LEPTIN PEPTIDES AND THEIR USE FOR TREATING NEUROLOGICAL DISORDERS
20200255492 ยท 2020-08-13
Inventors
Cpc classification
C07K14/5759
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
International classification
Abstract
A method of treating a neurological disorder comprising administering a leptin peptide fragment comprising amino acids located within the region of amino acids 116-125 of leptin is disclosed. The leptin peptide fragment preferably comprises up to 30 amino acids, and/or wherein the leptin peptide fragment comprises one or more amino acids located between amino acids 116-122 of leptin, for example the sequence X.sub.1CX.sub.2LPX.sub.3X.sub.4 wherein X.sub.1 is selected from G or S; X.sub.2 is selected from S, H or P; X.sub.3 is selected from Q, H, W, L, P or R and X4 is selected from T, A, or V (SEQ ID NO:14) or the sequence SCHLPWASGL (SEQ ID NO:22). The neurological disorder can include those which would benefit from treatment through cognitive enhancement and/or neuroprotection, such as age-associated memory impairment or loss, mild cognitive impairment, and Alzheimer's disease, and can include Parkinson's disease, frontotemporal dementia, progressive supranuclear palsy, Pick's disease, corticobasal degeneration, alcoholic dementia, (DLB) dementia with Lewy bodies, Picks' disease, thalamic dementia, hippocampal sclerosis, Hallervorden-Spatz, multiple system atrophy, tauopathies, subacute aterioscleroitic encephalopathy (Binswanger's disease), amyloid angiopathy, vasculitis, prion diseases, and paraneoplastic syndromes. The invention also includes a pharmaceutical formulation for this method, which can include the peptide in the form of a cyclic peptide or a peptide conjugate.
Claims
1.-34. (canceled)
35. A method of treating a neurological disorder comprising administering to a subject in need, a leptin peptide fragment comprising at least 4 consecutive amino acids located within the region of amino acids 116-125 of leptin.
36. The method according to claim 35, wherein the leptin peptide fragment comprises up to 30 amino acids.
37. The method of claim 35, wherein the leptin peptide fragment comprises one or more amino acids located between amino acids 116-122 of leptin.
38. The method of claim 35, wherein the leptin peptide comprises the sequence:
X1CX2LPX3X4 wherein X1 is selected from G or S; X2 is selected from S, H or P; X3 is selected from Q, H, W, L, P or R and X4 is selected from T, A, or V (SEQ ID NO:14).
39. The method according to claim 35, wherein the leptin peptide comprises amino acids selected from the group consisting of 116-121 of leptin, 117-122 of leptin, 117-125 of leptin, 118-123 of leptin, 119-124 of leptin, 120-125 of leptin, and 116-130 of leptin.
40. The method of claim 35, wherein the leptin peptide fragment comprises a sequence derived from human leptin.
41. The method of claim 35, wherein the leptin peptide fragment has the sequence SCHLPWASGL (SEQ ID NO:22).
42. The method of claim 35, wherein the leptin peptide fragment comprises 1, 2, or 3 deletions, modifications, substitutions and/or additions to the amino acid sequence.
43. The method of claim 35, wherein the leptin peptide fragment is in the form of a cyclic peptide.
44. The method of claim 35, wherein the peptide is in the form of a peptide conjugate, wherein the leptin peptide fragment is conjugated to another peptide, or non-peptide molecule.
45. The method of claim 44, wherein the other peptide or non-peptide molecule is a biologically or pharmaceutically active agent.
46. The method of claim 35, wherein the neurological disorder is a disorder which would benefit from treatment through cognitive enhancement and/or neuroprotection.
47. The method of claim 35, wherein the neurological disorder is selected from the group consisting of age-associated memory impairment or loss, mild cognitive impairment, and Alzheimer's disease.
48. The method of claim 35, wherein the neurological disorder is selected from the group consisting of Parkinson's disease, frontotemporal dementia, progressive supranuclear palsy, Picks disease, corticobasal degeneration, alcoholic dementia, (DLB) dementia with Lewy bodies, Picks' disease, thalamic dementia, hippocampal sclerosis, Hallervorden-Spatz, multiple system atrophy, tauopathies, subacute aterioscleroitic encephalopathy (Binswanger's disease), amyloid angiopathy, vasculitis, prion diseases, and paraneoplastic syndromes.
49. A pharmaceutical formulation comprising a leptin peptide fragment comprising at least 4 consecutive amino acids located within the region of amino acids 116-125 of leptin.
50. The pharmaceutical formulation according to claim 49, wherein the leptin peptide fragment comprises up to 30 amino acids.
51. The pharmaceutical formulation according to claim 49, wherein the leptin peptide fragment comprises one or more amino acids located between amino acids 116-122 of leptin.
52. The pharmaceutical formulation according to claim 49 comprising the sequence:
X1CX2LPX3X4 wherein X1 is selected from G or S; X2 is selected from S, H or P; X3 is selected from Q, H, W, L, P or R and X4 is selected from T, A, or V (SEQ ID NO:14).
53. The pharmaceutical formulation according to claim 49, wherein the leptin peptide fragment comprises amino acids selected from the group consisting of 116-121 of leptin, 117-122 of leptin, 117-125 of leptin, 118-123 of leptin, 119-124 of leptin, 120-125 of leptin, and 116-130 of leptin.
54. The pharmaceutical formulation according to claim 49, wherein the leptin peptide sequence is derived from human leptin.
55. The pharmaceutical formulation according to claim 49, wherein the 116-125 region of leptin has the sequence SCHLPWASGL (SEQ ID NO:22).
56. The pharmaceutical formulation according to claim 49, comprising 1, 2, or 3 deletions, modifications, substitutions and/or additions to the amino acid sequence.
57. The pharmaceutical formulation according to claim 49, wherein the leptin peptide fragment is in the form of a cyclic peptide.
58. The pharmaceutical formulation according to claim 49, wherein the peptide is in the form of a peptide conjugate, wherein the leptin peptide fragment is conjugated to another peptide, or non-peptide molecule.
59. The pharmaceutical formulation according to claim 58, wherein the other peptide or non-peptide molecule is a biologically or pharmaceutically active agent.
60. A cyclic peptide or peptide conjugate comprising a leptin peptide fragment comprising at least 4 consecutive amino acids located within the region of amino acids 116-125 of leptin.
61. The cyclic peptide or peptide conjugate according to claim 60, wherein the leptin peptide fragment comprises up to 30 amino acids.
62. The cyclic peptide or peptide conjugate according to claim 60, wherein the leptin peptide fragment comprises one or more amino acids located between amino acids 116-122 of leptin.
63. The cyclic peptide or peptide conjugate according to claim 60, comprising the sequence:
X1CX2LPX3X4 wherein X1 is selected from G or S; X2 is selected from S, H or P; X3 is selected from Q, H, W, L, P or R and X4 is selected from T, A, or V (SEQ ID NO:14).
64. The cyclic peptide or peptide conjugate according to claim 60, wherein the leptin peptide fragment comprises amino acids selected from the group consisting of 116-121 of leptin, 117-122 of leptin, 117-125 of leptin, 118-123 of leptin, 119-124 of leptin, 120-125 of leptin, and 116-130 of leptin.
65. The cyclic peptide or peptide conjugate according to claims 60, wherein the leptin peptide sequence is derived from human leptin.
66. The cyclic peptide or peptide conjugate according to claim 60, wherein the 116-125 region of leptin has the sequence SCHLPWASGL (SEQ ID NO:22).
67. The cyclic peptide or peptide conjugate according to claims 60, comprising 1, 2, or 3 deletions, modifications, substitutions and/or additions to the amino acid sequence.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] The present invention will now be further described by way of example, with reference to the following figures.
