Leptin-based hexamers facilitate memory and prevent amyloid-driven AMPA receptor internalisation and neuronal degeneration

Key pathological features of Alzheimer's disease (AD) include build-up of amyloid β (A β ), which promotes synaptic abnormalities and ultimately leads to neuronal cell death. Metabolic dysfunction is known to influence the risk of developing AD. Impairments in the leptin system have been detected in AD patients, which has fuelled interest in targeting this system to treat AD. Increasing evidence supports pro-cognitive and neuroprotective actions of leptin and these beneficial effects of leptin are mirrored by a bioactive leptin fragment (leptin 116– 130 ). Here we extend these studies to ex - amine the potential cognitive enhancing and neuroprotective actions of 8 six-amino acid peptides (hexamers) derived from leptin 116– 130 . In this study, we show that four of the hexamers (leptin 116– 121, 117– 122, 118– 123 and 120– 125 ) replicate the ability of leptin to promote α - amino- 3- hydroxy- 5- methyl- 4-isoxazolepropionic acid (AMPA) receptor trafficking and facilitate hippocampal synaptic plasticity. Moreover, the pro-cognitive effects of the hexamers were verified in behavioural studies, with the administra - tion of leptin 117– 122 enhancing performance in episodic memory tasks. The bioactive hexamers replicated the neuroprotective actions of leptin by preventing the acute hippocampal synapto-toxic effects of A β , and the chronic effects of A β on neuronal cell viability, A β seeding and tau phosphorylation. These findings provide further evi - dence to support leptin and leptin-derived peptides as potential therapeutics for AD


| INTRODUC TI ON
Alzheimer's disease (AD) is a degenerative brain disorder that leads to pronounced cognitive impairments.Key features of AD are the accumulation of amyloid beta (Aβ) and hyper-phosphorylation of tau that result in the formation of amyloid plaques and neurofibrillary tangles, respectively.Toxic forms of Aβ are produced by proteolytic processing of amyloid precursor protein (APP).Acute exposure to oligomeric Aβ promotes disruption of hippocampal synaptic function and chronic Aβ treatment leads to neuronal death (Malekizadeh et al., 2017).Numerous studies indicate that soluble Aβ oligomers interfere with activity-dependent synaptic plasticity resulting in impairments in hippocampal-dependent memory (Morley & Farr, 2014;Shankar et al., 2008).Indeed, induction of long-term potentiation (LTP) at CA1 synapses is blocked after treatment with Aβ, whereas acute exposure to Aβ facilitates induction of hippocampal long-term depression (LTD; Walsh et al., 2002;Shankar et al., 2008).Insertion and removal of AMPA receptors from synapses are crucial for hippocampal synaptic plasticity (Collingridge et al., 2004), and Aβ also interferes with AMPA receptor trafficking (Hsieh et al., 2006;Liu et al., 2010), which is likely to contribute to Aβ-driven impairments in hippocampal synaptic plasticity.
The risk of developing AD increases significantly with age.
However, clinical evidence indicates that lifestyle and dietary factors also influence AD risk (Cunnane et al., 2020;Livingston et al., 2020;Stranahan & Mattson, 2012), with AD risk elevated in individuals with mid-life obesity.Food intake and body weight are regulated by the hormone leptin and circulating leptin levels correlate directly with body fat content (Maffei et al., 1995).Thus, mid-life obesity and the associated increase in body fat content elevates plasma leptin levels ultimately resulting in leptin resistance (Friedman, 2014).Consequently, AD risk is likely to be markedly altered in the obese, leptin-resistant state.Alterations in leptin function occur in AD as significant reductions in leptin levels have been detected in AD patients (Power et al., 2001) and in transgenic mice with familial AD mutations (Fewlass et al., 2004).
