IT202000005074A1 - ADAM10 endocytosis inhibitory peptides and related uses in the treatment of Alzheimer's disease - Google Patents

Description

ADAM10 endocytosis inhibitory peptides and their uses in the treatment of Alzheimer's disease

The present invention relates to new cellular peptides permeable to cell membranes capable of interfering with the CAP2 / actin association and inhibiting the endocytosis of the ADAM10 enzyme. Are they also? pharmaceutical compositions comprising the endocytosis inhibitory peptides of ADAM10 and the related uses in the medical field for the treatment and / or prevention of diseases in which there is an increase or an accumulation of the peptide? -amyloid, such as for example the disease of Alzheimer's, head trauma and post-traumatic stress disorder.

Alzheimer's disease (AD)? a progressive neurodegenerative disease characterized by memory and cognitive function deficits. Initially, mild short-term memory changes appear, but as the disease progresses, symptoms worsen to the point of loss of executive functions. The immense social and economic ramifications of Alzheimer's disease have promoted the study of the mechanisms of the disease and the development of therapeutic agents for its treatment. However, there are currently no drugs available that can block the progression of the disease. Current therapeutic treatments temporarily relieve symptoms without stopping the inexorable progression of the disease.

The development of effective treatments? been limited by the lack of knowledge of the underlying mechanisms of the disease. Indeed, ? It is possible to develop therapies capable of actually modifying disease progression only when the molecular basis of the disease is thoroughly understood.

In the case of Alzheimer's disease, genetic and pathological evidence supports the amyloid hypothesis, which indicates the? -Amyloid (A?) Peptide as a key element of the disease capable of triggering a cascade of events leading to synaptic dysfunction, taupathy and neuronal death [1]. TO? derives from the amyloid precursor protein (APP) thanks to the concerted action of BACE1 and? -secretase. Alternatively, APP can? be cut by ADAM10 in the domain corresponding to A ?. The activity? of ADAM10 therefore prevents the formation of A? and generates the long neurotrophic fragment sAPP ?.

The pathogenesis of Alzheimer's disease? characterized by a cascade of sequential events, for which acting on a therapeutic target located upstream of the cascade would lead to a therapy capable of modifying the course of the disease; on the contrary, acting on a downstream target would only lead to symptom improvement without acting on progression [2].

ADAM10 (A disintegrin and metalloproteinase 10)? the? -secretase responsible for the proteolytic cleavage of the amyloid precursor protein (APP) which prevents the generation of A ?. Furthermore, in neurons, ADAM10 functions as a sheddase of various adhesion molecules, such as N-cadherin. Therefore, in a framework pi? broad, ADAM10 makes a contribution in the control of the morphology of the dendritic spines and of the plasticity? synaptic-dependent activity. Therefore, given the key role played by ADAM10, this enzyme represents a potential target to influence the early degeneration of dendritic spines in the early stages of AD.

First of all, ADAM10 competes with BACE1 for the cutting of APP, so to enhance the activity? of ADAM10 significantly reduces A? generation, as demonstrated in neuronal cultures [3] and in transgenic AD model animals [4].

Second, the sAPP fragment? of APP derived from the cut of ADAM10 has properties? neurotrophic and neuroprotective. sAPP? ? necessary for maintaining integrity? dendritic in the hippocampus [5]. Also, sAPP? can interfere with the APP-dependent activation of the signaling path of JNK (JUN N-terminal kinase) and therefore can? have an anti-apoptotic effect [6]. The effect on neurogenesis? been demonstrated by the increased proliferation of neural precursors in the subventricular zone, a neurogenic niche, following intraventricular injection of sAPP? [7]. Furthermore, ADAM10 expression potentiates neurogenesis in the adult hippocampus, which? reduced by late-onset AD-associated mutations [8]. A gene therapy approach aimed at overexpressing sAPP? in the brain thanks to the use of adeno-associated viruses has explored the therapeutic potential of this strategy in improving the pathological characteristics of AD in animals that already? they showed signs of the disease and amyloidosis. The results indicate that sAPP? can mitigate cognitive and memory deficits, influencing long-term enhancement (LTP) of synapses and density? of thorns in AD animals [9]. The sAPP fragment? it also induces the recruitment of microglia and the activation of TREM2 and the insulin-degrading enzyme (IDE), a protease produced by microglia which, in addition to neprilysin [10], contributes to the clearance of A? [11], improving the pathology related to amyloidosis.

Furthermore, the reduction of ADAM10 leads to an increase in the membrane levels of its substrate, the cellular prion protein (PrPC) [12], which? been shown to function as a receptor and mediator of neurotoxicity? in various proteinopathies such as AD. Indeed, ? has been shown that PrPC? a neuronal receptor of the oligomers of A? [13,14]. Therefore, an increase in activity? enzyme of ADAM10 would reduce the levels of this A receptor? limiting its toxic effects at the synaptic level. Furthermore, the processing of PrPC determines the formation of the neuroprotective fragment N1 which reduces the activation of the p53 pathway [15] and at the extracellular level it binds and neutralizes A? toxic [16].

The main concern of developing a therapy aimed at increasing activity? by ADAM10? relating to the large number of ADAM10 substrates that have been identified [17]. However, the increase in the levels of ADAM10 and, consequently, the increase in its activity? enzymatic, are surprisingly well tolerated in both mice [4, 8] and humans [18].