[0072]
[0073]
[0074]
[0075]
[0076]
[0077]
[0078]
[0079]
[0080]
[0081]
[0082]
[0083]
[0084]
[0085]
[0086]
[0087]
[0088]
[0089]
[0090]
[0091]
[0092]
[0093]
[0094]
[0095]
[0096]
[0097]
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0098]
[0099]
[0100]
[0101]
[0102]
[0103]
[0104]
[0105]
[0106]
[0107]
[0108]
[0109] Methods
[0110] Primary Neuronal Culture
[0111] Hippocampal cultures were prepared from neonatal Sprague Dawley rats as before (O'Malley et al. 2007). Briefly, neonatal Sprague Dawley rats (1-3 days old) were killed by cervical dislocation in accordance with Schedule 1 of the United Kingdom Government Animals (Scientific Procedures) Act, 1986 and the hippocampi removed. After washing in HEPES buffered saline (HBS) comprising (mM): NaCl 135; KCl 5; CaCl.sub.2 1; MgCl.sub.2 1; HEPES 10; D-glucose 25 at pH 7.4; cells were treated with protease Type X and Type XIV (0.5 mg/ml; Sigma) for 25 min at room temperature. Dissociated cells were plated onto sterile dishes (Falcon 3001) treated with poly-1-lysine (20 g/ml; 1-2 h) and maintained in MEM with serum replacement-2 (Sigma) in a humidified atmosphere of 5% CO2 and 95% O2 at 37 C. for up to 3 weeks.
[0112] Human Neural Cell Line SH-SY5Y
[0113] The human neuroblastoma cell line, SH-SY5Y (ECACC, UK) was maintained in Dulbecco's Modified Eagle Medium supplemented with glucose (4500 g/l) and 10% (v/v) cosmic calf serum (Fisher Scientific, UK) at 37 C. in a humidified atmosphere of 5% CO2, 95% air and allowed to reach 70-80% confluence before seeding. Cells (passage 10-18) were plated at 10 000 cells per well in 96 well tissue culture plates (Nunc, VWR, UK) and at a density of 210.sup.5 cells in 35 mm dishes for protein extraction. To induce differentiation, cells were cultured in DMEM supplemented with glucose (4500 g/l), 1% (v/v) cosmic calf serum and 10 M retinoic acid for 5 days. Thereafter they were incubated in DMEM supplemented with glucose (4500 g/l), serum replacement 2 (2%; Sigma, UK) and 18 M 5-fluorodeoxyuridine to inhibit proliferation of undifferentiated cells. The 50% of the medium was changed every 2-3 days and pharmacological treatment was carried out 7 days after switching to this medium. Reagents used were (from Sigma UK unless stated) 0.1-10 nM human leptin and leptin (116-130); 0.1-10 nM leptin (22-56; Bachem; Switzerland); 10 nM leptin (116-121) leptin (117-122), leptin (124-129) or leptin (125-130; all Severn Biotech, UK); 5 M copper chloride; 10 mM A.sub.1-42, 5 M WP1066 or 50 nM wortmannin. All treatments were added to the culture at the same time and survival assays were carried out after 96 h treatment. Protein samples for signaling ELISA were extracted 3 h after exposure to the relevant reagents and for biomarker expression after 72 hours.
[0114] Cell Survival Assays
[0115] The concentration of lactate dehydrogenase (LDH) in the culture medium or the mitochondrial activity within cells was used to monitor the level of cell death, as previously (Oldreive and Doherty 2010). In certain experiments, a Crystal violet assay was used to assess total cell number. Cells were fixed in neutral buffered formalin and washed 3 times in PBS prior to staining with 0.01% crystal violet acetate for 5 min. Plates were washed 5-10 times in dH2O and cells solubilised in 100 l dimethyl sulfoxide (DMSO) before reading the absorbance on a Biohit BP100 plate reader. For all assays, data is expressed as percentage relative to control, untreated wells to normalize for differences in plating density between individual experiments.
[0116] ELISA for Cell Signalling Pathways and Phosphorylated Tau
[0117] Protein from cultures was extracted into 500 l Tris-buffered saline containing protease inhibitor cocktail, and a Bradford assay used to determine protein concentration. Samples were diluted to give equal loading onto ELISA plates. Commercially available ELISA kits were used in accordance with the manufacturer's instructions to determine the ratio of pan-STAT3 to phospho-STAT3 (Sigma, UK) and pan-Akt to phospho-Akt (Sigma, UK) and the levels of alpha-tubulin and phosphorylated tau (Sigma, UK). Each protein sample was run in duplicate using samples derived from at least 5 biological repeats.
[0118] Surface Labelling of AMPA Receptors
[0119] To monitor GluA1 surface expression, immunocytochemistry was performed on hippocampal cultures (7-14 DIV) as before (Moult et al. 2010). Neurons were treated with agents for 20 min at room temperature (20-22 C.) before incubation with an antibody against an N-terminal region of GluA1 (sheep anti-GluR1; in house antibody against synthetic peptide (RTSDSRDHTRVDWKR) corresponding to 253-267 residues of GluR1; 1:100; Moult et al. 2010) at 4 C. Neurons were then fixed with 4% paraformaldehyde (5 min) before adding an appropriate fluorescently labelled secondary.
[0120] Electrophysiology.
[0121] Parasagittal hippocampal slices (300 m) were prepared from either P13-21 or 12-24-week-old male Sprague Dawley rats as previously (Moult et al. 2010). Brains were rapidly removed and placed in ice-cold artificial CSF (aCSF; bubbled with 95% O.sub.2 and 5% CO.sub.2) containing the following (in mm): 124 NaCl, 3 KCl, 26 NaHCO.sub.3, 1.25 NaH.sub.2PO.sub.4, 2 CaCl.sub.2, 1 MgSO.sub.4, and 10 d-glucose. Once prepared, parasagittal slices were allowed to recover at room temperature in oxygenated aCSF for 1 h before use. Slices were transferred to a submerged chamber maintained at room temperature and perfused with artificial cerebrospinal fluid at 2 ml min.sup.1. Standard extracellular recordings were used to monitor evoked field excitatory postsynaptic potentials (fEPSP) from stratum radiatum. The Schaffer collateral-commissural pathway was stimulated (constant voltage; 0.1 ms) at 0.033 Hz, using a stimulus intensity that evoked a peak amplitude 50% of the maximum. Synaptic potentials were low pass filtered at 2 kHz and digitally sampled at 10 kHz. The fEPSP slope was measured and expressed relative to baseline. Synaptic records are the average of 4 consecutive responses and stimulus artefacts are blanked for clarity. Recordings were made using an Axopatch 200B amplifier and analyzed using LTP v2.4 software. In synaptic plasticity studies, the degree of potentiation was calculated 30-35 min after HFS and expressed as a percentage of baselinestandard error of mean (SEM).
[0122] Hippocampal Neuron Culture
[0123] Hippocampal cultures were prepared as previously (Moult et al, 2010). Neonatal Sprague-Dawley rats (1-3 d old) were neonatal Sprague-Dawley rats were killed by cervical dislocation in accordance with the UK Animals (Scientific Procedures Act) 1986 legislation. Hippocampi were removed, and after washing in HEPES-buffered saline comprising (mM) 135 NaCl; 5 KCl; 1 CaCl2; 1 MgCl2; 10 HEPES; and 25 D-glucose at pH 7.40-4, were treated with papain (1.5 mg ml21; Sigma-Aldrich) for 20 min at 37 C. Dissociated cells were plated onto sterile dishes (35 mm diameter; Greiner Bio-One, Kremsmun-ster, Austria) treated with poly-D-lysine (20 g ml.sup.1; 1-2 h). Cultures were maintained in serum replacement medium (SR2; Sigma-Aldrich) in a humidified atmosphere of 5% CO2 and 95% O2 at 37 C. for up to 2 wk.
[0124] Immunocytochemistry.
