Patent Application
20200255492
The present invention relates to leptin peptide fragments and methods of using such peptide fragments in treating or protecting from neurological conditions, including use in cognitive enhancement and/or neuroprotection.
Alzheimer's disease (AD) is a complex, progressive brain disorder that results in profound cognitive deficits, particularly in memory. Accumulation of toxic amyloid plaques and neurofibrillary tangles comprising hyper-phosphorylated tau are key pathological features of AD. Proteolytic processing of APP and generation of toxic amyloid beta (Aβ) is linked to aberrant synaptic function and neuronal degeneration.
Clinical evidence indicates that diet and lifestyle are major risk factors for developing AD and disruption to metabolic systems is linked to AD (Stranahan and Mattson 2012). The metabolic hormone leptin regulates energy homeostasis, but also markedly influences hippocampal synaptic function. Indeed, leptin-insensitive rodents exhibit impaired hippocampal LTP and spatial memory (Li et al. 2002). Moreover, direct administration of leptin into the hippocampus facilitates learning (Wayner et al. 2004), whereas hippocampal dendritic morphology, glutamate receptor trafficking and synaptic plasticity are significantly altered after leptin treatment (Shanley et al. 2001; O'Malley et al. 2007; Moult et al. 2010; Luo et al. 2015). Clinical studies indicate that aberrant leptin function is associated with an increased risk of AD, as AD patients exhibit significantly lower leptin levels than normal (Power et al. 2001). Individuals with lower circulating leptin also have a greater risk of developing AD (Lieb et al. 2009). Additionally, leptin levels are markedly reduced in rodent models with familial AD mutations (APPSwe; PSIM146V; Fewlass et al. 2004). In cellular models of AD, leptin treatment prevents the aberrant effects of Aβ, as leptin reverses the ability of Aβ to inhibit LTP and facilitate LTD in hippocampal slices (Doherty et al. 2013) and it prevents Aβ-driven internalization of the AMPA receptor subunit, GluA1 in hippocampal neurons (Doherty et al. 2013). Chronic intra-cerebroventricular injection of leptin also reverses Aβ-induced impairments in LTP in vivo (Tong et al. 2015). In cell survival assays, leptin protects against Aβ-induced toxicity as neuronal viability is increased after leptin treatment. Moreover the cortical expression of 2 AD-linked biomarkers, endophilin 1 and phosphorylated tau, are enhanced in leptin-insensitive Zucker fa/fa rats (Doherty et al. 2013). Thus there is growing evidence that leptin-based therapies may be beneficial in AD.
However, using leptin therapeutically may not be the best approach due to its widespread central actions. One possibility is to develop small molecules that mimic leptin action. Indeed, studies have found that specific fragments of the leptin peptide are bioactive and mirror the anti-obesity effects of leptin (Grasso et al. 1997; Rozhayskaya-Arena et al. 2000). Indeed, application of a C-terminal fragment of leptin (amino acids 116-130) or a shorter fragment (116-121) to leptin-deficient ob/ob mice reduced food intake and body weight (Grasso et al. 1997; Rozhayskaya-Arena et al. 2000; Grasso et al. 2001). However, another leptin fragment (22-56) is also bioactive as significant reductions in food intake have been observed after leptin (22-56) administration (Samson et al. 1996). Thus, it is feasible that different parts of the leptin molecule are active in the CNS. However, the cognitive enhancing and neuroprotective properties of the bioactive leptin fragments are unknown.
In a first aspect there is provided a leptin peptide fragment or variant thereof for use in a method of treating or preventing development of a neurological disorder.
The leptin peptide fragment of the present invention may be up to 30, 25, 20, 15, or 10 amino acids in length and comprises amino acids located within the region of amino acids 116-125 of leptin (see the table below adapted from Peptides. 2012 December; 38(2); 326-336). Typically the leptin peptide is at least 4, 5, 6 or 7 amino acids in length.
As can be seen from the above, leptin sequences (using the conventional 1-letter amino acid code employed in the table and used throughout herein) are highly conserved in the region which the present invention is directed to.
In one embodiment, the leptin peptide fragment includes sequences located between amino acids 116-122. Based on the above, a consensus sequence for amino acids 116-122 may be as follows:
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).
