposure of hippocampal neurons to Ab 1-42 leads to an increase in Kv3.4 protein expression [32]. These results highlight the crucial role played by selective voltage-dependent potassium channels in the aetiology of Ab-induced toxicity, although the identity of the Kv subunits modulated by Ab depends on different examined neurons. In addition to these in vitro evidence, Pan and co-workers [35] reported a significant increased expression of Kv2.1, Kv1.4 and Kv4.2 subunits after intracerebroventricular (i.c.v.) injection of Ab [25][26][27][28][29][30][31][32][33][34][35] in the rat hippocampus and cerebral cortex.
On the other hand, potassium channel abnormalities have been reported in both neural and peripheral tissues of AD patients. In particular, K + channel dysfunction has been demonstrated in fibroblasts [36] and platelets [37] of AD patients and post-mortem studies showed alterations of K + channel expression in the brain [38,39]. Moreover, an aberrant glutamate-dependent modulation of Kv1.3 channels was recently demonstrated in T lymphocytes from AD patients [40].
Taken together, these findings demonstrate that alteration of Kv channel subunits expression and activity are involved in learning and memory dysfunction and in AD. We recently demonstrated that SP is able to significantly reduce in vitro the Ab-induced overexpression of Kv subunits [41]. Based on these results, in the present study we investigated whether treatment with SP can help the recovery from memory dysfunction induced by i.c.v. infusion of Ab [25][26][27][28][29][30][31][32][33][34][35] in rats, and whether this potential protective effect could be related to the SP modulation of Kv channel subunits expression.

Animals
Subjects. Male Sprague-Dawley rats (280-320 g at the time of surgery; Charles River Laboratories, Calco, Italy) were group housed and maintained in a temperature-controlled environment (20uC61uC) under a 12-h light/12-h dark cycle (0700-1900 h lights on) with food and water available ad libitum.

Ethics Statement
All procedures involving animal care or treatments were approved by the Italian Ministry of Health (Rome, Italy) and performed in compliance with the guidelines of the European Communities Council Directive of 24 November 1986 (86/609/ EEC). All efforts were made to minimize animal suffering and to reduce the number of animals used.

Morris Water Maze
Task procedures. Twenty eight days after surgery, the rats were handled 1 minute per day for 3 days before training on the MWM task. The water maze was a circular tank, 1.83 m in diameter and 0.58 m in height, filled with water (23-24uC) to a depth of 20 cm. A transparent Perspex platform (20-25 cm) was submerged 2.5 cm below the surface of the water in the northwest quadrant of the maze during training and could not be seen by the rats. The maze was located in a room containing several visual cues. The experiments were performed accordingly to the procedure previously described [43][44][45].
Spatial Training (Acquisition). Day 1-3. Rats were given a daily training session of 4 trials (60 sec each one) for 3 consecutive. On each trial, the animal was placed in the tank facing the wall at one of the four designated start positions and allowed to escape onto the hidden platform. If an animal failed to find the platform within 60 seconds, it was manually guided to the platform. The rat was allowed to remain on the platform for 15 seconds and was then placed into a holding cage for 25 seconds until the start of the next trial. The time each animal spent to reach the platform was recorded as the escape latency.
Probe (Memory retention). Day 4. The retention of the spatial training was assessed 24 hours after the last training session. Rats were returned to the water maze for a 60-second probe trial (in which the platform was removed) starting from a new position different from the starting points used during acquisition. The parameter measured from the probe trial was the time spent in the quadrant containing the platform during training (target quadrant).
Reversal learning. Day 5-8. Rats were given a daily training session of 5 trials (60 sec each one) for 4 consecutive days. The platform position was changed every day (day 5 north, day 6 northwest, day 7 northeast, day 8 northwest). On each trial, the animal was placed in the tank facing the wall at one of the four designated start positions and allowed to escape onto the hidden platform. If an animal failed to find the platform within 60 seconds, it was manually guided to the platform. The rat was allowed to remain on the platform for 10 seconds and was then placed into a holding cage for 15 seconds until the start of the next trial. The time each animal spent to reach the platform was recorded as the escape latency.
Behavioral data from the training, probe and reversal learning were acquired and analyzed using an automated video-tracking system (Smart, Panlab, Harvard Apparatus). The escape latency for the spatial training and reversal learning working memory and the amount of time rats spent in the target zone in the probe test were analyzed.