[0125] Immunocytochemistry was conducted on primary hippocampal cultures (7-14 DIV). Prior to labelling with antibodies, neurons were washed with HBS containing glycine (0.01 mM) and incubated with agents for 1 h at room temperature. Cultures were fixed with 4% paraformaldehyde for 5 min, then permeabilized with 0.1% Triton X-100 for 5 min. To label endogenous tau, neurons were incubated with a rabbit anti-tau (Ab-217; Genscript) primary antibody (1:200 dilution) at room temperature for 30 min, followed by incubation with an Alexa Fluor555 donkey anti-rabbit (1:250, Invitrogen) secondary antibody for an additional 30 min duration. To assess the % co-localisation of tau at synapses, neurons were dual labelled with an antibody against the synaptic marker, PSD-95 (mouse anti-PSD-95; 1:500, Thermo Fisher) followed by application of an Alexa Fluor488 goat anti-mouse secondary antibody (1:500, Invitrogen).
[0126] Phosphorylated tau was labelled with primary antibody rabbit anti-tau (Ab-396) polyclonal antibody (1:500, Gen Script) corresponding to phosphorylation site of serine 396 (Y-K-S.sup.P-P-V). To visualise p-tau labelling, neurons were then incubated for 30 min with Alexa fluor555 donkey anti-rabbit IgG secondary antibody (1:250, Thermo Fisher Scientific). For synaptic localisation of p-tau, a secondary primary antibody was then added to compare p-tau localization relative to the synaptic marker, PSD95 (mouse anti-PSD-95, 1:500, Thermo Fisher Scientific) for 30 min. PSD-95 labelling was visualized by incubating with a goat anti-mouse Alexa Fluor 468-conjugated antibody (1:500; Thermo Fisher Scientific) for 30 min.
[0127] Imaging and Analyses
[0128] A Zeiss LSM510 confocal microscope was used for image acquisition and analysis. Images were obtained using a 10-s scan speed in single tracking mode or multi-tracking mode for dual labelling experiments. The intensity of immunostaining was measured off-line using Lasersharp software (Zeiss Lasersharp), whereby analysis lines of 50 m length were drawn along randomly selected dendritic regions. All data were obtained from at least three different cultures from different animals. Imaging conditions including illumination intensity and photomultiplier gains were kept constant between treatments for each experiment. In addition, data were normalised relative to the mean fluorescence intensity obtained from control neurons. For synaptic co-localisation experiments, tau and PSD-95 positive immunostaining were compared. The number of tau-positive sites that co-localized with PSD-95 positive sites were counted and expressed as a percentage of total positive sites (peaks of intensity).
[0129] All data are presented as meanSEM. Statistical analyses were performed using a Student independent t test for comparison of means (for % co-localization) and a one-way ANOVA with Tukey's post hoc test for comparisons between multiple groups. A value of p<0.05 was considered significant.
[0130] Cell Culture
[0131] The human neuroblastoma cell line, SH-SY5Y (ECACC, UK) was maintained in Dulbecco's Modified Eagle Medium supplemented with glucose (4500 g/l) and 10% (v/v) cosmic calf serum (Fisher Scientific, UK) at 37 C. in a humidified atmosphere of 5% CO2, 95% air and allowed to reach 70-80% confluence before seeding. Cells (passage 10-18) were plated at 210.sup.5 cells on 13 mm borosilicate class coverslips or at 10 000 cells per well in 96 well tissue culture plates (Nunc, VWR, UK) prior to treatment. Reagents used were (from Sigma UK unless stated) 1 nM human leptin, leptin (116-121), leptin (117-122) and leptin (117-125); 0.0001-1 nm murine leptin fragments leptin (116-121), leptin (117-122) and leptin (116-130); 100 M 6-hydroxydopamine; 10 M CuCl.sub.2; 1 mM A.sub.1-42.
[0132] Thioflavin S Staining
[0133] After 96 hours, cultures were fixed for 15 minutes in neutral buffered formalin, washed in phosphate buffered saline and stained with 0.05% Thioflavin S for 10 minutes prior to washing in phosphate buffered saline and mounting for visualisation on a fluorescent microscope. Relative fluorescent intensity per cell was determined using post hoc image analysis with Fiji software. Quantitative data is presented as the mean fluorescence per cell relative to control untreated cells.
[0134] Determination of Cell Viability
[0135] The concentration of lactate dehydrogenase (LDH) in the culture medium or the mitochondrial activity within cells was used to monitor the level of cell death, as previously (Oldreive and Doherty 2010). A Crystal violet assay was used to assess total cell number. Cells were fixed in neutral buffered formalin and washed 3 times in PBS prior to staining with 0.01% crystal violet acetate for 5 min. Plates were washed 5-10 times in dH2O and cells solubilised in 100 l dimethyl sulfoxide (DMSO) before reading the absorbance on a Biohit BP100 plate reader. For all assays, data is expressed as percentage relative to control, untreated wells to normalize for differences in plating density between individual experiments.
[0136] Mitored Analysis of Mitochondrial Morphology
[0137] Cells were treated with pre-warmed cell culture medium containing 1 M MitoRed ((9-[2-(4-Methylcoumarin-7-oxycarbonyl)phenyl]-3,6-bis(diethylamino)xanthylium chloride) and returned to the incubator for 45 minutes. Following fixation in NBF for 15 minutes, cells were washed 3 times in PBS. Coverslips were mounted in fluorescence mountant onto microscope slides and imaged on the Zeiss Axio MR2 microscope. The mean mitochondrial area and index of fragmentation (number of mitochondria: total area of mitochondrial material) were calculated using Fiji.
[0138] Results
[0139] Leptin (116-130) Facilitates NMDA Receptor-Dependent Hippocampal Synaptic Plasticity
[0140] It is known that NMDA receptors contribute little to basal excitatory synaptic transmission but synaptic activation of NMDA receptors is crucial for LTP induction at hippocampal synapses (Bliss and Collingridge 1993). Previous studies indicate that lep-tin enhances the magnitude of activity-dependent LTP in acute hippocampal slices (Oomura et al. 2006) and following direct administration into the hippocampus in vivo (Wayner et al. 2004). We have also shown that leptin facilitates NMDA receptor-dependent synaptic plasticity as leptin promotes conversion of short term potentiation (STP) to hippocampal LTP (Shanley et al. 2001). In order to compare the effects of leptin and the leptin fragments on synaptic plasticity in juvenile hippocampal slices (P13-21), a primed burst stimulation paradigm (5 trains of 8 stimuli at 100 Hz; Rose and Dunwiddie 1986) was used to induce STP, which returned to baseline levels within 30-35 min (n=4;
[0141] Leptin (116-130) Induces Synaptic Plasticity at Adult Hippocampal CA1 Synapses
[0142] We have shown that leptin regulation of excitatory synaptic transmission is age-dependent. Thus in contrast to its effects in juvenile tissue, leptin (25 nM) induces a novel form of LTP in adult hippocampus (Moult and Harvey 2011). In order to verify if the leptin fragments mirror leptin action in adult tissue, the effects of the leptin fragments were examined in hippocampal slices from adult (12-24 weeks) rats. In accordance with previous studies (Moult et al. 2010; Moult and Harvey 2011), application of leptin (25 nM; 20 min) to adult slices rapidly enhanced synaptic transmission (to 18813% of baseline; n=4; P<0.01; data not shown) which was sustained for the duration of recordings. Synaptic transmission was also markedly increased (to 14013% of baseline; n=5; P<0.05;
[0143] Leptin (116-130), But Not Leptin (22-56) Enhances the Surface Expression of GluA1
[0144] Trafficking of AMPA receptors to and away from synapses is crucial for various forms of activity-dependent synaptic plasticity (Collingridge et al. 2004). Our studies indicate that leptin regulates AMPA receptor trafficking as leptin promotes trafficking of GluA1 to hippocampal synapses (Moult et al. 2010). Moreover the ability of leptin to induce LTP in adult hippocampus requires the delivery of GluA1 to synapses by leptin (Moult et al. 2010). Thus, to assess if the leptin fragments also influence AMPA receptor trafficking processes, the surface expression of GluA1 was assayed in cultured hippocampal neurons (Moult et al. 2010). In agreement with previous studies, application of leptin (50 nM; 15 min) increased surface GluA1 expression to 1847%; (n=36; P<0.001;