Considering the high potential of ADAM10 as a therapeutic target for AD, the authors of the present invention have studied the cellular mechanisms responsible for regulating activity. of ADAM10 in neuronal cells.

In this type of cell, ADAM10? located in the synapses, and? present both in the presynaptic vesicles [19], and enriched in the density? postsynaptic (PSD) of excitatory synapses [20, 21].

At the post-translational level, the activity? of ADAM10 as an enzyme? regulated by its distribution inside the cell and therefore by those mechanisms that determine its localization at the synaptic level. This aspect ? fundamental for the activity? of ADAM10 why? does the enzyme cut its substrates only when? properly inserted into the plasma membrane [20, 22]. The post-synaptic localization of ADAM10? controlled by the interaction of its cytoplasmic portion with specific binding partners, in particular, with the proteins SAP97 and AP2 [20, 23]. Specifically, SAP97 regulates the insertion of ADAM10 into the synaptic membrane, regulating its transport from the Golgi vesicles present in the dendrites [24], while the interaction with the AP2 heterotetrameric complex induces the removal of ADAM10 from the synaptic membrane itself [23].

It has been shown over the years that these trafficking mechanisms are compromised in patients with AD.

In particular, ? A reduction of the ADAM10 / SAP97 interaction and a concomitant association of ADAM10 to AP2 were observed in AD patients compared to healthy control subjects [24, 25]. These alterations lead to a reduction in the activity / synaptic localization of ADAM10.

Taken together, these data suggest that defects in the mechanisms that regulate the synaptic localization of ADAM10 can be considered a feature of the early stages of AD, when synaptic dysfunction occurs that precedes the stage of overt disease, and a potential pharmacological target placed at all. beginning of what? the pathogenetic cascade.

In light of the importance of the cytoplasmic domain of ADAM10 in regulating its activity, the authors of the invention identified a protein called cyclase-associated protein 2 (CAP2) as a new protein partner of ADAM10 by two-hybrid screening.

CAP2? a member of the CAP family, which are highly conserved proteins during evolution capable of regulating various functions related to the cytoskeleton such as cell migration, cell movement or vesicle trafficking and endocytosis [26]. Two homologs of CAP family proteins have been described in mammals: CAP1 and CAP2. CAP1? expressed in almost all cells, while the expression of CAP2? limited to a specific number of tissues, including the brain [27, 28], suggesting that CAP2 can be have unique roles, particularly in neurons.

Pi? specifically, the authors of the present invention have identified that the last 24 amino acids of CAP2 correspond to the binding domain of the protein to actin. By interfering with the association between CAP2 and actin? possible to increase the synaptic localization of ADAM10, why? the endocytosis of the enzyme is reduced.

Cell membrane permeable peptides (CPPs) being small polypeptide molecules that easily cross the plasma membrane, are tools capable of specifically interfering in protein-protein interactions (PPIs), both in vitro and in vivo, and are therefore effective tools for manipulate biological pathways.

The authors of the present invention have therefore developed peptides characterized by high permeability. cellular that are able to specifically interfere with the CAP2 / actin interaction by reducing the endocytosis of the ADAM10 enzyme and stabilizing its localization in synapses and activity? physiological. The peptides identified include a sequence derived from the HIV TAT protein (YGRKKRRQRRR) that translocates across plasma membranes and allows the transport of conjugated peptides, a space sequence and a sequence of 10 amino acids from amino acid 467 to amino acid 476 of the C- moiety. terminal of CAP2. The use of this type of CPP peptides? been already? exploited in clinical trials for acute neurological diseases [29].

Therefore, the subject of the present invention is a peptide characterized by an amino acid sequence of formula (I):

YGRKKRRQRRRGGSGLVTX1X2X3EIX4X5 (I)

in which:

X1? selected from the group consisting of L-amino acids characterized by neutral polar or negatively charged side chains;

X2, X4 and X5, the same or different from each other, are chosen from the group consisting of the L-amino acids characterized by neutral apolar side chains;

X3? chosen from the group consisting of L-amino acids characterized by non-polar or polar neutral side chains.

According to a preferred embodiment of the present invention said L-amino acids are characterized by polar neutral side chains are selected from the group consisting of glutamine, asparagine, serine, tyrosine, threonine and cysteine. In a further embodiment said L-amino acids characterized by negatively charged chains are glutamic acid or aspartic acid. Still according to a preferred embodiment of the present invention said L-amino acids are characterized by apolar neutral side chains are selected from the group consisting of glycine, leucine, isoleucine, alanine, phenylalanine, methionine, proline, tryptophan and valine.

In a particularly preferred embodiment of the peptide according to the invention, X2, X4 and X5, the same or different from each other, are selected from the group consisting of alanine, methionine, proline or valine. Preferably X2? proline or valine; X4? methionine or alanine; X5? alanine or glycine.

According to a particularly preferred embodiment of the peptide according to the invention X1? glutamic acid or threonine.

In a further preferred embodiment of the peptide according to the invention X3? alanine or threonine.

Still preferably, the peptide for use in the medical field according to the invention comprises or consists of the amino acid sequence YGRKKRRQRRRAGAGLVTEPAEIMA (SEQ ID NO: 1) or the amino acid sequence YGRKKRRQRRRAGAGLVTTVTEIAG (SEQ ID NO: 2).