Prospective studies have found a link between low leptin levels and an increased risk of AD (Lieb et al., 2009).Strengthening the hypothesis that leptin function correlates with AD and the severity of cognitive impairment, a recent meta-analysis of the 48 epidemiological studies concluded that AD patients had lower blood leptin than cognitively normal individuals and that lower leptin was associated with higher degree of cognitive impairment (Garcia-Garcia et al., 2022).Collectively this suggests that failure to maintain circulating leptin levels within the normal physiological range and/or dysfunctions in the leptin system significantly elevates AD risk.
In addition to playing a role in the hypothalamic control of energy homeostasis, leptin has a major impact on hippocampal excitatory synaptic function.Rodents with insensitivity to leptin display impaired hippocampal synaptic plasticity and spatial memory (Li et al., 2002), whereas rodent performance in hippocampal-specific memory tasks is enhanced after leptin administration (Wayner et al., 2004).In cellular studies, rapid pro-cognitive effects of leptin occur, with significant alterations in glutamate receptor trafficking and excitatory synaptic strength at hippocampal synapses (Luo et al., 2015;Moult et al., 2010;Shanley et al., 2001).However, the effects of leptin extend beyond its cognitive enhancing actions, as leptin protects against various toxic stimuli including Aβ (Doherty et al., 2013;Guo et al., 2008).Leptin also inhibits the aberrant effects of Aβ on hippocampal synaptic plasticity and it blocks Aβdriven synaptic removal of AMPA receptors (Doherty et al., 2013;Malekizadeh et al., 2017;Tong et al., 2015), suggesting that leptinbased treatments may be beneficial in AD.
As leptin is a large peptide with widespread actions, use of smaller molecules that mirror leptin action may be a better therapeutic approach.Indeed, specific fragments of the leptin molecule display CNS activity (Grasso et al., 1997;Rozhavskaya-Arena et al., 2000), and we have shown that one leptin fragment (leptin 116-130 ) mirrors the pro-cognitive and neuroprotective properties of whole leptin (Malekizadeh et al., 2017).Here we extend these studies to show that specific hexamer peptides derived from leptin 116-130 mirror the neuroprotective and pro-cognitive actions of leptin and leptin 116-130 .
These findings have important implications for the use of leptinbased peptides to treat AD.

| Preparation of amyloid β
The lyophilised powder was solubilised in dimethyl sulphoxide (DMSO) and diluted to a working concentration.For cell viability and biochemical assays, Aβ (ab120301; Abcam) was prepared as before as Aβ displays toxicity when in a β sheet conformation (Simmons et al., 1994).Briefly, lyophilised powder was solubilised in DH 2 0 to a concentration of 1 MM in PBS and incubated for 24 h at 37°C prior to further dilution to working concentration

| Quantification of expression of p-tau by ELISA
Protein was extracted into 500 μl tris-buffered saline containing protease inhibitor cocktail (Set II, Merck) as before (Ren et al., 2008).Bradford assay was used to determine protein concentration.Samples were diluted to give equal loading, and expression levels were determined using rabbit anti-p-tau (ser-396, 1:2000; RRID:AB1575880, Genscript) antibodies, followed by detection using an appropriate HRP-conjugated secondary antibody (RRID:AB_390191, Sigma) and 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System (T8665, Sigma).Each protein sample was run in duplicate (technical repeats), and absorbance was measured on a Biohit BP100 plate reader.Experiments were carried out on five separate occasions, representing five separate plate downs and thus biological repeats.
2.1.4| Quantification of amyloid after seeding SY-SY5Y cells were plated onto 13 mm borosilicate glass coverslips (CC7672-7548, CC7672-7458; VWR) and differentiated, prior to seeding with 1 μM Aβ 1-42 and treated with 10 nM leptin, or fragments for 96 h and fixed in neutral-buffered formalin (NBF; HT501128, Sigma) for 15 min before washing with phosphatebuffered saline (PBS; P4417, Sigma).500 μl of Thioflavin-S solution (T1892, Sigma; 0.05% in PBS) was added for 10 min before three 5 min washes with PBS.Coverslips were mounted and sealed on glass slides.Slides were imaged using a Zeiss Axio Imager M2 microscope with Apotome.2 at 63× magnification and analysed using Fiji (Schindelin et al., 2012).Five separate experimental plate downs were established with duplicate coverslips from which 5-10 fields of view across the 2 coverslips were imaged, thus 15-35 fields of view were collected for each condition in total.