Conveniently the peptide sequence may comprise, consist essentially of, or consist of amino acids 116-121, 117-122, 117-125, 118-123, 119-124, 120-125, or 116-130.
In certain embodiments the invention is directed to human therapies and so the leptin peptide fragment may be based on the human sequence. The human leptin sequence and numbering is shown below, with amino acids 116-125 highlighted:
In certain embodiments the neurological disorder is a disorder which would benefit from treatment through cognitive enhancement and/or neuroprotection.
The term “neurological disorder” refers to disorders of the nervous system that result in impairment of neuronal mediated functions and includes disorders of the central nervous system (e.g., the brain, spinal cord) as well as the peripheral nervous system.
The invention relates to leptin peptide fragments for use in methods of treating acute, chronic and prophylactic treatment of neurologic and neurodegenerative diseases, attenuation of acute or chronic neuronal damage in neurological disease (“neuroprotection”), and prophylaxis of neurological diseases.
The leptin peptides fragments may be useful for enhancing cognitive function and/or synaptic plasticity in vivo, e.g., for the treatment and/or prevention of memory impairment in mammalian subjects such as humans. In particular, the leptin peptide fragments may be useful for the treatment and/or prevention of age-associated memory impairment or loss, mild cognitive impairment, and Alzheimer's disease. In one embodiment the leptin peptide fragments may be useful for short term enhancement of cognitive function and/or synaptic plasticity. Cognitive enhancement may in addition include enhancement of synaptic plasticity.
“Cognition” generally refers to the process of obtaining, organizing, and using knowledge. Enhancing cognitive function refers to enhancing any aspect of this process, e.g., learning, the performance of mental operations, the storage and/or retrieval of information or thoughts (memory), and/or preventing a decline from a subject's current state. Numerous standardized tests can be used to evaluate cognitive function. Such tests can be used to identify subjects in need of enhancement of cognitive function and/or to monitor the effects of treatment. Suitable tests include, but are not limited to, the Mini-Mental Status Exam (Folstein, 1975), components of the PROSPER neuropsychological test battery (Houx, 2002), etc. Family history, age, and other factors may also be used to identify subjects in need of enhancement of cognitive function.
“Synaptic plasticity” is defined as the ability of a synapse to change its strength in response to a pattern of stimulation (i.e., one or more electrical or chemical stimuli), wherein the alteration in strength typically outlasts the event that triggers it. A synapse that exhibits this property is said to be plastic, or to display synaptic plasticity. A neural network in which some or all of the synapses exhibit plasticity is also said to exhibit synaptic plasticity. Synaptic plasticity may be considered to exist at the level of the presynaptic terminal, the postsynaptic terminal, or both. Thus a synapse is said to exhibit presynaptic plasticity if presynaptic strength is altered in response to a pattern of stimulation. A synapse is said to exhibit postsynaptic plasticity if postsynaptic strength is altered in response to a pattern of stimulation, and/or if the probability that an action potential will be generated in response to a second pattern of stimulation is altered as a result of a first pattern of stimulation.
“Synaptic strength” of a given synapse may be assessed by measuring one or more indicators of presynaptic strength, postsynaptic strength, or both. In general, presynaptic strength refers to properties including (i) the amount of neurotransmitter released in response to a pattern of stimulation; and/or (ii) the probability of neurotransmitter release in response to a pattern of stimulation. The product of (i) and (ii) provides an overall measure of presynaptic strength. Postsynaptic strength refers to properties including (i) the size of the postsynaptic current or potential induced by a fixed amount of neurotransmitter or other stimulus, e.g., an electrical stimulus; and/or (ii) the probability of firing of an action potential for a fixed amount of input. Overall synaptic strength reflects a combination of presynaptic and postsynaptic strength. Overall synaptic strength may be determined by combining measures of presynaptic and postsynaptic strength (e.g., by adding, multiplying, etc.). Alternatively, overall synaptic strength may be measured directly, e.g., by stimulating individual presynaptic neuron(s) and recording the evoked response at the corresponding postsynaptic neuron(s). For purposes of the present invention, a synapse will be said to increase its synaptic strength if it increases its presynaptic strength or its postsynaptic strength, or both. A synapse will be said to decrease its synaptic strength if it decreases its presynaptic strength or its postsynaptic strength, or both. One of ordinary skill in the art will recognize that other parameters indicative of synaptic strength may be used, and parameters may be combined in various ways to arrive at a measurement of synaptic strength. One of ordinary skill in the art will also appreciate that a variety of measurement techniques may be applied to assess parameters associated with synaptic strength.