The present invention also relates to the use of a peptide as defined above in the prevention and / or treatment of pathologies in which there is an increase or accumulation of the peptide? -amyloid, said pathologies being chosen from the group consisting of Alzheimer's disease, head trauma, post-traumatic disorder from stress. In particular, Alzheimer's disease (AD)? caused by an increase in the? -amyloid peptide, while in head trauma and post-traumatic stress disorder the increase in the? -amyloid? peptide? consequential to trauma or stress.

The term prevention is used here with reference to acute events such as head trauma, where treatment with the peptide according to the invention could prevent the increase in amyloid which is observed and which could cause an increased risk of dementia in subjects with risk, such as boxers.

The peptides according to the invention can be prepared by solid phase peptide synthesis based on Fmoc chemistry or by recombinant expression.

The present invention also contemplates the peptido-mimetic variants of said peptides, that is molecules that while maintaining the structure and bioactivity? of the original molecule show greater stability? (given by a better resistance to proteolysis and / or to physical and chemical degradation). These variants may result from the incorporation of non-naturally occurring amino acid residues in place of L-amino acids, such as, by way of example but not limited to, D-amino acids and / or non-proteinogenic amino acids. For example, the amino acids in proteins are all in L configuration but? it has been shown that the peptides synthesized with amino acids in the D configuration show a greater protection towards the activity? proteolytic of endogenous proteases and therefore a longer half-life. For this reason the synthesis of the peptides according to the invention with amino acids in configuration D can? be considered a valid and functional variation of the original peptide sequences.

It also constitutes? object of the present invention is a pharmaceutical composition comprising at least one peptide as defined above as active principle, together with one or more? pharmacologically acceptable excipients and / or adjuvants.

Preferably said pharmaceutical composition? formulated to be suitable for systemic administration by intravenous infusion.

According to an alternative embodiment of the present invention, the pharmaceutical composition according to the invention? suitable for oral or intranasal administration.

A further object of the present invention is a pharmaceutical composition for use in the prevention and / or treatment of pathologies in which an increase or accumulation of the? -Amyloid peptide occurs, said pathologies being selected from the group consisting of Alzheimer's disease, trauma cranial, post-traumatic stress disorder.

The present invention will come now described by way of illustration, but not of limitation, according to a preferred embodiment with particular reference to the attached figures, in which:

- Figure 1 shows the results of different immunoprecipitation assays in pi? complexes to evaluate the actual formation of complexes between CAP 2 and ADAM10. (A) In rat homogenate? An immunoprecipitation was performed with the antibody for CAP2 in order to evaluate the formation of the immune complex with ADAM10. CAP2, and not CAP1,? capable of specifically precipitating ADAM10 and not other members of the ADAM family, such as ADAM22. (B) ADAM10 interacts with CAP2 as demonstrated by a proximity ligation assay (PLA). The PLA signal? distributed along the GFP-positive dendrites. (C) COS-7 transfected with ADAM10 and mycCAP2. Immunofluorescence analysis reveals that in the absence of CAP2, ADAM10 exhibits diffuse perinuclear staining, whereas when CAP2 is expressed, ADAM10? localized in cytoplasmic aggregates. (D) Coimmunoprecipitation conducted by homogenate of HEK293 cells transfected with ADAM10-HA and Myc CAP2 96 ?. Deletion of the first 1-96 amino acids of CAP2 prevents interaction with ADAM10.

- Figure 2 shows the results of the study of the interaction between CAP2 and actin. (A) Actin interacts with the C-terminal tail of CAP2. GST CAP2 fusion proteins, which contain the Glutathione-S-transferase (GST) and CAP2 sequences, and the GST-CAP2 452? Deletion mutant, lacking the last 24 C-terminal amino acids of CAP2, were incubated with hippocampal homogenate in a pull-down assay. GST proteins were stained by Coomassie staining. The deletion of the last 24 amino acids prevents the binding with actin suggesting that this? the sequence responsible for the interaction between CAP2 and actin.

(B) Analysis of in vitro experiments aimed at evaluating the depolymerization rate of actin filaments in the presence of the recombinant CAP2 protein, indicate that both CAP2 and the CAP2 452 mutant? increase the depolymerization rate of F-actin. Deletion of the actin binding domain does not change the actin depolymerization dynamics.

(C) Coimmunoprecipitation experiments conducted using extracts of HEK293 cells stably transfected with ADAM10-HA and transiently transfected with EGFP-CAP2 or with EGFP-CAP2 452 ?. The CAP2 452? Mutant, lacking the actin binding domain,? able to bind ADAM10, indicating that in the CAP2 sequence the binding sites of ADAM10 and actin are different.

- Figure 3 shows the results of the study on the relevance of the actin binding domain of CAP2 in the cellular localization of ADAM10. (A) COS-7 cells were co-transfected with ADAM10 and EGFP-CAP2 or the EGFPCAP2 452? Mutant, lacking the actin binding domain. The expression of the EGFPCAP2 452 mutant? significantly increases the insertion of ADAM10 at the plasma membrane level; (B) EGFPCAP2452 ?, the actin binding domain mutant, significantly increases the degree of colocalization of ADAM10 with the postsynaptic compartment marker PSD-95. (C) Have antibody uptake assays been performed on hippocampal neurons transfected with TacADAM10-RAR and with myc-CAP2 or with the myc-CAP2 452 mutant? (devoid of the actin binding domain). The figure shows representative images in which you can? note that the expression of the mutant myc-CAP2452? significantly reduces the internalization coefficient of ADAM10.