No labelling was observed after incubation with secondary antibodies alone.A Zeiss LSM 510 confocal microscope was used for image acquisition and 488-nm laser line was used to excite the Alexa 488 fluorophore.Images were obtained in single-tracking using a 15-s scan speed and the intensity of staining was determined offline using LaserSharp software (Carl Zeiss).Analysis lines (50 μm) were drawn along randomly selected dendritic regions and mean fluorescence intensity of staining was calculated for each dendrite (McGregor et al., 2017).Data were obtained from at least four randomly selected cells for each condition, and all data were obtained from at least three different cultures from different animals.Within a given experiment, all conditions, including illumination intensity and photomultiplier gains were kept constant.To quantify data obtained from separate days, data were normalised relative to mean fluorescence intensity in control neurons.
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 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 the baseline.
LTP was induced using a high-frequency stimulation paradigm (100 Hz, 1 s).Recordings were made using an Axopatch 200B amplifier and analysed using LTP v2.4 software (Anderson & Collingridge, 2007).For studies comparing the actions of leptin hexamers on synaptic transmission, the mean slope (average of 5 min recording) of fEPSPs obtained during the 5 min period immediately prior to hexamer addition was compared with that after 25-30 min exposure.In LTP studies, the degree of potentiation was calculated 30-35 min after HFS and expressed as a percentage of baseline ± standard error of mean (SEM).For all experiments, each n value represents an individual slice taken from a separate animal.

| Subjects and design
41 C57/BL6 mice (6-8 months; 25 male and 16 female) were used.Sample sizes were determined using G-power and data from Malekizadeh et al. (2017) which examined the effects of leptin on similar types of memory.Malekizadeh et al. (2017) used group sizes of 14 per group which produced a η 2 of 0.181 for the critical group comparison in the ANOVA.G-power confirmed that this produced a large effect size (f = 0.47) and so the same group sizes were used in the current study.Mice were housed in groups of 3-5 and kept on a

| Episodic-like memory (Object-Place-Context; OPC)
Mice were habituated to the testing box (30 × 20 × 20 cm) for 3 days (1 group habituation, 2 days individual habituation).The box could be configured with 2 different sets of contextual features using floor and wall inserts (blue with a plain floor v green/white stripes with a mesh floor) and mice were habituated to both contexts (10 min sessions) on each day.Objects were household items approximately the size of the mice in at least 1 dimension.Mice then underwent object recognition, object-place and object-context training (4 days each) as before (Wilson et al., 2013).Mice received the IP injections 30 min prior to testing.OPC testing was carried out as before (Vandrey et al., 2020) and consisted of 3 stages; 2 sample phases and a test phase (Figure 5b).Each phase was 3 min duration and exploration behaviour was scored using in-house software (Observe, University of St Andrews).Between all phases, the box was cleaned.
Testing was recorded to allow offline scoring of behaviour.The discrimination ratio (DR) was calculated by subtracting the amount of time exploring the familiar object from the time exploring the novel object and dividing by the total exploration time.Total exploration in sample and test phases were compared between groups.Object location, object used at test, order of contexts and context at test were all counterbalanced.

| Spontaneous alternation T-maze
Mice were tested individually for 15 min on a T-maze with arms 16 cm in length.Each mouse was placed on the central arm and exploration was recorded and scored using custom-written software (Observe, University of St Andrews).In the spontaneous alternation task, a correct response was defined as animals choosing the arm that they have least recently visited (e.g.ABC or BAC), non-alternation is when the animal returns to an arm it has visited previously before completing a full sequence (e.g.ACA, BAB, AAB etc.).Performance is quantified as the proportion of alternation, which is the number of successful alternation sequences divided by the total number of attempts (successful + unsuccessful).

