TFP5,a Peptide Inhibitor of Aberrant and Hyperactive Cdk5/p25,Attenuates Pathological Phenotypes and Restores Synaptic Function in CK-p25Tg Mice

Animals

In this study, we used bitransgenic (CK-p25Tg) mice (males and females) in which forebrain CK-p25 expression was tightly regulated by the tet on/off system facilitated by the CamKII (CK) promoter [16]. The tet-off system was induced by dietary alteration in doxycycline-supplemented food in which CK-p25 expression was suppressed in the presence of doxycycline (Fig. 1). Calmodulin-dependent protein kinase II (CaMKII or CK) is a promoter used to activate the expression of the CK-p25 gene in the transgenic mice. It was shown that CK-p25Tg mice develop significant neuronal loss in the cortex and hippocampus and display neurodegeneration and pathological tau hyperphosphorylation when p25 expression is induced. These mice are identified as p25Tg OE. High p25 expression and elevated hippocampal cdk5 activity has been observed within 2 weeks after induction.

All mice were developed and raised in the presence of doxycycline (1 mg/g in food). To induce CK-p25 expression, the mice were fed a normal diet. To inhibit p25 production, the mice were again fed a doxycycline diet. All experimental cohorts were treated under the same conditions and fed doxycycline diet for the same period of time. Note that all animal procedures were performed in accordance with the NIH animal care committee’s regulations.

Design and synthesis of TFP5

TFP5 is a truncated portion of p35 (activator of Cdk5), which extends 24 aa residues in length (Lys254–Ala277) conjugated with an 11-aa modifying peptide derived from the trans-activator domain of TAT protein at the C terminus (to facilitate passage through the blood-brain barrier), while FITC, fluorescein isothiocyanate (a green fluorescent tag) with linker GGG, was attached at the N terminus (to serve as a marker). A scrambled peptide (Scb) was used as a control to TFP5 (sequence shown below). Peptide 2.0 (Chantilly, VA, USA) commercially synthesized both TFP5 and Scb peptides which were used after dissolving both in saline or double distilled water.

Sequences used were as follows:

TFP5, FITCGGGKEAFWDRCLSVINLMSSKMLQINAYARAARRAARR;

Scb peptide, FITCGGGGGGFWDRCLSGKGKMSSKGGGINAYARAARRAARR.

Intraperitoneal (i.p.) injection paradigm

Five cohorts of mice were used: vehicle-injected wild-type (WT), CK-p25Tg+DOX, CK-p25Tg-DOX, TFP5-injected CK-p25Tg-DOX, and Scb-injected CK-p25Tg-DOX (Fig. 1). After 12 weeks, the three mutant cohorts were taken off DOX to activate the expression of CK-p25. All five cohorts were age-matched and WT, CK-p25Tg+DOX, CK-p25Tg-DOX were treated with i.p. injection of vehicle, while another group of -DOX mutant mice was injected with 40 mg/kg/d TFP5. As an additional control, a cohort of -DOX mutants was injected with 40 mg/kg/d Scb peptide. All five cohorts were injected 3 days/week on weeks 13–17 for a total of 18 injections. All the mice were subjected to behavior analysis on week 18 and the mice were euthanized on week 19 when brain tissue was harvested for biochemical analysis. Based on the observation that injection of 200 mg/kg/day of TFP5 into WT mice in 5XFAD studies was not toxic, had no effect on body weight, behavior, appearance, and [23], we decided to limit the use of the expensive peptides and did not inject peptide into WT and CK-p25Tg+DOX cohorts as controls in the various experiments.

Sample preparation and Western blot analysis

Lysates were created from PBS-perfused hemibrains in 10% w/v homogenization buffer [20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Nonidet P-40; and complete mini protease inhibitor (Roche, Basel, Switzerland)]. Western blot analysis was performed according to standard protocols.

Immunoprecipitation and kinase assay

Cdk5 was immunoprecipitated from the respective brain lysates using the polyclonal C8 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and kinase assays were conducted using TFP5 and Scb as described. All kinase assays were carried out as double blind experiments. As reported for the p25TgOE mutant, cdk5 activity assayed in this manner reflected cdk5/p25 rather than cdk5/p35 or other kinases [16].

Synaptic activity: long term potentiation

Details of the procedures for recording LTP and its response to TFP5 in -DOX off animals are included in legend to Fig. 5.

Behavioral analysis

As previously stated, the following behavioral studies were conducted with 18-week-old mice of five cohorts: vehicle-injected WT, CK-p25Tg+DOX, CK-p25Tg-DOX, TFP5-injected CK-p25Tg-DOX, and Scb-injected CK-p25Tg-DOX. The Scb-injected CK-p25Tg-DOX was used as a second control. For each group, ≥7–15 animals were tested. Note, all the behavioral studies were double blinded.

Spontaneous alternation Y-maze task

Spontaneous alternation was measured using the Y-maze as previously described [29, 30]. The Y-maze test assesses hippocampal-dependent spatial working memory of the brain. Each mouse was placed at the center of the symmetrical Y-maze and was allowed to explore freely through the maze over an 8-min period. The sequence and total number of arms entered were recorded. Percentage alternation is measured as follows: number of triads containing entries into all three arms/maximum possible alternations. Each of the five experimental cohorts of animals were tested once after the i.p. injection regimen.