- Figure 4 shows the results of the in vitro coimmunoprecipitation experiments between CAP2 and actin in patients with Alzheimer's disease (AD) vs controls (HC). (TO) ? was performed a coimmunoprecipitation from human hippocampal homogenate in which? An antibody directed against CAP2 was used and the presence of actin in the immune complex was checked. The association between CAP2 and actin? increased in AD patients compared to control subjects (HC).

(B)? was performed a coimmunoprecipitation from human hippocampal homogenate in which? An antibody directed against CAP2 was used and the presence of ADAM10 in the immune complex was checked. The association between CAP2? ADAM10 does not change significantly in AD patients compared to control (HC) subjects.

- Figure 5 shows the results of the study on the interaction between CAP2 and its binding partners ADAM 10 and actin in the hippocampus of APP / PS1 mice first at 6 months, when deposition of amyloid plaques begins, and then at 12 months , which correspond to a stage of overt pathology. (A) Representative images of a brightfield proximity ligation assay (PLA) assay between CAP2 and ADAM10 in different hippocampal regions of APP / PS1 and wild-type (WT) animals are shown on the left. To the right ? A representative image of the CA1 area of APP / PS1 animals of 6 months was reported. The graphs show the quantitative analyzes of the PLA data. A decrease in the association between CAP2 and ADAM10 is observed in APP / PS1 mice of 6 months of age. compared to WT animals, while in 12 and 15 month old animals there are no significant differences between APP / PS1 and WT mice.

(B) Coimmunoprecipitation assays were conducted from homogenates of 6- or 12-month-old WT and APP / PS1 mice. The homogenates were incubated with the anti-CAP2 antibody and? the presence of actin in the immunocomplex was verified. The association between CAP2 and actin? increased in APP / PS1 mice at 6 and 12 months compared to WT counterparts.

Figure 6 shows the results of the efficacy study in the interaction with the CAP2 / actin complex of the CPP peptides according to the invention. (A) To test the efficacy of the two cell permeable peptides (CPP) (PEPA (SEQ ID NO: 1) and PEPB (SEQ ID NO: 2) designed to interfere with the CAP2 / actin complex an assay of pull-down with the GST-CAP2 fusion protein Aliquots of mouse brain homogenate were incubated with GST-CAP2 fusion protein and with different concentrations (1? M or 10? M) of the PEPA or PEPB peptides or a inactive control peptide (SCR) The results of this in vitro test revealed that PEPB reduces the binding of actin to CAP2 at a concentration of 10? M.

(B) Primary cultures of hippocampal neurons were incubated with PEPA or PEPB or with the inactive SCR peptide at two different concentrations (1? M or 10? M) for 30 min. Coimmunoprecipitation experiments have shown that PEPB turns out to be the most? effective by reducing the bond between CAP2 and actin to a concentration of 10? M.

- Figure 7 shows the results related to the capacity? soluble cellular peptides (CPPs) of the invention to cross the blood brain barrier. WT mice were administered PEPB or the inactive SCR peptide or saline by intraperitoneal injection at a dose of 3 nmol / g. 24 hours after injection, the animals were sacrificed to conduct immunohistochemistry analyzes with a core dye (DAPI), a neuronal cell marker (MAP2) and an antibody that recognizes the TAT sequence of our peptide. Confocal microscope images showed that the anti-TAT antibody signal? present only in the hippocampus of animals treated with CPP but not with saline, confirming that CPPs can cross the blood brain barrier. Scale bar = 20? M.

- Figure 8, panel A shows the efficacy results of the peptides of the invention in reducing the formation of the CAP2 / actin complex in vivo in WT mice treated with a dose of 3 nmol / g of PEPA or PEPB or of inactive SCR peptide or saline, by intraperitoneal injection. After 24 hours the animals were sacrificed to conduct coimmunoprecipitation experiments from hippocampal homogenates. Six animals per experimental group were analyzed and the statistical analysis showed that PEPB? able to significantly reduce the interaction between CAP2 and actin. In panel (B) are shown the results on specificity? of the peptides of the invention by conducting a coimmunoprecipitation assay between actin and CAP1 (homologous protein of CAP2 and belonging to the CAP family) from hippocampal homogenates of WT mice treated with PEPA or PEPB or of inactive peptide SCR or saline solution. The data show that both peptides do not significantly modify the interaction between CAP1 and actin (analysis conducted on 6 animals per group).

- Figure 9 shows the efficacy results of the peptides of the invention in increasing the localization of ADAM10 in the synapses. (A) WT mice were treated with a 3 nmol / g dose of PEPA or PEPB or SCR inactive peptide or saline, by intraperitoneal injection. After 24 hours the animals were sacrificed and? the Triton-insoluble fraction (TIF) was purified which? enriched with postsynaptic proteins. Western Blot analysis performed hippocampal TIF samples of mice of the four experimental groups (saline, inactive peptide SCR, PEPA and PEPB) allowed to evaluate the synaptic levels of ADAM10 and the proteolytic cleavage of its N-Cadherin substrate by evaluating the relationship between the density? CTF-N-Cadherin optics (C-terminal fragment of N-cadherin resulting from the cut of ADAM10) normalized on density? N-Caderina optics. PEPB significantly increases the synaptic localization of ADAM10 without affecting N-cadherin cleavage. (B) ELISA assay results to assess sAPP fragment levels? which is released following the proteolytic cleavage of APP by ADAM10.