| Elevated plus maze
Mice were tested on a standard elevated plus maze consisting of a 4arm (35 cm in length) maze arranged in a '+' cross.Two opposing arms had high walls (20 cm) while the other two had no walls.Mice were tested individually with each testing session lasting 5 min (Haleem et al., 2015).

| Statistical analyses
In immunocytochemical, ELISA and cell viability studies, all statistical analysis was carried out using GraphPad PRISM 9 (Graph Pad Inc.).In all experiments, data are expressed as mean ± SEM.After normal distribution test, statistical analyses were performed using one-way analysis of variance (ANOVA) with Tukey's post hoc test for comparisons between multiple groups.p < 0.05 was considered significant.In electrophysiological studies, all statistical analyses were performed using Sigmaplot 14.5 (Systat Software) and all analyses were performed using repeated measures ANOVA for comparison between multiple groups.All behavioural data were analysed using SPSS 26.0 (IBM).Following Shapiro-Wilk testing for normality, OPC data (DR, total exploration) was examined with mixed factorial ANOVA with treatment group as between-subject factor and testing block (1st 4 days, last 4 days) as within-subject factor.Tukey's tests were used for post hoc comparisons.One-sample t-tests were used to compare performance to 0. T-maze and Plus maze data were analysed using one-way ANOVA.Finally, weight was analysed using mixed factorial ANOVA with group and day as between-and withinsubject factors respectively.No analysis for outliers was used and all statistical tests were two-tailed and used p < 0.05 significance threshold.

| Differential regulation of AMPA receptor trafficking by the leptin hexamers
Our previous studies indicate that leptin also regulates glutamate receptor trafficking as leptin stimulates insertion of the AMPA receptor subunit, GluA1 into synapses (Moult et al., 2010).Thus, to further verify hexamer bioactivity, the effects of the hexamers on AMPA receptor trafficking were examined by monitoring GluA1 surface expression in hippocampal neurons (Doherty et al., 2013;Moult et al., 2010).In agreement with previous studies  We next determined whether leptin 117-122 , mirrored leptin's known improvement in spatial memory (Oomura et al., 2006;Haleem et al., 2015), AMPA receptor trafficking is also detrimentally influenced in AD as Aβ causes GluA1 endocytosis (Hsieh et al., 2006;Liu et al., 2010); an effect alleviated by treatment with either leptin (Doherty et al., 2013) or leptin 116-130 (Malekizadeh et al., 2017).Thus, the neuro-protective actions of two of the bioactive hexamers, leptin 116-121 and leptin 117-122 were examined further, by probing the cell surface density of GluA1 in hippocampal neurons (Moult et al., 2010). In

| Leptin 116-121 and leptin 117-122 mirror the neuroprotective actions of leptin
Multiple strands of evidence support a role for leptin in protecting against neurotoxic stimuli (Doherty et al., 2008;Doherty et al., 2013;Guo et al., 2008), but only part of the C-D loop region, leptin 116-130 , is required to replicate this effect of the fulllength molecule (Malekizadeh et al., 2017).Initially, we treated cultures with the peptides to ensure that they did not have any significant effects on cell viability in the absence of a stressor and no significant effects were observed (Figure S2).observed for all four bioactive leptin hexamers (Figure 5c), whereas leptin 124-129 and leptin 125-130 were without effect.In parallel studies, analogous concentration-response profiles were obtained for leptin and the hexamers using an LDH assay (Figure 5d).et al., 2002).A one-way ANOVA revealed a significant effect of treatment (F (9,191) = 21.17

| DISCUSS ION
Although food intake and body weight are regulated by the hypothalamic actions of leptin (Spiegelman & Flier, 2001), the hippocampus is also a key CNS target for leptin.Increasing evidence indicates a procognitive role for leptin within this brain region, as leptin facilitates hippocampal synaptic plasticity and hippocampus-dependent learning and memory (Irving & Harvey, 2021;McGregor & Harvey, 2018).