[0145] As excitatory synaptic strength is governed by the density of AMPA receptors expressed at synapses, the effects on synaptic AMPA receptors was examined by comparing the colocalization between surface GluA1 and synaptophysin immunolabelling in hippocampal cultures (Moult et al. 2010). In agreement with previous studies (O'Malley et al. 2007; Moult et al. 2010), leptin (50 nM; 30 min) increased synaptophysin staining to 1449% of control (n=48; P<0.001) and it also enhanced the degree of colocalization between surface GluA1 and synaptophysin immunostaining from 435.4% to 624.4% (n=36; P<0.05;
[0146] Inhibition of the phosphatase, PTEN underlies leptin-driven trafficking of GluA1 to synapses (Moult et al. 2010). In order to determine if similar leptin dependent signalling cascades mediate the actions of leptin (116-130), the effects of pharmacological inhibition of PTEN with bisperoxovanadium (bpV; Schmid et al. 2004) were assessed in hippocampal cultures. Application of bpV (50 nM; 30 min) increased GluA1 surface expression to 1488% of control (n=36; P<0.001; FIG. 2F). In accordance with previous studies (Moult et al. 2010), leptin resulted in a significant increase in surface GluA1 labelling (1407.2%; n=36; P<0.001;
[0147] Leptin (116-130), But Not Leptin (22-56) Reverses A1-42 Inhibition of Hippocampal Synaptic Plasticity
[0148] Several studies indicate that soluble A oligomers impair activity-dependent synaptic plasticity, as exposure to A inhibits hippocampal LTP (Shankar et al. 2008; Li et al. 2009) and enhances LTD (Shankar et al. 2008). Moreover, our recent studies indicate that leptin reverses the detrimental effects of A1-42 on both LTP and LTD (Doherty et al. 2013). Thus we assessed if either of the fragments mirrored the protective actions of leptin on hippocampal synaptic plasticity. Initially we determined that application of leptin prevented the acute effects of .sub.A1-42 on LTP. In control slices, synaptic plasticity was induced using high frequency stimulation (HFS; 100 Hz 10 trains of 8 stimuli) which increased synaptic transmission to 1273.4% of baseline (n=8; P<0.01). Similarly in slices treated with the inactive peptide .sub.A42-1 (1 tiM; 40 min), an enhancement of synaptic transmission (1328.8% of base-line) was induced (n=5; P<0.05;
[0149] Leptin (116-130) Reverses A1-42-Induced LTD
[0150] It is known that oligomeric A promotes the induction of LTD (Shankar et al. 2008) and that exposure to a low concentration of leptin (10 nM) prevents facilitation of hippocampal LTD by A1-42 (Doherty et al. 2013). Thus we assessed if either of the leptin fragments mirror leptin action. Initially we verified that leptin prevented A1-42-induced LTD. In agreement with previous studies (Doherty et al. 2013), application of the subthreshold LFS paradigm failed to induce LTD in vehicle-treated slices (945.6% of baseline; n=5; P>0.05), whereas robust LTD (733.8% of baseline; n=5; P<0.001) was induced in A1-42-treated slices (
[0151] Leptin (116-130) Prevents A-Induced Internalization of GluA1
[0152] Previous studies indicate that A promotes internalization of the AMPA receptor subunit, GluA1 (Hsieh et al. 2006; Liu et al. 2010); an effect that is prevented by leptin (Doherty et al. 2013). To determine if the leptin fragments mirror this effect, the cell surface density of GluA1 was probed in cultured hippocampal neurons (Moult et al. 2010). In accordance with previous studies (Doherty et al. 2013), treatment with A.sub.1-42 (500 nM; 20 min) significantly attenuated (to 702% of control) GluA1 surface expression compared with control (A.sub.42-1-treated) hippocampal neurons (n=48; P<0.001;
[0153] We have demonstrated previously that leptin attenuates cortical neuronal death triggered by A.sub.1-42 or divalent copper ions (Doherty et al. 2013). To determine whether leptin (116-130) has neuroprotective actions, the effects of leptin (116-130) on the viability of differentiated human neural cells (SH-SY5Y) was examined after exposure to either 5 M CuCl2 or 10 M A.sub.1-42. Cells were treated with the toxin alone or with a range of concentrations (10-0.1 nM) of leptin or leptin (116-130). Determination of membrane leakage by LDH assay revealed a significant reduction in LDH release after treatment with either leptin or leptin (116-130; both 0.1-10 nM). Thus for CuCl.sub.2-treated cells, 10 nM leptin reduced LDH release by 39.52.73% compared with CuCl.sub.2 alone (n=5; P<0.001;
[0154] In parallel studies a crystal violet assay was used to verify these findings by assessing cell number. In CuCl.sub.2-treated cells, there was a concentration-dependent increase in the survival of cells treated with either leptin or leptin (116-130). Thus, treatment with leptin (0.1 nM) resulted in a 16.73.4% increase in cell number and this increased to 43.49.2% in the presence of 10 nM leptin (n=5; P<0.01). Similarly, exposure to 0.1 or 10 nM leptin (116-130) increased cell number by 27.810.6% and 39.913.5%, respectively (n=5;
[0155] As leptin (22-56) has biological activity in other systems, the specificity of the leptin (116-130) fragment in promoting cell survival was examined by determining whether leptin (22-56) inhibited neuronal death induced by A.sub.1-42. In contrast to leptin (116-130), treatment with leptin (22-56) had no effect on the viability of cells exposed to A.sub.1-42 (41.25.8% survival following A.sub.1-42 treatment and 48.69.3% in A.sub.1-42 with 10 nM leptin (22-56) treated cells; n=5; P>0.5; data not shown). These data reveal a potent neuroprotective effect of the leptin fragment (116-130) that is comparable to the survival actions of leptin. Moreover, this anti-apoptotic response is specific to leptin (116-130) as leptin (22-56) failed to influence neuronal viability.
[0156] The Neuroprotective Effects of Leptin (116-130) Involve Activation of STAT3 and PI3-Kinase-Dependent Signalling Pathways
[0157] Our previous studies indicate a crucial role for STAT3 and PI3-kinase/Akt signalling in the neuroprotective actions of leptin (Doherty et al. 2013). To determine whether leptin (116-130) acts via similar signalling cascades we examined the effects of pharmacological inhibitors of STAT3 (WP1066) or PI3-kinase (wortmannin). In A.sub.1-42-treated SH-SY5Y cells, application of either inhibitor significantly reduced the ability of leptin (116-130) to alleviate neuronal death. When neurons were treated with the STAT3 inhibitor, an 18.33.2% increase in LDH release in leptin (116-130) and A.sub.1-42-treated cultures was observed, which is similar to the 26.74.4% increase observed with A.sub.1-42 alone (n=5; P>0.5;
[0158] To verify that leptin (116-130) directly activates these signaling pathways, SH-SY5Y cells were exposed to 1 nM leptin (116-130; 3 h) or left untreated prior to protein extraction for ELISA. The ratio of phosphorylated STAT3 to pan STAT3 increased markedly following leptin (116-130) administration (n=3; P<0.01;
[0159] Leptin (116-130) Enhances Episodic-Like Memory
[0160] The current data demonstrate that leptin (116-130) enhances hippocampal synaptic plasticity mechanisms and has neuroprotective effects. To further assess its therapeutic potential we next asked if this fragment has similar cognitive enhancing properties to the whole leptin molecule. Previous studies indicate that leptin enhances hippocampal-dependent memory (Oomura et al. 2006; Farr et al. 2006), whereas resistance to lep-tin results in impaired spatial memory (Li et al. 2002). We used the object-place-context (OPC) recognition task which models human episodic memory, the first cognitive process to be compromised in the early stages of AD (Swainson et al. 2001). Performance on this task has been shown to be impaired in murine models of AD (Davis et al. 2013) and is compromised in animals with lesions of hippocampus (Langston and Wood 2010) and lateral entorhinal cortex (Wilson et al. 2013). The task is based on the object recognition paradigm and models the integrated aspect of human episodic memory by exposing rodents to novel combinations of objects, the spatial locations in which they are experienced and the contextual features of the environment (
[0161] Leptin (116-121) and Leptin (117-122) Facilitate Hippocampal LTP.