- Figure 10 shows the results of the acute treatment of WT and APP / PS 1 mice with the peptides of the invention to evaluate their therapeutic potential. (A) To evaluate the therapeutic potential of PEPB, 6-month-old WT and APP / PS1 mice were treated by daily intraperitoneal injections of PEPB or the inactive SCR peptide (3 nmol / g) for 18 days. Behavior tests were conducted after two weeks of treatment to assess the effect on cognitive deficits. After 18 days of treatment, the animals were sacrificed to conduct biochemical and morphological analyzes of the dendritic spines. (B) To evaluate the effectiveness of the treatment? A coimmunoprecipitation assay was performed to determine the level of association between CAP2 and actin. The analysis showed that there? a greater association between CAP2 and actin in transgenic mice treated with the inactive peptide SCR compared to WT animals and that treatment with the PEPB peptide is able to restore the levels of the interaction to values similar to those found in WT animals. (C) Western blot analysis of the postsynaptic fraction (TIF) reveals a reduction in the synaptic localization of ADAM10 in APP / PS1 mice compared to WT animals. Treatment with PEPB significantly increases synaptic levels of ADAM10 in both WT animals and APP / PS1 mice. The cut of N-cadherin by ADAM10 is not modified

- Figure 11 shows the results of the ELISA assays to evaluate the levels of sAPP?, which derives from the proteolytic cleavage of APP by ADAM10, and of amyloid peptide A? 1-42 in the hippocampus of APP / PS1 mice in order to determine the effect of PEPB treatment on APP metabolism. Did the results obtained demonstrate an increase in sAPP levels? (panel A) and a concomitant reduction in A levels? (panel B) in the hippocampus of APP / PS1 treated with PEPB compared to APP / PS1 mice treated with SCR.

- Figure 12, panel A shows the results of the density analysis of dendritic spines in the hippocampus to evaluate the effect of PEPB treatment on synapse loss in APP / PS1 animals. The analysis showed that in APP / PS1 animals there is a reduction in density. of dendritic spines compared to WT animals. PEPB treatment of APP / PS1 animals significantly increases density. of dendritic spines bringing it back to values similar to those measured in WT animals. In panel B, the results of the NORT test concerning the effect on the cognitive performance of the animals are reported. The data obtained confirm that APP / PS1 mice show a clear cognitive dysfunction compared to WT, as they cannot distinguish the new object from the familiar one. The significant difference between exploration time of the new object compared to the familiar one found in APP / PS1 animals treated with PEPB indicates an improvement in cognitive performance in this test when compared to APP / PS1 mice treated with the inactive SCR peptide.

In order to better illustrate the invention, the following examples are now provided which are to be considered illustrative and not limitative thereof.

EXAMPLE 1: Study on the capacity of CAP2 to bind actin and affect the localization of ADAM10

The identification by two-hybrid screeing of the CAP2 protein as a new protein partner of ADAM10 have been confirmed by evaluating the effective association between ADAM10 and CAP2 in pi? complex.

As shown in Figure 1A, CAP2? able to precipitate ADAM10 and the association? specific, since? CAP2 not? able to interact with ADAM22. Furthermore, as shown by an immunoprecipitation assay, ADAM10 does not? able to precipitate CAP1, the protein homologous to CAP2, demonstrating the specificity? of the ADAM10 / CAP2 complex.

In Figure 1B, the expression of CAP2 in COS7 cells is found to modify the cellular distribution of ADAM10, concentrating the enzyme in intracellular clusters of CAP2. To verify the presence and therefore the valence of this bond also in hippocampal neurons deriving from primary rat cultures, the interaction ADAM10 with CAP2? confirmed by proximity ligation assay (PLA) (Figure 1C). The PLA signal? present when ADAM10 and CAP2 are located at a distance of less than 40 nm.

Co-immunoprecipitation experiments, conducted by lysates of HEK293 cells stably transfected with ADAM10-HA and transiently transfected with the full-length form of CAP2 or with the missing deletion mutant of the first 96 aa (Myc-CAP2 96?), demonstrated that the N-terminal region CAP2 (1-96) is involved in the interaction with ADAM10 (Figure 1D).

? reported in the literature that CAP are proteins capable of influencing the dynamics of actin.

Specifically, CAP2 directly interacts and binds actin through its C-terminal domain.

It has been shown by the authors of the present invention that deletion of the region extending from 452-476 to (CAP2 452?) Completely abolishes the binding of the protein with actin (Figure 2A).

CAP2? a protein capable of accelerating the rate of actin depolymerization, as demonstrated by Total-internal reflection fluorescence (TIRF) microscopy experiments (Figure 2B).

Contrary to expectations, the lack of actin binding domain does not affect activity. of CAP2 on the speed? depolymerization of actin itself, indicating that this C-terminal domain is not? important for the effect of CAP2 on actin dynamics (Figure 2B).