Primary cultures
Hippocampal cultures were prepared from brain of embryos Sprague-Dawley rats (Charles River) at embryonic day 17-18 (E17/E18), as previously reported [46]. Briefly, hippocampus was dissected out in Hanks' balanced salt solution buffered with Hepes and dissociated via trypsin treatment. Cells were plated at 1610 6 cells on 3.5-cm dishes precoated with poly-L-lysine. After 2 days of culturing in neurobasal medium with B-27 supplement (0.5 mM Lglutamine, 1% antibiotic penicillin/streptomycin), half of the medium was changed every 3-4 days. All experimental treatments were performed on 12-day in vitro (DIV) cultures in Neurobasal +K B27 fresh medium.

Preparation of enriched membrane protein extracts and Western blot analysis
Rat cortex and hippocampus membranes were prepared by using a simplified version of protocols previously described [35]. All procedures were performed at 4uC, and all solutions contained a mixture of protease inhibitors (1 mg/ml leupeptin and 16 protease inhibitor mixture) to minimize proteolysis. By using a glass homogenizer, tissues were homogenized in a hypoosmotic buffer containing 50 mM Tris HCl (pH 7.4). Nuclei and debris were pelleted by centrifugation at 1,000 g for 10 min. The supernatant was then centrifuged at 100,000 g for 1 hr and the resulting pellet was resuspended in lysis buffer (1% NP40, 50 mM Tris-HCl (pH 8) and 0.5 M Na 2 EDTA. The protein concentration of the resulting enriched membranes was then determined by using Bio-Rad protein assay solution with bovine serum albumin (BSA) as a standard. Solubilized membranes were stored frozen at 280uC until use.
For Western blot analyses, equal amounts of proteins (50 mg/ lane) were loaded and run on standard 4-12% sodium dodecyl sulfate (SDS)-polyacrylamide gels in MOPS electrophoresis buffer according to manufacturer protocols (NuPage). After staining with Ponceau S to verify uniformity of protein loads/transfer, the membranes were analyzed for immunoreactivity. Incubation with primary antibodies was performed overnight at 4uC (b-actin, Sigma 1:5000, rabbit anti Kv1.4, Kv2.1, and Kv4.2, Sigma 1:500). Incubation with secondary antibodies peroxidase-coupled antimouse or anti rabbit (Sigma) was performed for 1 h at room temperature. Immunoreactivity was developed by enhanced chemiluminescence (ECL system; Amersham, Arlington Heights, IL) and visualized by autoradiography. For analysis of the Western blotting data, densitometric analysis was performed using Scan Analysis software.