Synaptic plasticity is believed to be essential for the processes involved in learning and memory. Thus compositions that enhance synaptic plasticity are of use for the treatment of individuals (subjects) suffering from any of a variety of conditions in which cognitive function, e.g., memory and/or learning is impaired. The compositions are also useful to prevent the onset of such conditions. These conditions include, but are not limited to, those known as “benign senescent forgetfulness”, “age-associated memory impairment”, “age-associated cognitive decline”, “mild cognitive impairment”, Alzheimer's disease, dementias (associated with any of a number of causes), attention-deficit disorder, etc. The compositions and methods of the invention may also find use to enhance the cognitive function, e.g., memory and/or learning capacity of normal individuals, i.e., individuals not suffering from any clinically recognized condition or disorder. They may be useful on a short-term basis or may be administered chronically.
AD may be diagnosed according to the National Institute of Neurological and Communicative Disorders and Stroke—Alzheimer's Disease and Related Disorders Association criteria for a clinical diagnosis of probable Alzheimer's disease, imaging and various biomarkers (e.g., levels of tau protein in cerebrospinal fluid). In addition, individuals with dominant mutations in the amyloid precursor protein, PS1, or PS2 genes are at increased risk of AD. It has also been found that the risk of developing AD is greater in individuals with the ε4 allele of the gene encoding ApoE. Such individuals may be particularly appropriate candidates for therapy with the compositions described herein.
The term “neuroprotection” refers to prevention or a slowing in neuronal degeneration, including, for example, neuronal death and/or axonal loss.
The term “Subject”, as used herein, refers to an individual to whom a leptin peptide fragment is to be delivered. Preferred subjects are mammals, particularly domesticated mammals (e.g., dogs, cats etc.), primates, or humans. The subject may be a human being, e.g., a human being suffering from or at risk of a neurological disease or condition such as age-associated memory loss, mild cognitive impairment, or Alzheimer's disease. Typically the subject will be administered a leptin peptide fragment comprising a sequence which is based on the subjects endogenous leptin sequence. Thus, a human may be administered a leptin peptide fragment comprising sequence based on the human leptin sequence. However, this should not be construed as being essential.
Mild cognitive impairment (MCI) refers to the transitional zone or time period between normal aging and mild dementia. Criteria for the diagnosis of MCI may include subjective and objective memory impairment, normal cognitive and activities of daily living (ADL), and the absence of any specific criteria for dementia. The cognitive impairment may be amnestic (memory) or involve any other isolated cognitive domain that is greater than expected for normal aging. The patient and family may have insight into the impairment, but the patient is still able to function adequately with ADL. The objective memory function detected by neuro-psychological tests usually 1.5 SD below the average performance of individuals with similar age and education. MRI of the brain may reveal mild atrophy of the hippocampus and entorhinal cortex while neuropathologic studies can reveal some early features of dementia. Thus, while subjects with MCI have a condition that differs from normal aging and are likely to progress to dementia at an accelerated rate, not all patients progress to dementia. Finally, most subjects with MCI that convert to dementia have elevated levels of CSF tau protein.
Cognitive decline may occur in various other neurological diseases which have dementia as a symptom and which may have either a genetic predisposition (chromosome 17), contain Lewy bodies or tau proteins. For example, mutations of tau occur in families with FTDP-17 (frontal temporal dementia linked with Parkinson's disease). This syndrome is characterized by widespread NFT formation associated with tau, in the absence of amyloid deposits. Thus, abnormalities of tau structure and function produces progressive, severe neuronal degeneration and death. Additional dementing illnesses 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. Those skilled in the art will recognize that these diseases are not Alzheimer's disease or an MCI condition, but may be treated in accordance with the present invention.