In cellular studies, rapid effects of leptin on glutamate receptor trafficking and dendritic structure have been observed that likely contribute to the pro-cognitive properties of leptin (Irving & Harvey, 2014).Additionally, leptin significantly impacts neuronal viability, as leptin enhances the survival of central and peripheral neurons and protects neurons against various toxic stimuli (Weng et al. 2007; et al., 2008;Guo et al., 2008).
A correlation between plasma leptin levels and AD risk has been detected in clinical studies (Lieb et al., 2009;Power et al., 2001), and increasing evidence supports the notion that targeting the leptin system is beneficial in AD models (Doherty et al., 2013;Farr et al., 2006;Fewlass et al., 2004).However, other studies have found no link between leptin levels and the incidence of Alzheimer's disease or cognitive decline in AD patients (Teunissen et al., 2015).However, in this study, the average age of the AD patients was 63 and therefore this may reflect differences between the late and early onset forms of the disorder.However, the whole leptin molecule is not required for its protective actions as a smaller leptin fragment, leptin 116-130 , is as effective as leptin in preventing the toxic effects of Aβ on hippocampal synapses and neuronal viability (Doherty et al., 2013;Malekizadeh et al., 2017).Here we extend those studies to provide compelling evidence that two specific leptin hexamers, Previous studies have demonstrated that leptin acts as a potential cognitive enhancer as it facilitates synaptic plasticity at hippocampal CA1 synapses (Malekizadeh et al., 2017;Oomura et al., 2006;Shanley et al., 2001).Similarly, treatment of hippocampal slices with four of the hexamers (leptin 116-121 , leptin 117-122 , leptin 118-123 or leptin 120-125 ) resulted in a significant increase in the magnitude of LTP compared to control LTP.It is well known that activitydependent alterations in the synaptic density of AMPA receptors are key for maintaining the increase in synaptic efficacy associated with LTP (Collingridge et al., 2004).Moreover, we have shown that leptin regulates the movement of the AMPA receptor subunit, GluA1 to and away from hippocampal synapses (Moult et al., 2010); a process mirrored by leptin 116-130 (Malekizadeh et al., 2017).Here we show that the four hexamers that facilitate LTP, also increase the surface To assess whether the hexamer-induced changes in synaptic plasticity have a significant impact on learning and memory, the effects of one hexamer, leptin 117-122 were compared to whole leptin and a saline control on a series of behavioural tasks which assessed episodic-like memory, spatial memory and anxiety-like behaviour.
Consistent with our previous findings (Malekizadeh et al., 2017) leptin improved performance on the OPC task.We extend these findings to show that leptin 117-122 produces a similar facilitation of episodic-like memory.Given recent findings showing that increased performance on object exploration tasks correlates with memory discrimination in humans (Sivakumaran et al., 2018), this provides evidence of improved memory in rodents.The OPC task assesses integration of object, place and context information and consequently provides a model of episodic memory.Given that a deficit in episodic memory is one of the first symptoms of AD, these data suggest that leptin 117-122 might be a useful therapeutic target.Future studies could aim to examine whether the hexamers rescue similar episodiclike memory deficits reported in transgenic models of AD (Davis et al., 2013).The fact that chronic administration of both leptin and leptin 117-122 improved memory over a 3-week period also suggests that the memory facilitation is persistent.