[0162] To determine if the leptin hexamers influence the magnitude of activity-dependent synaptic plasticity, standard extracellular recording techniques were used to assess the effects of leptin (116-121, 117-122, 118-123, 120-125, 124-129 and 125-130) on the magnitude of LTP induced by high frequency stimulation (100 Hz, 1 s) in acute hippocampal slices. In control slices, application of the HFS protocol resulted in robust LTP (11710.8% of baseline; n=5; p<0.05;
[0163] Leptin (116-121) and Leptin (117-122) Increase the Surface Expression of GluA1 in Hippocampal Neurons.
[0164] We have shown that leptin (116-130) increases the surface expression of GluA1 in hippocampal neurons (Malekizadeh et al, 2016), which mirrors the action of the whole leptin molecule. Thus to determine if the leptin hexamers are also capable of influencing AMPA receptor trafficking processes, the effects of leptin (116-121, 117-122, 118-123, 120-126, 122-128, 124-129 and 125-130) on the surface expression of GluA1 was assessed using immunocytochemical approaches in hippocampal neurons. Application of 10 nM leptin (116-121) for 15 min increased GluA1 surface expression to 1469% of control (p<0.001; n=36;
[0165] To determine whether the leptin hexamers described above had neuroprotective actions, the effects of leptin (116-121, 117-122, 124-129 and 125-130) on the viability of differentiated human neural cells (SH-SY5Y) were examined after exposure to 10 M A1-42. Cells were treated with the toxin alone or 10 nM of leptin, leptin (116-130) or the listed hexamers. Determination of membrane leakage by LDH assay revealed a significant reduction in LDH release after treatment with either leptin or leptin (116-130; both 10 nM). Furthermore, leptin (116-121) and leptin (117-122) mirrored this neuroprotective effect but leptin (124-129) and leptin (125-130) did not. Thus for A31-42-treated cells, 10 nM leptin and 10 nM leptin (116-130) significantly reduced LDH release by 25.32.5% and 24.0+2.2*% respectively compared with A.sub.1-42 alone (n=11; P<0.001;
[0166] To confirm the findings of the LDH assay, cells were treated with A.sub.1-42 alone or with a 10 nM of leptin, leptin (116-130) or the listed hexamers, and mitochondrial activity, as a measure of cell viability, determined by MTT assay. A significant increase in mitochondrial activity was detected following treatment with either leptin or leptin (116-130; both 10 nM). Furthermore, leptin (116-121) and leptin (117-122) mirrored this neuroprotective effect but leptin (124-129) and leptin (125-130) did not. Thus for A.sub.1-42-treated cells, 10 nM leptin and 10 nM leptin (116-130) increased mitochondrial activity by 32.65.2% and 40.9+5.8% respectively compared with A.sub.1-42 alone (n=11; P<0.001;
[0167] Leptin (116-121) and Leptin (117-122) Mirror the Ability of Leptin and Leptin (116-130) to Reduce the Expression of the AD-Linked Biomarker Phosphorylated Tau (p-Tau)
[0168] Hyper-phosphorylation of tau is the underpinning mechanism of the development of neurofibrillary tanglesone of the key pathological feature of AD. Human SH-SY5Y neuronal cells were exposed to A1-42 alone or in combination with 10 nM leptin, leptin (116-130), leptin (116-121), leptin (117-122), leptin (124-129) or leptin (125-130). Protein was extracted for ELISA assay and the ratio of p-tau to the house-keeping protein -tubulin determined (
[0169] Previous studies have shown that exposure to amyloid beta (A) promotes phosphorylation of tau, and that leptin protects against this aberrant action of A in our models of AD [6]. Recent evidence indicates that treatment with oligomeric A increases translocation of tau to synapses and this has been linked to synaptic dysfunction and ultimately cognitive impairments.
[0170] In accordance with existing data, we have set up and characterised a cellular model in hippocampal neurons that mirrors the aberrant trafficking of tau to synapses. In this model chronic treatment with A results in increased expression of tau at dendrites and specifically trafficking of tau to synapses where it is likely to interfere with normal excitatory synaptic transmission (see
[0171] Previous studies have indicated that phosphorylation of tau is a key event that occurs in AD, and tau phosphorylation is also linked to its increased expression at synapses. In accordance with this, exposure to A increases the dendritic levels of p-tau, and in particular the synaptic levels of p-tau are increased after exposure to A (
[0172] Murine Leptin116-130
[0173] Previously we have shown neuroprotection in models of Alzheimer's Disease (AD)[1].
[0174] In accordance with the existing data we have demonstrated that leptin116-130 prevents the accumulation of amyloid beta following seeding of cultures with amyloid (
[0175] We have expanded upon our AD data to consider the potential of leptin116-130 to demonstrate neuroprotection in an in vitro model of ischemic stroke. Thus, an emerging model of stroke related neuronal death is the deprivation of serum and glucose from cultures of neural cells [New Ref]. Under these conditions we see significant neuroprotection by leptin116-130 (
[0176] In a cellular Parkinson's Disease (PD) model, leptin116-130 prevents mitochondrial swelling and clumping in response to 6-hydroxydopamine (6-OHDA;
[0177] Taken together these data strengthen the findings that leptin116-130 can protect against neuronal death and dysfunction in AD. Excitingly we have also expanded upon this existing knowledge to reveal the potential for a more general beneficial effect in other neurodegenerative conditions.
[0178] Murine Leptin Hexamers Based on Leptin.sub.116-130
[0179] The data presented above reported pro-survival effects of leptin116-121 and leptin117-122 in AD models.
[0180] Building on these findings we have evaluated the potential for these hexamer fragments to ameliorate amyloid propagation after initial seeding with 1 M A1-42. We have demonstrated that leptin116-121 and leptin117-122 prevent the accumulation of amyloid beta following seeding of cultures with amyloid (
[0181] Further to this we have evaluated the effects of these hexamer peptides on episodic memory in mice. There is an 8 fold increase in episodic memory performance following treatment with leptin116-121 or leptin117-121 (
[0182] Blood samples taken 24 hours after injection showed no significant alterations in circulating leptin levels following any of the treatments revealing no long-term effects of the hexamers on endogenous leptin production (
[0183] Thus hexamer fragments of leptin.sub.116-130 mirror the effects of leptin and of leptin.sub.116-130 validating their further investigation as potential small peptide therapeutics.
[0184] Humanised Leptin Fragments Based on leptin.sub.116-130
[0185] As all fragments tested above have been based on murine leptin116-130, we have designed and synthesised 3 human leptin fragments, hleptin117-125, hleptin116-121 and hleptin117-122 (
[0186] Using thioflavin S staining as before, we have demonstrated that hleptin117-125, hleptin116-121 and hleptin117-122 prevent the accumulation of amyloid beta following seeding of cultures with amyloid (
[0187] Furthermore both hleptin117-125 and hleptin116-121 prevent neuronal loss in vitro in response to either 10 M amyloid betal-42 (
[0188] These data reveal that humanised forms of target sequences within leptin116-130 demonstrate potent neurobeneficial effects in vitro. Crucially these sequences are amenable to peptide modification via halogenation, which the murine sequence is not (see below) and therefore open the possibility of peptide modification and stabilisation by this route.
[0189] Peptide Modification
[0190] Halogenation and cyclisation: Target sequences for halogenation should ideally contain a tryptophan and there is no such residue in murine leptin.sub.116-130. Therefore, this work is focused on the human sequences, and to date 3 sequences (.sub.hleptin.sub.117-125, .sub.hleptin.sub.116-121, and .sub.hleptin.sub.117-122) containing a 7-bromo-tryptophan have been synthesised by Severn Biotech, UK. Second generation peptides with alternative bromo-tryptophans and/or which have been cyclised are also being synthesised.