As well as the leptin peptide fragments identified herein, the present invention extends to variants thereof. Variants include peptides with one or more substitutions, deletions and/or additions. Variants also include chemical modifications to the N-terminus, C-terminus and/or backbone amino acids, which do not substantially negatively alter the activity of the peptide. By substantially negatively alter the activity of the peptide means that the activity is not reduced by more than 10%, 5%, or 1% as compared the unmodified peptide. Of course such chemical modifications could have a positive effect on activity and any modifications which have a positive effect on activity are included.
A variant of the present invention includes a variant of a parent leptin peptide fragment having at least about 80%, 90% identity and most preferably at least about 95% identity to the parent molecule and which has cognitive enhancing and/or neuroprotective activity. In a preferred embodiment, the variant peptide has an amino acid sequence which differs by 3, 2 or 1 amino acid(s), from the leptin peptide fragments identified herein.
In one embodiment, the leptin peptide fragment variant comprises between one and three amino acid deletions (or additions) from the leptin peptide fragments identified herein, providing that the variant peptide still has cognitive enhancing and/or neuroprotective activity. Based on the teaching herein, the skilled addressee can easily test such variant peptides in order to determine whether or not they possess or are likely to possess cognitive enhancing and/or neuroprotective activity and hence be useful in accordance with the present invention. It is also possible through comparison with unmodified leptin peptide fragments to determine whether or not a modified peptide has an altered activity and by how much. Suitable examples of tests are described herein.
Variants also include peptide conjugates. Peptide conjugates may generally include a further biologically active agent being conjugated to the leptin peptide fragment, such as through the N or C-terminal amino acid of the leptin peptide fragment. The other biologically active agent may be a further peptide, for example, which may facilitate translocation of the leptin peptide fragment across the gut and/or blood brain barrier and/or may itself have cognitive enhancing and/or neuroprotective properties. A further envisaged conjugate may be the leptin peptide conjugated to itself, that is two leptide peptides of the present invention conjugated to another, by appropriate means.
Peptides composed of L-amino acids undergo rapid proteolysis in the gut, making oral administration, the method generally associated with the highest patient compliance, often problematic. Additionally, peptides degrade fairly rapidly in serum and therefore must be administered in large doses which often can cause numerous adverse side effects and serious toxicity. As peptides are expensive to manufacture, high dosage levels contribute significantly to the overall cost of peptide therapeutics. Furthermore, the flexibility of the peptide structure in solution is often associated with low biological activity and/or selectivity. Thus, it may be appropriate to modify the peptides of the present invention in order to address and/or obviate the above problems.
Some post-translational modification strategies have found use in peptide engineering The simplest and most commonly used approach is to include N-terminal acetylation and C-terminal amidation. Natural peptides may be halogenated, such as brominated or occasionally chlorinated, and it may be desired to modify the peptides of the present invention by halogenation of particular residues, such as tryptophan residues. Enhanced peptide stability may result from increase in size or hydrophobicity due to halogenation, or protect the peptide from degradation/oxidation. Incorporation of D-amino acids provides another useful approach for improvement of peptide stability. Alternatively, partial incorporation of D-amino acids may also improve peptide stability.
Another modification known to the skilled addressee, is to cyclize peptides. Following the pioneering work of R. Schwyzer [Ludecher, U., et al., Helv. Chim. Acta 54. 1637 (1971)] on gramicidin S, conformational restriction of peptides by medium and long range cyclization has been extensively employed. In addition to other modes of conformational restriction, such as configurational and structural alteration of amino acids, local backbone modifications, short-range cyclization etc., medium and long range cyclization [Hruby, V. J., Life Sci. 31, 189 (1982); Kessler, H., Angew. Chem. Int. Ed. Eng., 21, 512 (1982); Schiller, P. W., in the “Peptides”, Udenfriend, S., and Meienhofer, J. Eds., Volume 6 p. 254 (1984); Veber, D. F. and Freidinger, R. M., Trends in Neurosci. 8, 392 (1985); Milner-White, E. J., Trends in Pharm. Sci. 10, 70 (1989)] is used for the following purposes: biologically active peptides are cyclized to achieve metabolic stability, to increase potency, to confer or improve receptor selectivity and to control bioavailability. The possibility of controlling these important pharmacological characteristics through cyclization of linear peptides prompted the use of medium and long range cyclization to convert natural bioactive peptides into peptidomimetic drugs. Cyclization also brings about structural constraints that enhance conformational homogeneity and facilitate conformational analysis [Kessler, H., Angew. Chem. Int. Ed. Eng., 21, 512 (1982)]. Moreover, the combination of structural rigidification-activity relationship studies and conformational analysis gives insight into the biologically active conformation of linear peptides.