Immunofluorescence
After behavioral analysis, rats were anaesthetized with sodium pentobarbital and intracardially perfused with a saline solution followed by a 4% paraformaldehyde solution in phosphate buffer saline (PBS). Brains were post-fixed for 24 hours, transferred in 30% sucrose/PBS solution at 4uC until it sank and sectioned at a sliding freezing microtome (Leica, Wetzlar, Germany). Forty micrometers coronal sections were collected in 0.05% sodium azide/PBS in a culture well and stored at 4uC until usage. Double immunofluorescence was performed by sections incubation in a mix solution of the following primary antibodies: mouse anti-NeuN (dilution 1:100; Millipore) and rabbit anti-Kv1.4 (1:100; Sigma) in PBS containing 0.3% Triton-X for 2 days at +4 C. After three washes, sections were incubated in a mix of the following secondary antibodies: Alexa Fluor 488 conjugated donkey anti-mouse IgG and Alexa-Fluor 555 conjugated donkey anti-rabbit IgG (dilution 1:200; Molecular Probes, Eugene, OR, USA) for 2 hours at room temperature. The last step was three washes in PBS and 40 min incubation with the Hoescht solution (1 ng/ml, Molecular Probes, Invitrogen) for nuclei visualization. After that, sections were mounted on slides, air dried and coverslipped using gel mount (Biomeda Corp., Foster City, CA, USA).
Primary hippocampal neurons, after 48 hs experimental treatment, were washed in PBS and fixed in 4% paraformaldehyde (w/ v in PBS) for 15 min at room temperature. Fixed cells were washed in PBS, pH 7.4, permeabilized using 0.1% Triton X100-Tris-HCl, pH7.4 for 5 min and then incubated with primary polyclonal antibodies raised against the anti-Kv1.4 (1:200) subunit and mouse anti-NeuN (1:500) in PBS at 4uC overnight. Cells were then washed in PBS and incubated with a Alexa Fluor 488 (1:800) conjugated donkey anti-mouse IgG and Alexa-Fluor 555 conjugated donkey anti-rabbit IgG (1:500) for 60 min at room temperature. Nuclei were stained with Hoechst for 5 min at room temperature.
Immunofluorescence was examined under a confocal laser scanning microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany). Confocal acquisition settings were identical among the different experimental cases. For figure production, brightness and contrast of images were adjusted by applying the same values, and by taking care to leave a tissue fluorescence background for visual appreciation of the all fluorescence intensity features and to help comparison between the different experimental groups. For hippocampal cultures, neurons were selected and acquired by NeuN identification. Final figures were assembled by using Adobe Photoshop 7 and Adobe Illustrator 10. Boundaries and subdivisions of cortical and hippocampal structures were identified on the base of the Hoescht histofluorescence using a rat brain atlas (Paxinos). Image acquisitions were performed on frontal cortex (somatosensory area) and CA1 hippocampal region.

Image analysis
Image analysis was performed by using Imaris Suite 7.4H (Bitplane A.G., Zurich, Switzerland) software (surface and spot modules) on six different images derived from each experimental group. Image analysis was performed under visual control to determine thresholds that subtracts background noise and takes into account cellular structures. During image processing, the images were compared with the original raw data to make sure that no structures seen in the original data series were introduced or that structures present in the original data series were not removed. To evaluate the cell bodies and neuropil areas, their relative fluorescence intensity, vesicles diameters and relative fluorescence intensity, images were zoomed and two different masks were manually drawn. The first mask type was drawn using the NeuN channel by considering only cells clearly displaying a nucleus to selectively identify neurons. The second type was drawn only on neuropil portions by taking care to exclude cell bodies. After which the Hoescht and NeuN channels were deleted and measures were performed.

Statistical Analysis
Statistical analysis was performed using SPSS 11.0.0 for Windows (SPSS Inc., USA). All results are expressed as mean 6 SEM, with n the number of independent experiments. Results obtained from behavioral studies were analyzed with one-or twoway analysis of variance (ANOVA) with repeated measures when appropriate. Post hoc comparisons were performed using Tukey's test. Data from Western blot analysis and immunofluorescence were performed by ANOVA, followed by Tukey's test for multiple comparisons. The significance level was set at p,0.05 (*) and p,0.01 (**).