[0191] Discussion
[0192] It is well established that the hormone leptin circulates in the plasma and enters the brain via transport across the blood brain barrier. In the hypothalamus, leptin plays a major role in regulating food intake and body weight (Spiegelman and Flier 2001). However, the central actions of the hormone leptin are not restricted to the hypothalamus and the regulation of energy homeostasis. Indeed, a number of extrahypothalamic brain regions, including the hippocampus display high levels of leptin receptor expression (Irving and Harvey 2014). Leptin mRNA and protein are also highly expressed in the hippocampal formation (Morash et al. 1999) and emerging evidence suggests brain-specific production of leptin (Eikelis et al. 2006). Thus, it is likely that a combination of locally released leptin as well as peripherally derived leptin reach hippocampal synapses and can influence synaptic function. Indeed, numerous studies indicate that leptin has potential cognitive enhancing properties as it readily facilitates the cellular events underlying hippocampal learning and memory. Thus, leptin has rapid effects on activity-dependent synaptic plasticity, glutamate receptor trafficking and dendritic morphology (Irving and Harvey 2014). In addition, several studies have identified neuroprotective effects of leptin as the viability of central and peripheral neurons is markedly influenced by this hormone (Weng et al. 2007; Doherty et al. 2008; Guo et al. 2008). Recent clinical evidence has established a link between circulating leptin levels and the incidence of AD (Power et al. 2001; Lieb et al. 2009) that has fueled the possibility of using the leptin system as a novel therapeutic target in AD. Indeed, treatment of various AD models with leptin prevents the detrimental effects of A that occur at both early and late stages of the disease (Fewlass et al. 2004; Farr et al. 2006; Doherty et al. 2013). However, as leptin is a very large peptide, developing small leptin-like molecules may be a better therapeutic approach. Several fragments of the leptin peptide are biologically active and mirror the anti-obesity effects of leptin (Grasso et al. 1997; Rozhayskaya-Arena et al. 2000; Grasso et al. 2001). However, the cognitive enhancing and neuroprotective effects of the leptin fragments are not known. Here we provide the first compelling evidence that leptin (116-130), but not leptin (22-56), has a potent effect on hippocampal synaptic function as it promotes trafficking of AMPA receptors to synapses and facilitates hippocampal synaptic plasticity. Moreover in cellular models that mimic amyloid toxicity, leptin(116-130), but not leptin (22-56) prevents the aberrant effects of A on hippocampal synaptic function and neuronal viability. These findings indicate that one particular leptin fragment, namely (116-130), mirrors the beneficial actions of leptin in preventing the detrimental effects of A at the early and late stages of AD. Finally we have shown that the leptin fragment that enhances hippocampal synaptic plasticity and has neuro-protective effects, namely leptin (116-130), is also a cognitive enhancer as it improves performance on tests of episodic memory.
[0193] Here we show that, in accordance with previous studies (Shanley et al. 2001; Wayner et al. 2004), NMDA receptor-dependent synaptic plasticity is enhanced by leptin as treatment with leptin promoted conversion of STP into a persistent increase in synaptic transmission in juvenile hippocampal slices. Similarly exposure to the leptin fragment (116-130) readily facilitated synaptic plasticity as an increase in synaptic strength was evident in leptin (116-130), but not leptin (22-56)-treated slices. We have shown that the efficacy of excitatory synaptic transmission is also regulated by leptin in adult hippocampus (Moult et al. 2010). In agreement with this, application of either leptin or leptin (116-130) to adult hippocampal slices resulted in the induction of a persistent increase in synaptic transmission. In contrast, however leptin (22-56) failed to alter excitatory synaptic strength in adult hippocampus.
[0194] AMPA receptor trafficking is pivotal for activity-dependent synaptic plasticity (Collingridge et al. 2004) and leptin regulates trafficking of GluA1 to synapses (Moult et al. 2010). In this study, treatment with either leptin or leptin (116-130) increased GluA1 surface expression in cultured hippocampal neurons, whereas leptin (22-56) was without effect. In co-localization studies, the density of GluA1 subunits associated with synapses was increased after application of leptin or leptin (116-130), suggesting that leptin (116-130) parallels the actions of leptin by boosting the synaptic insertion of AMPA receptors. We have shown that leptin-driven trafficking of GluA1 involves inhibition of PTEN (Moult et al. 2010) Similarly in this study, the ability of leptin (116-130) to influence GluA1 trafficking involves inhibition of PTEN, as application of the PTEN inhibitor bpV blocked the increase in GluA1 surface expression induced by leptin (116-130) in hippocampal neurons. These data indicate that like leptin, treatment with leptin (116-130) promotes GluA1 trafficking to hippocampal synapses via inhibition of PTEN. Thus, overall these data indicate that the leptin fragment (116-130) mirrors the actions of leptin as it markedly influences the cellular events underlying learning and memory by regulating AMPA receptor trafficking.
[0195] It is known that A inhibits the induction of hippocampal LTP (Shankar et al. 2008), and this detrimental effect of .sub.A1-42 is reversed by leptin (Doherty et al. 2013). Similarly, leptin (116-130) reversed the acute effects of .sub.A1-42 in this study as synaptic plasticity was readily induced in hippocampal slices exposed to leptin (116-130) and A.sub.1-42. Contrastingly, application of leptin (22-56) failed to prevent the detrimental effects of A.sub.1-42 as no increase in synaptic strength was induced after exposure to A.sub.1-42 and leptin (22-56). However, in slices exposed to leptin (22-56) post-tetanic potentiation (PTP) and some STP was observed after HFS, suggesting that this fragment may influence the transient enhancement of synaptic strength induced by HFS. As PTP and STP are thought to involve presynaptic expression mechanisms (Zucker and Regehr 2002; Lauri et al. 2007), it is feasible that leptin (22-56) can act pre-synaptically to influence glutamate release mechanisms.
[0196] Several studies indicate that A.sub.1-42 also facilitates the induction of hippocampal LTD (Shankar et al. 2008; Li et al. 2009), and this effect is also reversed by leptin (Doherty et al. 2013). In accordance with these findings, treatment with leptin reduced the magnitude of LTD in A.sub.1-42-treated slices. Similarly, leptin (116-130), but not leptin (22-56) attenuated the effects of A.sub.1-42 as the magnitude of LTD was significantly decreased in the presence of leptin (116-130). Moreover, application of either leptin or leptin (116-130) inhibited A.sub.1-42-driven AMPA receptor removal from hippocampal synapses, whereas treatment with leptin (22-56) was without effect. Thus overall these data demonstrate that leptin (116-130) mirrors the actions of leptin in counteracting the detrimental acute effects of A.sub.1-42 on hippocampal synaptic function.
[0197] Evidence is growing that leptin has neuroprotective actions in various models of neurodegenerative disease. In Parkinson's disease models, treatment with leptin protects dopaminergic neurons from various toxic insults (Weng et al. 2007; Doherty et al. 2008), whereas in AD models of amyloid toxicity, leptin increases neuronal viability via activation of STAT3 and PI3-kinase signalling (Doherty et al. 2008; Guo et al. 2008; Doherty et al. 2013). In this study, leptin and leptin (116-130) enhanced the survival of human neural (SH-SY5Y) cells treated with either A.sub.1-42 or Cu.sup.2+. Conversely no change in cell viability was evident after treatment with leptin (22-56), thereby providing further evidence that leptin (116-130) but not leptin (22-56) mirrors the protective actions of leptin.
[0198] In these studies, we reveal that signalling via PI3-kinase and STAT3 is essential for leptin (116-130)-mediated neuroprotection as selective inhibition of these pathways eliminated the protective effects of leptin (116-130). Moreover, direct activation of key components of PI3-kinase and STAT3 signalling pathways was observed following administration of leptin (116-130). As stimulation of both PI3-kinase (Doherty et al. 2013; Doherty et al. 2008) and STAT3 signalling cascades (Doherty et al. 2013; Guo et al. 2008) mediate the neuroprotective actions of leptin, these data indicate that leptin (116-130) is activating the same signalling pathways as the full length leptin peptide to induce neuronal survival. This provides further evidence that leptin (116-130) is mirroring the neuronal effects of leptin.