Conformationally restricted peptides containing medium and long range cyclizations have been mainly prepared following the same modes of cyclization of homodetic and heterodetic natural peptides. These include: a side-chain to side-chain cyclization (usually the formation of a lactam ring and/or an —S—S— bond through cyclization of functional groups already present in the native sequence or by substitution of other amino acids with Glu and Lys or Cys respectively); b end to end cyclization (previously called backbone to backbone cyclization [Manesis, N. J. and Goodman, M., Org. Chem., 52, 5331 (1987)]) and c side-chain to end groups cyclization.
Another mode of cyclization includes side-chain to amino end and side-chain to carboxyl end. The exact location, type and size of the ring (which can also be controlled by “spacers” [Manesis, N. J. and Goodman, M., Org. Chem., 52, 5331 (1987)]) to achieve maximum selectivity and activity is determined mainly by Structure-Activity-Relationship (SAR) considerations in conjunction with conformational analysis.
In another embodiment the peptides of the present invention may be cyclized by use of an enzyme cleavable linker. In this manner, the N-terminal amino group and the C-terminal carboxyl group of the peptide is linked via a linker, or the C-terminal carboxyl group of the peptide is linked to a side chain amino group or a side chain hydroxyl group via a linker, or the N-terminal amino group of said peptide is linked to a side chain carboxyl group via a linker, or a side chain carboxyl group of said peptide is linked to a side chain amino group or a side chain hydroxyl group via a linker. Useful linkers include 3-(2′-hydroxy-4′,6′-dimethyl phenyl)-3,3-dimethyl propionic acid linkers and its derivatives and acyloxyalkoxy derivatives linkers—see for example U.S. Pat. No. 5,672,584.
The present invention, therefore, can be extended to include cyclizing the peptide of the invention with a compound (i.e. a “linker”) which is (a) capable of being reacted with the peptide in a cyclizing reaction scheme to produce a cyclic peptide and optionally (b) capable of re-linearizing the peptide by means of in vivo enzymes to linearize the peptide.
Other methods of cyclizing the peptides of the present invention include the methods described in WO2014001822 and WO2016071422 to which the skilled reader is directed and the entire contents of which are hereby incorporated herein by way of reference.
Thus, in a further aspect there is provided a cyclic form of a peptide as described herein. There is also provided pharmaceutical formulations comprising such cyclic peptides, as well as their use in method of treating the diseases and conditions discussed herein.
The invention further provides a method of treating a neurological disorder in a subject, such as a disorder which would benefit from treatment through cognitive enhancement and/or neuroprotection, comprising administering to the subject an effective amount of a leptin peptide fragment as described herein.
An “effective amount” of a leptin peptide fragment refers to the amount of leptin peptide fragment which is sufficient to elicit a desired biological response. As will be appreciated by those of ordinary skill in this art, the absolute amount of a particular agent that is effective may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the target tissue, etc. Those of ordinary skill in the art will further understand that an “effective amount” may be administered in a single dose, or may be achieved by administration of multiple doses. A desired biological response may be, for example, (i) an increase in synaptic plasticity; (ii) an improvement in a task requiring cognitive function, e.g., improved performance on a test that measures learning and/or memory; (iii) a slowing in the rate of decline in cognitive function (e.g. neuroprotection), e.g., as measured by performance on a test that measures learning and/or memory. An effective amount in humans may be less than 10 □m, such as less than 100 nm.
Different dosing regiments may likewise be administered, typically at the discretion of the medical practitioner. As the leptin peptide fragments are based on a natural molecule (i.e leptin) it is expected that the fragments are likely to display low toxicity and allow for at least daily administration although regimes where the compound(s) is (or are) administered more infrequently, e.g. every other day, weekly or fortnightly, for example, are also embraced by the present invention.