Morris Water Maze
To examine whether treatment with SP can result in recovery from memory deficit induced by i.c.v. injection of Ab 25-35 , the MWM task was carried out 4 weeks after Ab 25-35 inoculation (Figure 1a). Rats were early infused with Ab 25-35 or its vehicle and administered i.p. with SP or its vehicle for 7 days starting from the day of the surgery. After 4 weeks all groups were trained for 3 consecutive days on spatial training procedure of 4 trials session per day. During these sessions, rats had to learn to localize a hidden platform set always in a fixed place. Figure 1b shows that all rats, regardless of any treatment, were equally able to acquire the cognitive task. Indeed, ANOVA for repeated measures (with trials as repeated measures) revealed a trial effect (F (11,418)  The effect of Ab 25-35 inoculation on long-term memory was analyzed 24 h after the last acquisition day on a single 60 secprobe trial (Figure 1c,d). During the probe test the platform was removed from the water maze tank and the time spent in the target quadrant where the platform was previously located was measured as a parameter of long-term memory retention. Oneway ANOVA did not reveal any statistical significant Ab [25][26][27][28][29][30][31][32][33][34][35] (F (1,38) = 0.045; P = 0.84), and SP (F (1,38) = 2.28; p = 0.14) treatment effect, or a statistically significant interaction between Ab 25-35 and SP treatments (F (1,38) = 2.26; p = 0.14). Post hoc comparisons performed on the interaction revealed a statistically significant difference between Ab 25-35 /Sal and Ab 25-35 /SP treated rats (p,0.05), thus showing that SP administration is able to reverse the impairing effects induced by Ab on long-term memory retention.
Twenty-four hours after the probe test, rats were tested for reversal learning capabilities during a daily session of 4 trials for 4 consecutive days. The hidden platform were relocated in a new position every day, thus rats had to learn to quickly adjust their searching strategy. Figure 1e shows the effect of SP administration on reversal learning performances. ANOVA for repeated measures revealed a trial effect (F (15,570) = 11.77; p,0.0001) and a statistically significant interaction between trials and Ab 25-35 treatment (F (15,570) = 2.03; p,0.012) but did not reveal any statistically significant interaction between trials and SP treatment Results obtained by Western blot analysis confirmed the previously reported up-regulation of Kv1.4 and Kv2.1 in hippocampus and Kv4.2 in cerebral cortex. However, SP was able to modify only the increased expression of Kv1.4 protein, while SP treatment did not change Kv2.1 and Kv4.2 protein levels in both tissues ( Fig. 2a and 2b).
Western blot analysis revealed that Kv1.4 protein was increased by about 5 times in hippocampus (Fig. 3a) and 3 times in cerebral cortex (Fig. 3b)  Image analysis confirmed these qualitative observations (Figure 4a and 4b).
Effect induced by SP on the Ab-dependent overexpression of Kv1. 4