Open-field task

The open-field task was used to assess exploratory behavior and locomotor activity, as described previously [31]. The testing apparatus was made of PVC, with a square 42-×42-cm surface area and 31-cm-high walls. Mice were placed individually in the square open field area for a 20-min session and were monitored by an automated tracking system (Digipro; Accuscan Instruments Inc., Columbus, OH, USA). The exploratory activity and anxiety of all five cohorts were examined. For each group, the animals were tested once after i.p. injections.

Rotarod task

The rotarod task assesses the ability of an animal to keep its balance on a rotating rod and measures the animal’s balance, coordination, and motor ability. Rotarod assay was performed on 18-week-old mutant and WT mice as previously described [32]. The animal was placed on a cylinder which begins to rotate at a slowly increasing speed. The duration of time before it fell was recorded. For each group, the animals were tested thrice after i.p. injections.

Statistics

All statistical analyses of behavioral experiments are reported as means±SE, where n≥7–15 for each treated condition, as previously stated. Student’s t test was used to make comparisons between control and TFP5-treated mice. Values of p≤0.05 were considered statistically significant. Except where indicated all other statistical analyses were also as student T-tests.

Reduction in hyperactivation of CDk5 activity post TFP5 treatment

The proof of concept in this instance requires that the TFP5 peptide does, indeed, successfully and specifically target (i.e., inhibit) overexpression of p25-induced Cdk5 hyperactivity, reduce AD-like pathology, improve abnormal behavior and promote survival. Previous studies have shown that a regimen of i.p. injections of the TFP5 peptide successfully passed the blood-brain barrier and reduced cdk5/p25 hyperactivity and AD phenotypes in a 5XFAD mouse model. This transgenic mouse expresses five human mutant genes involved in AβPP processing which may confound the phenotype. The CK-p25Tg mouse, however, is a better model for p25 overexpression; within six weeks after p25 induction it develops significant neuronal loss in cortex and hippocampus and displays neurodegeneration and pathological tau hyperphosphorylation [16, 32]. The entire phenotype is a result of specific overexpression of p25. To assess the effect of the peptide, brain tissue lysates harvested from the five cohorts (induced and/or treated), were immunoprecipitated with a cdk5 antibody and assayed for cdk5 activity (Fig. 2A). The results show the cdk5 activity levels increase more than 2.5 fold after p25 induction (in Fig. 2B, compare lane 3 with lane 1, 2). This is consistent with the evidence of p25 overexpression as reported [16]. Treatment with TFP5, however, shows a significant reduction in activity (almost 40%; compare lane 4 with lane 3) which more closely resembles the CK-p25Tg+DOX cdk5 activity or that of the WT control; the scrambled peptide control, however, had no effect on the hyperactivity which was similar to the untreated p25TgOE mice. These data show that TFP5 treatment inhibits hyperactive cdk5 and suggest that TFP5 treatment of the p25TgOE mouse may also prevent the AD-like phenotypes induced by cdk5 hyperactivation.

TFP5 inhibits tau and neurofilament hyperphosphorylation

Hyperphosphorylated tau and neurofilaments contribute to NFTs, a hallmark of AD pathology [1, 34]. Tau contains multiple target sites for phosphorylation by several kinases, the principal ones being cdk5 and GSK3-β [35–38]. Brains of CK-p25Tg mice, when induced, exhibit high levels of tau and neurofilament-H/M phosphorylated at PHF sites Ser202, Ser404, and Thr205 [16]. To determine whether the TFP5 peptide would affect tau phosphorylation, we prepared Western blots of brain lysates from treated and untreated cohort mice using tau site- specific phospho-antibodies (Fig. 3A, B). Antibody SMI31 detected phosphorylated neurofilaments (p-NF-H/M) and also detects phospho-tau at 64 kDa. Western blot results of the brain lysates from CK-p25Tg mice show that of the four cohorts only the untreated p25TgOE mutant (lane 3) displayed increased levels of p-NF-H/M and p-Tau (25–50%) as detected by SMI 31, AT8, and pTau 404 antibodies, respectively. Brain lysates from TFP5-treated p25TgOE mice, however, showed significant reduction in both p-NF-H/M and p-Tau levels (lane 4, Fig. 3A-B). Actin antibody was employed to verify equal loading of the samples.

TFP5 reduces neuro-inflammation

The AD brain in humans and in AD mouse models is also characterized by neuro-inflammation involving activated astrocytes and microglia [39–41]. Activation of astrocytes parallels the accumulation of Aβ and microglia; the immune cells of the brain are activated when in proximity to senile plaques. Inflammation is a key early phenotype of the p25TgOE mutant after p25 induction [22, 42] and is ameliorated by the expression of transfected CIP, a larger truncated fragment containing the P5 sequence. Enhanced neuro-inflammation (almost five-fold) after p25 induction in brains of p25Tg OE mutants is significantly reduced (approximately 20–50%) upon TFP5 treatment as displayed in Western blots using GFAP antibody for activated astrocytes, Iba-1 for activated microglia and CD 86 for neuro-inflammation (Fig. 4 A, B). Equal loading of the samples was confirmed by immunoblotting with actin antibody.