As previous studies have shown that leptin facilitates spatial memory (Oomura et al., 2006;Haleem et al., 2015), we assessed whether leptin 117-122 has a similar effect relative to leptin and a saline control.We found a small but significant increase in spatial memory performance on a spontaneous alternation task which has been shown to be hippocampus-dependent (Ainge et al., 2007;Dudchenko 2004).This is consistent with the current findings of facilitation of hippocampal synaptic plasticity and suggests that one driving factor behind enhanced performance in the OPC task is an improved memory for spatial features of an environment.Although anxiety-like behaviour can impact performance on behavioural tasks, particularly those that are spontaneous, we found no significant difference between groups on plus-maze performance demonstrating that changes in memory performance are not attributable to changes in behaviour induced by anxiety.Although we have no direct evidence that the hexamers cross the blood brain barrier in our behavioural studies, given that previous studies have shown that leptin and fragments cross the blood brain barrier (Grasso et al., 1997;Lee et al., 2008), and the behavioural effects of leptin and the hexamers are strikingly similar, the most feasible explanation is that the hexamer also readily accesses the brain.This is also consistent with the facilitation of synaptic plasticity by the hexamers that we observed.
Aβ interferes with excitatory synaptic function at hippocampal CA1 synapses, such that acute exposure to Aβ promotes endocytosis of the AMPA receptor subunit GluA1 (Hsieh et al., 2006).
Our previous studies indicate that leptin and leptin 116-130 prevent this synapto-toxic effect of Aβ in hippocampal neurons (Doherty et al., 2013;Malekizadeh et al., 2017).Likewise, in hippocampal neurons treated with leptin 116-121 or leptin 117-122 the ability of Aβ to internalise GluA1 was markedly reduced, suggesting that the hexamers replicate the protective actions of leptin by preventing the harmful effects of Aβ on AMPA receptor trafficking.
Accumulating evidence indicates a neuroprotective role for leptin in models that mirror the neuronal degeneration that occurs in diseases like AD (Doherty et al., 2008;Guo et al., 2008).For instance, in models that reproduce the toxic events associated with chronic exposure to Aβ, leptin attenuates the magnitude of cell death, by activating pro-survival signalling cascades such as PI3 kinase and STAT3 (Doherty et al., 2008;Doherty et al., 2013;Guo et al., 2008).In line with previous studies, we show that the viability of human SH-SY5Y cells is markedly reduced after chronic treatment with Aβ 1-42 (Malekizadeh et al., 2017) (Greco et al., 2008;Malekizadeh et al., 2017;Zhang et al., 2016), via inhibition of GSK3β.
Here we show that only a short six amino acid sequence derived from leptin 116-130 is sufficient to replicate these effects on tau phosphorylation.There is clear evidence that leptin modulates Aβ levels, as the activity of β secretase, a key enzyme in the production of Aβ 1-42 , is reduced by leptin (Fewlass et al., 2004).In AD, amyloid deposits typically appear with a hierarchical spatial distribution, suggesting propagation of the peptide between different brain areas (Braak & Braak, 1991;Thal et al., 2002) and in support of this several studies have demonstrated transmission of Aβ between cells both in vivo and in vitro (Gouras et al., 2010;Nath et al., 2012).The release of intracellular amyloid is well established as the source of amyloid plaques (Calhoun et al., 1999;Zhao et al., 1996).Here we show that chronic treatment of SH-SY5Y cells with a low dose of Aβ 1-42 results in amyloid seeding, and that exposure to leptin, leptin 116-130 , leptin 116-121 or leptin 117-122 prevented generation of more amyloid.
Together these data present compelling evidence that leptin 116-121 and leptin 117-122 are as effective as leptin and leptin 116-130 in reducing tau phosphorylation and amyloid accumulation.