[0199] These studies demonstrate that the 116-130 fragment of the leptin molecule enhances hippocampal synaptic plasticity and has neuroprotective effects. As such this fragment is a very interesting therapeutic target to treat memory dysfunction and protect against neurodegeneration in the early stages of AD. To test the functional implications of the effects of leptin 116-130 we examined the effects of acute doses of this fragment on a test of episodic-like memory. This test is particularly appropriate as it models the type of memory that is first compromised in AD. Performance on the task has been shown to be impaired in rodents with damage to the lateral entorhinal cortex (Wilson et al. 2013), the first region to be damaged in AD, and the hippocampus (Langston and Woods, 2010). It has also been shown that the triple transgenic murine model of AD show impaired performance on this task at 6 months of age (Davis et al. 2013). The current data demonstrate powerful cognitive enhancing effects of both leptin and leptin (116-130) as both groups performed significantly better than controls on the OPC task. This is the first time that leptin has been shown to enhance the specific type of memory that degrades in AD and the fact that this cognitive enhancement is also produced by leptin (116-130) suggests that this fragment is a viable tool to treat memory dysfunction caused by damage to the hippocampal-entorhinal network. Recent studies indicate that administration of leptin also protects against A-induced impairments in spatial memory tasks (Tong et al. 2015). Thus, it is feasible that administration of leptin (116-130) will also mirror the effects of leptin and protect against the chronic effects of A on hippocampal-dependent learning and memory.
[0200] The current experiments demonstrated enhancement of memory for object-place-context associations. Enhancement of this hippocampal-dependent task is consistent with our findings showing enhancement of hippocampal synaptic plasticity but it remains a possibility that leptin 116-130 may also enhance simpler forms of recognition memory such as object recognition or object-place recognition. These simpler forms of recognition memory are dependent on other areas of the medial temporal lobe network and so future work could examine whether the cognitive enhancement is specific to the hippo-campus or also affects the surrounding cortical inputs. One other consideration is the anxiolytic properties of leptin that have been reported in both normal (Liu et al. 2010) and chronically stressed rats (Lu et al. 2006). Reduced anxiety could potentially affect performance on the spontaneous recognition tasks as less anxious animals may explore more freely. This was not found to be the case in the current study as the levels of exploration in both sample and test phases of the OPC experiment were not different between groups. This is not surprising as animals had extensive handling and pre-training before the OPC test and so levels of anxiety would have been very low in all animals. In conclusion, these data indicate that the leptin (116-130) fragment mirrors the cognitive enhancing effects of leptin as it promotes trafficking of the AMPA receptor subunit GluA1 to synapses, facilitates hippocampal synaptic plasticity and improves performance in an episodic-like memory task. In addition, leptin (116-130) counteracts the detrimental effects of A.sub.1-42 on hippocampal synaptic function and neuronal viability in various cellular models of amyloid toxicity.
[0201] To further refine the precise sequence of leptin (116-130) that is required for its leptin-mimetic effects, hexamer peptides of the molecule were generated by peptide scanning. The potential for these to elicit leptin-like biological effects was tested in vitro.
[0202] Two specific leptin hexamers (116-121; 117-122) are effective in mirroring the cognitive enhancing effects of leptin, as treatment of hippocampal slices with either hexamer results in facilitation of hippocampal LTP. In contrast, leptin (124-129) and leptin (125-130) failed to alter the magnitude of LTP suggesting that the N-terminal region of leptin (116-130) is the bioactive region. AMPA receptor trafficking is also critical for hippocampal synaptic plasticity and leptin and leptin (116-130) potently regulate the trafficking of the AMPA receptor subunit, GluA1 (Moult et al, 2010; Malekizadeh et al, 2016). Similarly, exposure of hippocampal neurons to either leptin (116-121) or leptin (117-122) increased the surface expression of GluA1, thereby mirroring the effects of leptin. Contrastingly, treatment with either leptin (124-129) or leptin (125-130) had no effect on GluA1 surface expression in hippocampal neurons.
[0203] In accordance with the data above, both leptin (116-121) and leptin (117-122) attenuated A.sub.1-42-mediated cell death as effectively as either leptin or leptin (116-130). Thus both LDH and MTT assays confirmed that the bioactive region of leptin (116-130) lies in the N-terminal end of the molecule as neither leptin (124-129) nor leptin (125-130) mirrored the neuroprotective effects of leptin or leptin (116-130). Similarly only leptin (116-121) and leptin (117-122) mirrored the leptin or leptin (116-130)-mediated attenuation of p-tau upregulation in response to A.sub.1-42. Neither leptin (124-129) nor leptin (125-130) had any significant effect.
[0204] Taken together the work on the hexamer peptides further refine the sequence required to elicit a leptin-mimetic response, isolating it to the N-terminal of fragment leptin (116-130). Our findings not only reinforce the consensus that the leptin system is an important therapeutic target in AD, but also establish that leptin (116-130), and smaller hexamer fragments of this molecule, may be useful in the development of leptin-mimetic agents for therapeutic use.
REFERENCES
[0205] All references cited herein are hereby incorporated by reference in their entirety. [0206] 1. Anderson W W, Collingridge G L. 2007. Capabilities of the WinLTP data acquisition program extending beyond basic LTP experimental functions. J Neurosci Methods. 162: 346-356. [0207] 2. Bliss T V, Collingridge G L. 1993. A synaptic model of memory: long-term potentiation in the hippocampus. Nature. 361: 31-39. [0208] 3. Clarke R E, Zola S M, Squire L R. 2000. Impaired recognition memory in rats after damage to the hippocampus. J Neurosci. 20: 8853-60. [0209] 4. Collingridge G L, Isaac J T, Wang Y T. 2004. Receptor trafficking and synaptic plasticity. Nat Rev Neurosci. 5:952-962. [0210] 5. Davis K E, Eacott M J, Easton A, Gigg J. 2013. Episodic-like memory is sensitive to both Alzheimer's-like pathological accumulation and normal ageing processes in mice. Behav Brain Res. 254:73-82. [0211] 6. Doherty G H, Beccano-Kelly D, Yan S D, Gunn-Moore F J, Harvey J. 2013. Leptin prevents hippocampal synaptic disruption and neuronal cell death induced by amyloid . Neurobiol Aging. 34:226-237. [0212] 7. Doherty G H, Oldreive C, Harvey J. 2008. Neuroprotective actions of leptin on central and peripheral neurons in vitro. Neuroscience. 154:1297-1307. [0213] 8. Eacott M J, Norman G. 2004. Integrated memory for object, place, and context in rats: a possible model of episodic-like memory? J Neurosci. 