Regarding the study of this binding domain,? it has been shown that, in coimmunoprecipitation experiments conducted by the homogenate of HEK293 cells expressing ADAM10-HA and transfected with EGFP-CAP2 or EGFP-CAP2 452? , EGFP-CAP2 452 ?, not? able to bind actin but? still able to associate with ADAM10. These data indicate that the CAP2 domains responsible for association with ADAM10 with actin are not overlapping (Figure 2C). Given the importance of the actin cytoskeleton and CAP proteins in protein trafficking and their endocytosis,? It was evaluated whether CAP2 and, in particular, its actin binding domain, played a relevant role in the cellular localization of ADAM10.

Therefore, ? It was decided to evaluate the insertion of ADAM10 into the plasma membrane in COS7 transfected with ADAM10 and with CAP2 or with the mutant without the actin binding domain (CAP2 452?). Quantitative analysis demonstrated that the mutant lacking the actin binding domain significantly increases the localization of ADAM10 in the plasma membrane, while CAP2 wt does not affect the level of the protein in the membrane (Figure 3A). The result ? been confirmed in neuronal cells in which? been seen that the colocalization between ADAM10 and PSD-95? increased in neuron transfected with the CAP2 452 mutant, while full-length CAP2 does not regulate synaptic levels of ADAM10 (Figure 3B).

Since? the actin binding site of CAP2 influences the membrane levels of ADAM10,? it was evaluated whether the protein endocytosis process could be involved [23]. Was an internalization test performed in neurons, in which TacADAM10-RAR and Myc CAP2 or its mutant Myc CAP2452? have been over-expressed. The CAP2 mutant significantly altered the ADAM10 endocytosis rate, while the total CAP2 length did not modulate ADAM10 internalization (Figure 3C). Therefore, these data suggest that CAP2? actively involved in ADAM10 endocytosis, probably anchoring the protein to the actin cytoskeleton.

EXAMPLE 2: Study on the modulation of the CAP2 / actin interaction in Alzheimer's disease

To understand the role of the interaction between CAP2 and actin in AD, autopsy human hippocampal samples from AD patients at Braak stage 4 and 5 and healthy controls (HC), provided by the Netherland Brain Bank, were used.

Coimmunoprecipitation experiments showed that the association between CAP2 and actin? increased in AD patients compared to controls (Figure 4A), while there are no significant changes in the CAP2-ADAM10 interaction in AD patients compared to controls (Figure 4B).

In order to assess whether the interaction between CAP2 and its binding partners ADAM10 and actin? modified in the different phases of the pathology,? The interaction between the two proteins in the hippocampus of APP / PS1 mice was analyzed first at 6 months, when the deposition of amyloid plaques begins, and then at 12 months, which correspond to a stage of overt disease.

The results revealed that the interaction between CAP2 and ADAM10? reduced only in the early stages of the disease, at 6 months, in the hippocampus of APP / PS1 mice compared to wild-type (WT) animals, indicating that the association between CAP2 and ADAM10? impaired in the early stages of AD (Figure 5A). Furthermore, to evaluate whether the link between CAP2 and actin was altered in APP / PS1 mice in the different stages of the disease considered above,? An immunoprecipitation technique was again performed, precipitating CAP2 in the hippocampal tissue of APP / PS1 mice and revealing actin by western blot. It was discovered that the CAP2 association with actin? increased at different stages of disease progression (Figure 5B). These data are consistent with data obtained in the hippocampus of AD patients indicating that the link between CAP2 and actin? involved in the pathogenesis of AD and could serve as a potential drug target. EXAMPLE 3: Design and validation of the peptdes according to the invention in vitro and in vivo

Since the actin binding domain of CAP2 controls the endocytosis of ADAM10 and, therefore, its localization in the membrane, two cell permeable peptides (CPP) PEP A (SEQ ID NO: 1) and PEP B have been designed (SEQ ID NO: 2) capable of interfering with the CAP2 / actin association and an SCR scramble control peptide (SEQ ID NO: 3). CPPs comprise a peptide derived from the HIV TAT protein, TAT 47-57, which crosses plasma membranes and enables the transport of conjugated peptides, called effectors.

These CPPs have been designed in order to specifically interfere with the actin / CAP2 interaction and, consequently, reduce the endocytosis of the enzyme. So ? possible to increase levels and activity? of ADAM10 in the synapses to limit the generation of A ?. CPPs, being small polypeptide molecules that easily cross the plasma membrane, are tools capable of specifically interfering in protein-protein interactions (PPIs), both in vitro and in vivo, and are therefore effective tools for manipulating biological pathways.

Two CPPs called PEPA (YGRKKRRQRRRAGAGLVTEPAEIMA - SEQ ID NO: 1) and PEPB (YGRKKRRQRRRAGAGLVTTVTEIAG - SEQ ID NO: 2) were designed and tested both in vitro and in vivo.

The design and the design of the two peptides? was carried out using the approach called "computer-aided peptide design". In detail, the design? was performed taking into account the last 24 amino acids of CAP2 previously identified to be directly involved in the stabilization of the interaction with actin. As the C-terminal region of CAP2 (24 amino acids) is too long to be synthesized and used as a small peptide, we performed a three-dimensional (3D) structure prediction study that this portion can be synthesized and used as a small peptide. acquire in solution, with the aim of detecting the elements of the secondary structure pi? stable in the C-terminal region of CAP2. On the basis of bioinformatic predictions and available X-ray structures of homologous proteins, the last 10 amino acids (ie from amino acid 467 to amino acid 476) have been identified as the largest region from a structural point of view. stable in solution of the C-terminal portion of CAP2 having a? high probability? to form a ?? - helix.