TFP5 treatment restores normal synaptic activity and LTP

It has been shown that forebrain-specific overexpression of p25 results in the impairment of LTP in mouse hippocampus [32]. Moreover, a recent study indicates that chronic p25 expression is responsible for synaptic dysfunction in the 5XFAD mouse model of AD. Therefore, we sought to address whether TFP5 could affect p25-mediated synaptic dysfunction in the brain of the p25TgOE mouse. To do this, we examined LTP at Schaffer-collateral synapses in hippocampus of p25TgOE mice with TFP5, scrambled peptides or PBS administration. Three-month-old CKp25 male mice were fed with regular diet (off-DOX) for the onset of p25 expression. During the off-DOX period (6 weeks), we intraperitoneally injected the mice with TFP5, scrambled peptide (Scb), or PBS three times a week with 40 mg/kg of animal weight. Electrophysiological experiments were carried out at 18 weeks of age. After stable recording of baseline at least 20 min, we applied two times theta-burst stimulation (TBS) at 10-s intervals. Consistent with a previous study [43], we observed significantly impaired LTP in hippocampal slices from PBS-injected CK-p25Tg mice compared to littermate controls (Fig. 5A). We found that TFP5 administration completely restored hippocampal LTP whereas scrambled peptide failed to attenuate LTP deficits in these animals. The quantitation of these data shown in Fig. 5B indicates that TFP5 effectively inhibits p25-mediated synaptic dysfunction in the brain of the p25TgOE mouse.

TFP5 inhibits NMDA-induced cdk5/p25 activation in mouse hippocampal slices

It has been shown that glycine or NMDA stimulation of hippocampal slices induces elevated cdk5/p25 activity within a few minutes. The uptick in activity is presumed to result from increased availability of p25. If true, then our hypothesis predicts that treatment of hippocampal slices with TFP5 should successfully prevent hyperactive cdk5 under these conditions. The experiment, including scrambled peptide as control, is shown in Fig. 6A and indeed shows that TFP5 successfully inhibited cdk5 hyperactivity induced by stimulation.

A more compelling negative control is illustrated in a knock-in mutant mouse model in which endogenous p35 is replaced by a calpain resistant mutant of p35 (Δp35KI) to prevent p25 generation. These mutants, however, exhibit normal endogenous cdk5/p35 activity. Unable to produce p25, these mice should show endogenous levels of cdk5/p35 activity unresponsive to TFP5 pretreatment (100μM for 30 min) and in Fig. 6B we note that the modest reduction seen in activity after TFP5 treatment is not significant. We conclude that TFP5 selectively inhibits aberrant hyperactive cdk5/p25 activity but not cdk5/p35 physiological activity essential for neural development and function. It should be further noted that crossing the Δp35KI mutant with the 5XFAD AD mutant resulted in reduction of Aβ, synaptic depression, and cognitive impairment. Expression of the calpain resistant gene was sufficient to reduce the expression of p25 in the hybrid brain.

Spatial working memory deficit is rescued in p25TgOE mutants after TFP5 treatment

The mice overexpressing p25 (CK-p25Tg) display a spatial working memory deficit due to neuronal loss induced by AD-like pathology [16, 32]. Spatial working memory of CK-p25Tg and WT mice was tested measuring spontaneous alternation in the Y-maze (Fig. 7A). A spontaneous alternation value <45% indicates significant spatial working memory deficit.

The injection paradigm was amended for the behavioral experiments and the spontaneous alternation was studied for the five cohorts after 6 weeks off DOX (Fig. 7A, D). Using the second injection paradigm, WT mice displayed normal alternation performance in the Y maze, whereas p25TgOE mice performed poorly (<45%). The peptide- treated CK-p25Tg+DOX+T mice performed well with a spontaneous alternation value well over 50%, comparable in value to the WT and CK-p25Tg+DOX mice, while the untreated and Scb-treated groups showed similar poor spontaneous alternation performance (Fig. 7A).

TFP5 treatment improves other behavioral defects induced by p25 overexpression

Induction of p25 after DOX removal results in a 40% reduction in fall latency (roto-rod test) which is significantly restored after TFP5 treatment (Fig. 7B); again, we see that the scrambled peptide control has no effect on the abnormal behavior. The motor defects induced by p25 overexpression are prevented by TFP5 treatment. The results of the open field exploration test Fig. 7C) were unexpected; the p25 overexpressing animals (neurotoxic) exhibit hyperactive exploratory behavior compared to saline injected controls. This effect is not understood other than to suggest that some motor centers may be affected in response to high p25 levels. Nevertheless, TFP5 treatment succeeds in inhibiting that hyperactivity towards control values (compare red curve with green and blue). Here, too, scrambled peptide has no effect. It is interesting that the open field anxiety test (Fig. 7D) showed no significant difference among all the cohorts.