24:1948-1953. [0214] 9. Eikelis N, Wiesner G, Lambert G, Esler M. 2006. Brain leptin resistance in human obesity revisited. Regul Pept. 139:45-51. Farr S A, Banks W A, Morley [0215] 10. J E. 2006. Effects of leptin on memory processing. Peptides. 27:1420-1425. [0216] 11. Fewlass D C, Noboa K, Pi-Sunyer F X, Johnston J M, Yan S D, Tezapsidis N. 2004. Obesity-related leptin regulates Alzheimer's Abeta. FASEB J. 18:1870-1878. [0217] 12. Grasso P, Leinung M C, Ingher S P, Lee D W. 1997. In vivo effects of leptin-related synthetic peptides on body weight and food intake in female ob/ob mice: localization of leptin activity to domains between amino acid residues 106-140. Endocrinology. 138:1413-1418. [0218] 13. Grasso P, Rozhayskaya-Arena M, Leinung M C, Lee D W. 2001. [D-LEU-4]-OB3, a synthetic leptin agonist, improves hyperglycemic control in C57BL/6J ob/ob mice. Regul Pept. 101: 123-129. [0219] 14. Guo Z, Jiang H, Xu X, Duan W, Mattson M P. 2008. Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization. J Biol Chem. 283:1754-1763. [0220] 15. Hsieh H, Boehm J, Sato C, Iwatsubo T, Tomita T, Sisodia S, Malinow R. 2006. AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss. Neuron. 52: 831-843. [0221] 16. Irving A J, Harvey J. 2014. Leptin regulation of hippocampal synaptic function in health and disease. Phil Trans R Soc B. 369: 20130155. [0222] 17. Langston R F, Wood E R. 2010. Associative recognition and the hippocampus: differential effects of hippocampal lesions on object-place, object-context and object-place-context memory. Hippocampus. 20:1139-1153. [0223] 18. Lauri S E, Palmer M, Segerstrale M, Vesikansa A, Taira T, Collingridge G L. 2007. Presynaptic mechanisms involved in the expression of STP and LTP at CA1 synapses in the hippo-campus. Neuropharmacology. 52:1-11. [0224] 19. Li X L, Aou S, Oomura Y, Hori N, Fukunaga K, Hori T. 2002. Impairment of long-term potentiation and spatial memory in leptin receptor-deficient rodents. Neuroscience. 113:607-615. [0225] 20. Li S, Hong S, Shepardson N E, Walsh D M, Shankar G M, Selkoe D. 2009. Soluble oligomers of amyloid Beta protein facilitate hippocampal long-term depression by disrupting neuronal glutamate uptake. Neuron. 62:788-801. [0226] 21. Lieb W, Beiser A S, Vasan R S, Tan Z S, Au R, Harris T B, Roubenoff R, Auerbach S, DeCarli C, Wolf P A, et al. 2009. Association of plasma leptin levels with incident Alzheimer disease and MRI measures of brain aging. J Am Med Assoc. 302:2565-2572. [0227] 22. Liu J, Garza J C, Bronner J, Kim C S, Zhang W, Lu X Y. 2010. Acute administration of leptin produces anxiolytic-like effects: a comparison with fluoxetine. Psychopharmacology. 207 (4): 535-545. [0228] 23. Liu S J, Gasperini R, Foa L, Small D H. 2010. Amyloid-beta decreases cell-surface AMPA receptors by increasing intracellular calcium and phosphorylation of GluR2. J Alzheimers Dis. 21:655-666. [0229] 24. Luo X, McGregor G, Irving A J, Harvey J. 2015. Leptin induces a novel form of NMDA receptor-dependent LTP at hippocam-pal temporoammonic-CA1 synapses. eNeuro. 2 (3):1-7. [0230] 25. Lu X Y, Kim C S, Frazer A, Zhang W. 2006. Leptin: a potential novel antidepressant. Proc Natl Acad Sci USA. 103: 1593-1598. [0231] 26. Morash B, Li A, Murphy P R, Wilkinson M, Ur E. 1999. Leptin gene expression in the brain and pituitary gland. Endocrinology. 140:5995-5998. [0232] 27. Moult P R, Cross A, Santos S D, Carvalho A L, Lindsay Y, Connolly C N, Irving A J, Leslie N R, Harvey J. 2010. Leptin regulates AMPA receptor trafficking via PTEN inhibition. J Neurosci. 30:4088-4101. [0233] 28. Moult P R, Harvey J. 2011. NMDA receptor subunit composition determines the polarity of leptin-induced synaptic plasticity. Neuropharmacology. 61:924-936. [0234] 29. Oldreive C E, Doherty G H. 2010. Effects of tumour necrosis factor-alpha on developing cerebellar granule and Purkinje neurons in vitro. J Mol Neurosci. 42:44-52. [0235] 30. O'Malley D, MacDonald N, Mizielinska S, Connolly C N, Irving A J, Harvey J. 2007. Leptin promotes rapid dynamic changes in hippocampal dendritic morphology. Mol Cell Neurosci. 35: 559-572. [0236] 31. Oomura Y, Hori N, Shiraishi T, Fukunaga K, Takeda H, Tsuji M, Matsumiya T, Ishibashi M, Aou S, Li X L, et al. 2006. Leptin facilitates learning and memory performance and enhances hippocampal CA1 long-term potentiation and CaMK II phosphorylation in rats. Peptides. 27:2738-2749. [0237] 32. Power D A, Noel J, Collins R, O'Neill D. 2001. Circulating leptin levels and weight loss in Alzheimer's disease patients. Dement Geriatr Cogn Disord. 12:167-170. [0238] 33. Rose G M, Dunwiddie T V. 1986. Induction of hippocampal long-term potentiation using physiologically patterned stimulation. Neurosci Lett. 69:244-248. [0239] 34. Rozhayskaya-Arena M, Lee D W, Leinung M C, Grasso P. 2000. Design of a synthetic leptin agonist: effects on energy balance, glucose homeostasis, and thermoregulation. Endocrinology. 141:2501-2507. [0240] 35. Samson W K, Murphy T C, Robison D, Vargas T, Tau E, Chang J K. 1996. A 35 amino acid fragment of leptin inhibits feeding in the rat. Endocrinology. 137:5182-5185. [0241] 36. Schmid A C, Byrne R D, Vilar R, Woscholski R. 2004. Bisperoxovanadium compounds are potent PTEN inhibitors. FEBS Lett. 566:35-38. [0242] 37. Shankar G M, Li S, Mehta T H, Garcia-Munoz A, Shepardson N E, Smith I, Brett F M, Farrell M A, Rowan M J, Lemere C A, et al. 2008. Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med. 14:837-842. [0243] 38. Shanley L J, Irving A J, Harvey J. 2001. Leptin enhances NMDA receptor function and modulates hippocampal synaptic plasticity. J Neurosci. 21:RC186. [0244] 39. Simmons L K, May P C, Tomaselli K J, Rydel R E, Fuson K S, Brigham E F, Wright S, Lieberburg I, Becker G W, Brems D N, et al. 1994. Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro. Mol Pharmacol. 45: 373-379. [0245] 40. Spiegelman B M, Flier J S. 2001. Obesity and the regulation of energy balance. Cell. 104:531-543. [0246] 41. Stranahan A M, Mattson M P. 2012. Metabolic reserve as a determinant of cognitive aging. J Alzheimers Dis. 30:S5-13. [0247] 42. Swainson R, Hodges J R, Galton C J, Semple J, Michael A, Dunn B D, Iddon J L, Robbins T W, Sahakian B J. 2001. Early detection and differential diagnosis of Alzheimer's disease and depression with neuropsychological tasks. Dement Geriat Cogn Disord. 12:265-280. [0248] 43. Tong J Q, Zhang J, Hao M, Yang J, Han Y F, Liu X J, Shi H, Wu M N, Liu Q S, Qi J S. 2015. Leptin attenuates the detrimental effects of -amyloid on spatial memory and hippocampal later-phase long term potentiation in rats. Horm Behav. 73: 125-130. [0249] 44. Walsh D M, Klyubin I, Fadeeva J V, Cullen W K, Anwyl R, Wolfe M S, Rowan M J, Selkoe D J. 2002. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 416:535-539. [0250] 45. Wayner M J, Armstrong D L, Phelix C F, Oomura Y. 2004. Orexin-A (Hypocretin-1) and leptin enhance LTP in the dentate gyrus of rats in vivo. Peptides. 25:991-996. [0251] 46. Weng Z, Signore A P, Gao Y, Wang S, Zhang F, Hastings T, Yin X M, Chen J. 2007. Leptin protects against 6-hydroxydopamine-induced dopaminergic cell death via mitogen-activated protein kinase signaling. J Biol Chem. 282:34479-34491. [0252] 47. Wilson D I, Watanabe S, Milner H, Ainge J A. 2013. Lateral entorh-inal cortex is necessary for associative but not non associative recognition memory. Hippocampus. 23:1280-1290. [0253] 48. Zucker R S, Regehr W G. 2002. Short-term synaptic plasticity. Annu Rev Physiol. 64:355-405.