“Treating”, when used with respect to a desired therapeutic effect in a subject such as a human being, can include reversing, alleviating, inhibiting the progress of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder or condition. “Preventing” refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur.
The leptin peptide fragments of the invention may be administered at intervals during the time over which treatment is required or is deemed necessary. For example, the leptin peptide fragments can be administered 3-4 times daily, 1-2 times daily, every other day, weekly, etc. It may be preferred to maintain an effective concentration within the body over a time period during which treatment is desired. Since, in general, it is desirable to maintain cognitive function throughout life, the compounds may be administered indefinitely.
The peptides of this invention can exist as stereoisomers or mixtures of stereoisomers; for example, the amino acids which comprise them can have the configuration L-, D-, or be racemic independently of each other. Therefore, it is possible to obtain isomeric mixtures as well as racemic mixtures or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and on which isomers or isomeric mixtures are present. The preferred structures of the peptides of the invention are pure isomers, i.e., enantiomers or diastereomers.
For example, when it is stated that an amino acid, can be —S—, it is understood that the amino acid, is selected from —L—S—, —D—S— or mixtures of both, racemic or non-racemic. The preparation and processes described in this document enable the person skilled in the art to obtain each of the stereoisomers of the peptide of the invention by choosing the amino acid with the right configuration.
In the context of this invention, the term “amino acids” includes the natural amino acids codified by the genetic code as well as non-codified amino acids, whether they are natural or not. Examples of non-codified amino acids are, without restriction, citrulline, ornithine, sarcosine, desmosine, norvaline, 4-aminobutyric acid, 2-aminobutyric acid, 2-aminoisobutyric acid, 6-aminohexanoyc acid, 1-naphthylalanine, 2-naphthylalanine, 2-aminobenzoic acid, 4-aminobenzoic acid, 4-chlorophenylalanine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, cycloserine, carnitine, cystine, penicillamine, pyroglutamic acid, thienylalanine, hydroxyproline, allo-isoleucine, allo-threonine, isonipecotic acid, isoserine, phenylglycine, statin, B-alanine, norleucine, N-methyl amino acids, a-amino acids and [3-amino acids, among others, as well as their derivatives. A list of unnatural amino acids can be found in the article “Unusual amino acids in peptide synthesis” by D. C. Roberts and F. Vellaccio, in The Peptides, Vol. 5 (1983), Chapter VI, Gross E. and Meienhofer J., Eds., Academic Press, New York, USA or in the commercial catalogues of the companies specialized in the field.
Synthesis of the peptides of the invention, their stereoisomers, mixtures thereof and/or their pharmaceutically acceptable salts can be carried out according to conventional methods, known in the prior art, such as using solid phase peptide synthesis methods [Stewart J. M. and Young J. D., “Solid Phase Peptide Synthesis, 2nd edition”, (1984), Pierce Chemical Company, Rockford, Ill.; Bodanzsky M. and Bodanzsky A., “The practice of Peptide Synthesis”, (1994), Springer Verlag, Berlin; Lloyd-Williams P. et a “Chemical Approaches to the Synthesis of Peptides and Proteins”, (1997), CRC, Boca Raton, Fla., USA], synthesis in solution, a combination of the methods of solid phase synthesis and synthesis in solution or enzymatic synthesis [Kullmann W. “Proteases as catalysts for enzymic syntheses of opioid peptides”, (1980), J. Biol. Chem., 255(17), 8234-8238].
The peptides can also be obtained by fermentation of a bacterial strain, modified or unmodified, by genetic engineering to produce the desired sequences, or by controlled hydrolysis of the full length leptin molecule obtained from a suitable animal or human source, to release the leptin peptide fragments of the invention.
There is further provided an in vitro method of preparing a leptin peptide fragment as described herein, or modified form thereof.