Consequently, yes? decided to focus the study only on this portion of the protein. In general, secondary structure elements (SSE) play a key role in the interactions between proteins, and? it has been shown that the? -helices are the SSE pi? areas that make up the surfaces involved in protein-protein interactions. For this reason the sequence of the peptides? been modified in order to form stable SSEs in solution. Finally, to avoid the formation of non-functional interactions between the peptide residues and the positively charged amino acids of the TAT portion,? A short spacer connection sequence was added to space out the active peptide and the TAT sequence.

The TAT sequence, the space sequence and the sequence of the last 10 amino acids of CAP2 responsible for binding with actin therefore constitute these exemplary peptides of the invention (PEPA (SEQ ID NO: 1) and PEPB (SEQ ID NO: 2)) . In all the experiments carried out,? An inactive control peptide consisting of TAT and an inert protein sequence (ie the scramble peptide, SCR) has always been used.

In order to test the effectiveness of the two CPPs designed,? an in vitro test was performed first. A GST-pull-down assay was set up, using a GST-CAP2 fusion protein as a bait for actin. The association between GST-CAP2 and actin was evaluated in the presence of active CPP (PEPA and PEPB) or inactive CPP (SCR) at a concentration of 1? M or 10? M. With the western blot technique? The most effective CPP was shown to be PEPB (Figure 6A). As a second model, we used primary hippocampal neurons treated with active or inactive CPPs at the concentration of 1? M or 10? M for 30 minutes. As shown in Figure 6B, coimmunoprecipitations (Co-IPs) revealed that once again PEPB (10? M) si? demonstrated the sequence with pi? effectiveness.

After the positive results obtained from in vitro experiments, the peptides of the invention were tested in vivo.

First of all, ? the capacity has been verified? of our PPCs to cross the blood-brain barrier. To evaluate this, we administered inactive and active peptides at a dose of 3nmol / g to wild-type mice (WT, C57BL / 6); the negative control? The one injected with saline was considered, medium in which the peptides were dissolved. After 24 hours, the animals were sacrificed and perfused with 4% PFA. We conducted immunohistochemistry experiments by labeling 50µm coronal brain sections with a neuronal marker (MAP2), a nuclear marker (DAPI) and an antibody against the TAT sequence inserted in our CPPs (TAT). The TAT protein was only visible in animals treated with our inactive and active CPPs, but not in saline-treated mice, confirming penetration into the brain (Figure 7).

After checking the capacity? of PPCs to cross the blood brain barrier,? the efficacy in decoupling the formation of the complex between CAP2 / actin was evaluated. The treatment scheme was repeated, i.e. active CPPs (PEPA or PEPB) or inactive peptide (SCR) at a dose of 3 nmol / g were administered by intraperitonel injection to 8-week-old C57BL / 6 mice, considering the injection of a saline solution as a control. 24 hours after injection, the animals were sacrificed and the hippocampus rapidly dissected. The hippocampus was homogenized to purify the Triton-Insoluble fraction (TIF), which? enriched in postsynaptic proteins. Coimmunoprecipitation analyzes revealed that PEPB significantly reduced the interaction between CAP2 and actin in agreement with previous in vitro results, while PEPA did not affect the association (Figure 8A). The effectiveness of the PEPB? also specific since? it does not affect the binding of actin to CAP1, another member of the CAP protein family (Figure 8B).

To verify the efficacy of PPCs in vivo, levels and activity were analyzed. synaptic effect of ADAM10 towards its main substrates (APP and N-cadherin) after treatment with CPP. For this purpose, ? the TIF fraction was purified from the hippocampi by suitably dissected to determine the synaptic levels of ADAM10. As shown in Figure 9A, western blot analysis revealed that ADAM10 levels increased in the TIF of PEPB-treated mice compared to saline or SCR-treated mice.

To verify if the increase in the synaptic localization of ADAM10 modified the proteolytic cleavage of APP,? Was an ELISA assay used to assess the levels of the sAPP fragment? which is released following the proteolytic cleavage of APP by ADAM10.

Treatment with both active peptides increases the release of sAPP?, Indicating a shift in APP metabolism towards the non-amyloidogenic pathway mediated by ADAM10 (Figure 9B). Do the results of the ELISA assay show that PEPB significantly increases sAPP levels? in the hippocampus.

On the other hand, cutting another ADAM10 substrate such as N-cadherin is not? affected by treatment with the PEP B peptide (Figure 9A).

EXAMPLE 4: Chronic treatment of APP / PS1 mice with PEPB

In light of the data obtained on the CAP2 / actin association in APP / PS1 6-month-old mice, the next step? been the chronic treatment of WT and APP / PS1 animals, injected with the active peptide that si? demonstrated more? effective in previous tests, PEPB, and the inactive SCR peptide as a control.

The experiment? was designed considering an injection protocol of a daily dose of PEPB or SCR for 18 days. The functional results were evaluated using cognitive behavioral tests that evaluate the cognitive performance of the animals and the analysis of the morphology of the dendritic spines in the neurons of the hippocampus, since? the loss of dendritic spines? one of the first hallmarks in AD.

To determine the effectiveness of the treatment on our target,? was first evaluated the interaction between CAP2 and actin, then the levels and activity? synaptic of ADAM10.