A key area that remains to be addressed is how the leptin-based peptides interact with the leptin receptor (LepR).Based on its similarity to the IL-6 receptor, it has been proposed that leptin has three possible binding domains that enable interaction with LepR.Binding site I lies within the C terminus of helix D and works in concert with residues from the connection loop between helices A and B. Binding site II consists of residues from helices A and C whereas binding site III contains residues at the N-terminus of helix D (Peelman et al., 2004).None of these hypothetical LepR interaction sites are located within the leptin 116-130 sequence, identified as bioactive by us and others (Grasso et al., 1999;Malekizadeh et al., 2017) and on which the leptin hexamers are derived.However, the exact locations of binding sites I and II remain controversial (Greco et al., 2021) and therefore detailed in silico and empirical studies are needed to model leptin fragment/s interactions with LepR and to clarify further how native leptin interacts with its receptor.Evidence suggests that the long LepR isoform (LepRb) may not be required for leptin 116-130driven anti-obesity signalling as db/db mice that lack functional LepRbs still respond to leptin 116-130 (Grasso et al., 1999).The signalling pathways activated by leptin 116-130 mirror those activated by the full-length leptin molecule and include JAK/STAT, MAPK and PI3-kinase/Akt signalling (Jeremy et al., 2022;Lin et al., 2014;Malekizadeh et al., 2017).Consequently, this raises the question of how leptin-mimetic peptides are signalling and opens the possibility for involvement of other LepR isoforms.
As the emerging evidence that leptin and leptin-based peptides have a wide range of neuro-beneficial effects in empirical models of AD, their suitability as druggable entities should be considered.
Leptin's multi-faceted protective effects against various elements of AD pathology are well known, with beneficial effects demonstrated at very early stages of the disease, including effects on synaptic dysfunction (Doherty et al., 2013) and mitochondrial aberrations (Cheng et al., 2020) through to behavioural changes (Greco et al., 2010) and ultimately the loss of neurons.This study explores the potential for the small hexamer peptides to elicit beneficial changes on synaptic function, biomarker expression, episodic memory to cell death.It is prudent at this stage in the investigation of the neurological actions of these peptides to consider ways in which they can be modified to aid with the potential routes for administration and bioavailability, and to test the modified forms on the simpler models used here, prior to adopting more complex studies on transgenic animals.

14714159, 0 ,
Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jnc.15733by University of Dundee, Wiley Online Library on [22/12/2022].See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions)on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 12 h light/dark cycle (behavioural testing carried out during the light phase) with ad libitum access to food and water.All procedures were approved by the Animal Welfare Ethics Committee (University of St Andrews) and complied with national (Animal [Scientific Procedures] Act, 1986) and international (European Directive 2010/63/EU) legislation (Home Office project licence P53E784C4).Mice were randomly assigned to three treatment groups: saline (n = 14, 6 female), leptin (n = 13, 4 female) and leptin 117-122 (n = 14, 6 female).A random number generator was used to generate a random sequence of the numbers from 1 to 41.The first 14 numbered animals were assigned to the saline group, the next 13 to the leptin group and the final 14 to the hexamer group.Experiments were run blind to treatment group.100 μl of either saline, leptin or leptin 117-122 (7.8 nM/ml; Malekizadeh et al., 2017) were administered daily by IP injection during the 20-day testing period.This consisted of 4 days Object-Place-Context (OPC) tests, 4 days rest, 1 day T-maze, 3 days elevated plus maze, 4 days rest and 4 days OPC testing (Figure 5a).No inclusion/exclusion criteria were used in these studies.Weight was monitored throughout experiments and did not differ significantly between groups.
Testing started by placing a mouse in the centre of the maze facing an open arm.Exploration was recorded and scored using in-house software (Observe, University of St Andrews).Two measures of exploration were taken; number of entries into open and closed arms and time spent in open vs. closed arms.