Discussion
The present study provides the first in vivo evidence, to our knowledge, for a protective effect of SP against the cognitive impairment induced by infusion of Ab [25][26][27][28][29][30][31][32][33][34][35] in rats and it identifies Figure 1. Neuroprotective effects of SP on memory impairments induced by intracerebroventricular injection of Ab25-35. (a) Timeline and experimental design. All animals received an infusion (i.c.v.) of Ab [25][26][27][28][29][30][31][32][33][34][35] (2 mg/ml; 10 mL injection volume) or its vehicle (PBS 10 mL injection volume) and daily treated (7 days) with SP (50 mg/ml/Kg, i.p.) or its vehicle (saline solution 0.9%, i.p.). On the 31 st day after surgery rats were given a daily training session of 4 trials for 3 consecutive days (days 31 st -33 rd ). On the 34 th day after surgery the retention of the spatial training was assessed during a 1 min probe trial. On the 35 th day after surgery rats were given a daily training session of 5 trials for 4 consecutive days (days 35 th -38 th ).  Kv channel subunits as possible modulator of SP effects on memory rescue. To evaluate whether SP was able to prevent the cognitive dysfunction in a non-transgenic model of AD, we performed the MWM task, which is considered one of the best choice to investigate cognitive impairments in rodents. This task permits to evaluate the most affected cognitive processes in the onset symptoms associated with AD [47] and when based on a serial reversal procedure, is considered a useful tool to investigate episodic-like memory in rodents in respect to one-trial behavioral tasks [48]. In the present in vivo study, we found that all rats were equally able to learn and acquire the task and had similar motor and visual capabilities. However, we found that SP-treated rats show an ameliorated performance when long-term memory was tested, as highlighted by the fact that SP-treated rats remember better where the platform was during the training, since they spend more time swimming in the quadrant where the platform was (target quadrant) than Ab 25-35 -infused rats. Interestingly, we found that Ab 25-35 -injected rats present deficits in the reversal learning capabilities, which have been roughly compared to episodic-like memory in humans [48,49]. Impairment of episodic memory has been found to be a marker for future development of AD based on convergent data from asymptomatic AD-related mutation carriers, longitudinal studies of patients with mild cognitive impairment, and epidemiological studies of incident AD cases [50,51].
Notably, systemic SP treatment was able to improve long-term memory and reversal learning performances [20,24,52,53], confirming the notion that SP easily crosses the blood-brain barrier [54].
The present findings confirm the emerging notion that specific memory deficits, rather than a general learning dysfunction, may occur in the early onset of AD. Previous studies in transgenic mice, indeed, report that deficits in spatial memory but not in learning capabilities early occur [55][56][57]. Although the mechanisms underlying this phenomenon have still to be clarified, taken together these results are consistent with human data, reporting that the main clinical symptom of AD is the early memory impairment, which then develops in dementia during the progression of the pathology [58,59].
In the aetiology of Ab neuronal death and AD, recent evidence points to an increase in voltage-gated potassium (Kv) channel current and to an up-regulation of selective voltage-gated potassium channels subunits, depending on different examined neurons [33]. In addition to the well known neuroprotective properties of SP, we recently demonstrated that SP was able to decrease Ab 25-35 -induced neuronal death in rat cerebellar granule neurons through a selective regulation of the Kv4.2 and Kv4.3 channel subunits overexpressed by Ab 25-35 exposure [41]. We also demonstrated that, in the same neurons, SP stimulates nonamyloidogenic APP processing, thereby reducing the possibility of generation of toxic Ab peptides in brain [60]. It thus appears that the toxic effect of Ab and the neuroprotective effect of SP may be ascribed, at least in part, to their opposite actions on these currents. In support of this hypothesis is the evidence that i.c.v. injection of Ab 25-35 significantly impairs spatial memory of rats in the Morris water maze and increased the expression, both at mRNA and protein levels, of Kv2.1 and Kv1.4 in hippocampus and of Kv4.2 in cortex of Ab-treated rats [35].
In the present study, we confirmed the up-regulation of Kv2.1, Kv1.4, and Kv4.2 subunits in Ab treated rats.
However, SP was able to interfere only with Kv1.4 protein dysregulation. The diversity of Kv channels underlies much of the   variability in the active properties between different mammalian central neurons [61]. The different structure of these proteins could be responsible for the selective modulation of Kv1.4 upregulation, without affecting Kv2.1 and Kv4.2 expression. In addition, the activity of Kv channels can be modulated indirectly via signal transduction pathways leading to modifications of Kv channel function and SP receptor activation involved different transduction pathways [62].
As revealed by Western blot analysis, SP significantly reduced, in the hippocampus, the over-expression of Kv1.4 subunits induced by Ab treatment. In the whole cerebral cortex the mild Ab-induced increase in Kv1.4 subunits was not significantly affected by SP treatment.
Interestingly, when specifically analyzing the tissue distribution of this protein in cerebral cortex by immunofluorescence, we found that in Ab-treated rats, Kv1.4 expression level is increased in frontal cortex (somatosensory area), as compared to control rat brains. Notably, by immunofluorescence, we observed that SP was able to normalize the Kv1.4 subunits over-expression both in hippocampus and somatosensory cortex. The effect of SP on Kv1.4 subunit expression was further confirmed in cultured hippocampal neurons, indicating that SP is able to cross the bloodbrain barrier and directly act on hippocampal neurons.
We suppose that the increased expression of Kv1.4, Kv2.1, and Kv4.2 we found in Ab-treated rats could be at least partially responsible for the memory impairment detected in Ab-treated rats. Increased K + channel outward currents may indeed result in a decrease in neuronal excitability and K + channels have been proved to be involved in the regulation of cognitive processes and altered in AD [38,39]. Supporting our hypothesis, it has been demonstrated that i.c.v. injection of antisense oligodeoxyribonucleotide to Kv1.1, by reducing the expression of its particular intracellular mRNA target, provoked hippocampal-dependent memory loss in rat [63] and KV1.1 and KV1.3 channel blocker improves associative learning in rats [64].
On the whole, our results indicate that SP is able to modulate in vivo the expression of specific Kv channel subunits, known to be upregulated by Ab treatment. The antiamnesic effect of SP shown in our rat model of AD could be of clinical relevance for a better understanding of AD development and it might represents a potential disease-modifying agent.
Since the targeted drug delivery to the central nervous system as treatment of neurodegenerative disorders such as AD, is restricted due to the limitations posed by the blood-brain barrier, the availability of a natural neuropeptide able to easily cross the blood-brain barrier may be a valuable therapeutic tool for AD treatment.