Coimmunoprecipitation experiments conducted on the hippocampal homogenate showed that, after 18 days of treatment, PEPB? was able to restore the altered levels of interaction of the CAP2 / actin complex (Figure 10A).

Then ADAM10 levels in the TIF synaptic fraction were assessed. As shown in Figure 10B,? A significant increase in synaptic levels of ADAM10 was found in animals treated with PEPB.

Therefore, after 18 days of chronic treatment, we obtained our first result: PEPB reduces the CAP2-actin interaction leading to an increase in availability. synaptic of ADAM10. The second step? been evaluate the effect of the active peptide on the activity? by ADAM10. Has the release of sAPP been measured? using the ELISA technique.

As shown in Figure 11A, in the hippocampal brain homogenate of mice treated with PEPB, the levels of sAPP? have significantly increased, demonstrating cos? an increase in activity? enzymatic. In line with this result, we measured reduced levels of amyloid (A?) (Figure 11B).

After demonstrating that PEPB alters the synaptic localization of ADAM10 and increases its activity,? The morphology of the dendritic spines of the treated mice was evaluated. The activity? enzymatic of ADAM10? relevant for the remodeling of the dendritic spines, since? modulates the composition of different adhesion molecules [30].

We used DilDye lipophilic dye to visualize the dendritic spines. From images acquired under the confocal microscope? The number, length and width of the head of the dendritic spines of the hippocampal neurons were measured. The quantitative analysis in Figure 12A shows that PEPB treatment promotes an increase in density. of thorns. This result could be attributed to the increased release of the neurotrophic factor sAPPalpha.

To strengthen our data,? A behavioral test was performed to assess the cognitive function of the 6-month-old APP / PS1 mice. The animals underwent a novel object recognition test (NORT) behavioral test. The NORT test? was performed at the end of a 14-day treatment with SCR or PEPB. The NORT, based on the preference of animals to explore new objects, involves the hippocampus through processes of spatial recognition and memory, providing a solid reading of the potential cognitive deficit. The mice were exposed to two identical objects for 10 minutes and after 24 hours they were re-exposed to an identical object (FAMILY) and a completely different object (NEW). Analysis of the tests showed that wild-type mice prefer to explore the new object over the familiar object. while APP / PS1 mice treated with inactive peptide do not discriminate the two objects. ? Interestingly, in mice treated with PEPB? A clear preference for the new object over the familiar object was observed (Figure 12B), indicating that PEPB treatment restores the cognitive impairment of APP / PS1 mice.

Claims (16)

CLAIMS 1. Peptide for use in the medical field characterized by an amino acid sequence having the following formula (I): YGRKKRRQRRRGGSGLVTX1X2X3EIX4X5 (I) in which: X1? selected from the group consisting of L-amino acids characterized by neutral polar or negatively charged side chains; X2, X4 and X5, the same or different from each other, are chosen from the group consisting of the L-amino acids characterized by neutral apolar side chains; X3? chosen from the group consisting of L-amino acids characterized by non-polar or polar neutral side chains. 2. Peptide for use in the medical field according to claim 1, wherein said L-amino acids characterized by polar neutral side chains are selected from the group consisting of glutamine, asparagine, serine, tyrosine, threonine and cysteine. 3. Peptide for use in the medical field according to any one of claims 1-2, wherein said L-amino acids characterized by negatively charged side chains are glutamic acid or aspartic acid. 4. Peptide for use in the medical field according to any one of claims 1-3, wherein said L-amino acids characterized by apolar neutral side chains are selected from the group consisting of glycine, leucine, isoleucine, alanine, phenylalanine, methionine, proline , tryptophan and valine. Peptide for use in the medical field according to any one of claims 1-4, wherein X2, X4 and X5 equal or different from each other, are selected from the group consisting of alanine, methionine, proline and valine. 6. Peptide for use in the medical field according to any one of the preceding claims, wherein X1? glutamic acid or threonine. 7. Peptide for use in the medical field according to any one of the preceding claims, wherein X2? proline or valine. 8. Peptide for use in the medical field according to any one of the preceding claims, wherein X3? alanine or threonine. 9. Peptide for use in the medical field according to each of the preceding claims, wherein X4? methionine or alanine. 10. Peptide for use in the medical field according to each of the preceding claims, wherein X5? alanine or glycine. 11. Peptide for use in the medical field according to any one of the preceding claims, comprising or consisting of the amino acid sequence YGRKKRRQRRRAGAGLVTEPAEIMA (SEQ ID NO: 1). 12. Peptide for use in the medical field according to any one of the preceding claims, comprising or consisting of the amino acid sequence YGRKKRRQRRRAGAGLVTTVTEIAG (SEQ ID NO: 2). 13. Peptide according to any one of the preceding claims for use in the prevention and / or treatment of pathologies in which an increase or accumulation of the? -Amyloid peptide occurs, said pathologies being chosen from the group consisting of Alzheimer's disease, trauma cranial, post-traumatic stress disorder. 14. Pharmaceutical composition comprising at least one peptide according to any one of claims 1-12 as an active ingredient, together with one or more? pharmacologically acceptable excipients and / or adjuvants. 15. Pharmaceutical composition according to claim 14, which? suitable for systemic administration by intravenous infusion. 16. Pharmaceutical composition according to claim 14, suitable for oral or intranasal administration.