Figure 6a,b; p < 0.0001).For all experiments, n = 15-35 fields of view per condition from five separate plate downs.In the absence of Aβ 1-42 , thioflavin S-positive fluorescence was comparable across all

14714159, 0 ,
Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jnc.15733by University of Dundee, Wiley Online Library on [22/12/2022].See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions)on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License leptin 116-121 and leptin 117-122 , derived from leptin 116-130 also replicate the rapid effects of leptin on AMPA receptor trafficking and facilitation of hippocampal synaptic plasticity, suggesting that these hexamers mirror the synaptic actions of leptin.In contrast, the leptin hexamers 121-126 , 123-128 , 124-129 and 125-130 were found to be inactive.Further evaluation of two of the active hexamers (leptin 116-121 and leptin 171-122 ) established that these hexamers prevent the acute detrimental effects of Aβ on hippocampal synaptic function, as treatment with either hexamer prevents Aβ-driven internalisation of the AMPA receptor subunit, GluA1.Both hexamers replicate the neuroprotective characteristics of leptin as exposure to either leptin 116-121 or leptin 117-122 counteracts the chronic effects of Aβ on neuronal viability.This study highlights the cross-species potency of these neuroactive hexamers by using established in vitro models of AD and building on existing murine in vivo studies.This is essential for peptides where protein sequences per se diverge between species and even a single amino acid alteration could be crucial in determining whether efficacy is maintained.And yet early-stage testing of neuroactive compounds in humans is necessarily limited.Thus, we have shown potent neuroprotection following leptin 116-121 and leptin 1717-122 administration in amyloid-treated human neural cells coupled to the prevention of amyloid-driven synaptic deficits in rat hippocampal slices and improvements in episodic-like memory in mice.These data exhibit a robust cross-species response to leptin 116-121 and leptin 1717-122 across the mammalian taxon and validates the use of rodent models to further determine the neurobeneficial effects of these molecules.Collectively, these findings indicate that the beneficial actions of whole leptin are mirrored by leptin 116-121 and leptin 1717-122 as evidenced by the ability of both hexamers to prevent the aberrant effects of Aβ in various cellular models of AD.
expression of GluA1 in hippocampal neurons.Conversely, treatment with leptin 121-126 , 123-128 , 124-129 and 125-130 had no effect on GluA1 trafficking as no significant change in GluA1 surface expression was detected after exposure to these hexamers.Overall, these findings indicate that four of the hexamers (leptin 116-121 , leptin 117-122 , leptin 118-123 and leptin 120-125 ) mirror the effects of leptin on glutamate receptor trafficking and hippocampal synaptic plasticity.
Peptide-based natural hormone analogues present a simple way to target hormonal receptors such as LepR for pharmacological benefit.However, peptide-based therapeutics present several challenges including delivery difficulties and in vivo instability.Significant progress has been made in advancing the use of peptide therapeutics, with numerous strategies to enhance bioavailability being developed including C-terminal amidation or N-terminal acetylation (Di, 2015), incorporation of synthetic enantiomer amino acids(Weinstock et al., 2012), and peptide cyclisation(Hayes et al., 2021).Therefore, emerging strategies will allow further development of leptin peptides towards therapeutic leads but will require robust evaluation to ensure efficacy is maintained.As different leptin fragments have been linked with different aspects of leptin's biological functions, there is the potential for hexamer-based fragments to have reduced off-target effects compared to the full-length molecule.
hexamers, leptin 116-121 and leptin 117-122 , mirror the synaptic effects of whole leptin, via their ability to rapidly regulate AMPA receptor trafficking and enhance synaptic plasticity at hippocampal CA1 synapses.In addition, leptin 116-121 or leptin 117-122 prevent the acute effects of Aβ at hippocampal synapses, as Aβ-driven internalisation of GluA1 is inhibited by the two leptin hexamers.Treatment with either leptin 116-121 or leptin 117-122 also prevents the neuronal damage caused by chronic exposure to Aβ 1-42 and reduces Aβ-driven accumulation of AD-linked biomarkers.Leptin 117-122 also improves episodic-like and spatial memory relative to controls.Overall, these findings add further weight to the possibility of targeting the leptin system to treat AD, and it identifies that two leptin-based hexamers are highly potent leptin-mimetics that may provide the basis for the development of AD therapeutic agents in the future.