For use in methods according to the present invention, the leptin peptide fragments or a physiologically acceptable salt, solvate, ester or amide thereof described herein may be presented as a pharmaceutical formulation, comprising the leptin peptide fragment or physiologically acceptable salt, ester or other physiologically functional derivative thereof, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic and/or prophylactic ingredients. Any carrier(s) are acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
Examples of physiologically acceptable salts of the compounds according to the invention include acid addition salts formed with organic carboxylic acids such as acetic, lactic, tartaric, maleic, citric, pyruvic, oxalic, fumaric, oxaloacetic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids.
The determination of physiologically acceptable esters or amides, particularly esters is well within the skills of those skilled in the art.
It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the compounds described herein, which may be used in the any one of the uses/methods described. The term solvate is used herein to refer to a complex of solute, such as a compound or salt of the compound, and a solvent. If the solvent is water, the solvate may be termed a hydrate, for example a mono-hydrate, di-hydrate, tri-hydrate etc., depending on the number of water molecules present per molecule of substrate.
It will be appreciated that the compounds of the present invention may exist in various stereoisomeric forms and the compounds of the present invention as hereinbefore defined include all stereoisomeric forms and mixtures thereof, including enantiomers and racemic mixtures. The present invention includes within its scope the use of any such stereoisomeric form or mixture of stereoisomers, including the individual enantiomers of the compounds of formulae (I) or (II) as well as wholly or partially racemic mixtures of such enantiomers.
Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration e.g., by inhalation. The formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of active compound. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope. An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.
Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound is formulated in an appropriate release-controlling matrix, or is coated with a suitable release-controlling film. Such formulations may be particularly convenient for prophylactic use.
Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds.
Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles.
Injectable preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use. Alternatively, an active compound may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.
An active leptin peptide fragment may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly. Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.
Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.
As one possibility such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self-propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent. Suitable liquid propellants include propane and the chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Self-propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.
Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.
As a further possibility an active compound may be in the form of a solution or suspension for use in an atomizer or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.
Formulations suitable for nasal administration include preparations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.
It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical formulations described above may include, an appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases and the like.
Formulations suitable for topical formulation may be provided for example as gels, creams or ointments. Such preparations may be applied e.g. to a wound or ulcer either directly spread upon the surface of the wound or ulcer or carried on a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated.
Liquid or powder formulations may also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g. a wound or ulcer. Alternatively, a carrier such as a bandage, gauze, mesh or the like can be sprayed or sprinkle with the formulation and then applied to the site to be treated.
Therapeutic formulations for veterinary use may conveniently be in either powder or liquid concentrate form. In accordance with standard veterinary formulation practice, conventional water soluble excipients, such as lactose or sucrose, may be incorporated in the powders to improve their physical properties. Thus particularly suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients. These powders may either be added to animal feedstuffs, for example by way of an intermediate premix, or diluted in animal drinking water.
Liquid concentrates of this invention suitably contain the compound or a derivative or salt thereof and may optionally include a veterinarily acceptable water-miscible solvent, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol. The liquid concentrates may be administered to the drinking water of animals.
The present invention will now be further described by way of example, with reference to the following figures.
Methods
Primary Neuronal Culture
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; CaCl2 1; MgCl2 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 μg/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.
Human Neural Cell Line SH-SY5Y
The human neuroblastoma cell line, SH-SY5Y (ECACC, UK) was maintained in Dulbecco's Modified Eagle Medium supplemented with glucose (4500 μg/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 2×105 cells in 35 mm dishes for protein extraction. To induce differentiation, cells were cultured in DMEM supplemented with glucose (4500 μg/l), 1% (v/v) cosmic calf serum and 10 μM retinoic acid for 5 days. Thereafter they were incubated in DMEM supplemented with glucose (4500 μg/l), serum replacement 2 (2%; Sigma, UK) and 18 μM 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 μM copper chloride; 10 mM Aβ1-42, 5 μM 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.
Cell Survival Assays
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 μl 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.
ELISA for Cell Signalling Pathways and Phosphorylated Tau
Protein from cultures was extracted into 500 μl 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.
Surface Labelling of AMPA Receptors
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.
Electrophysiology.
Parasagittal hippocampal slices (300 μm) 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% O2 and 5% CO2) containing the following (in mm): 124 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgSO4, 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−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 baseline±standard error of mean (SEM).
Hippocampal Neuron Culture
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 μg ml−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.
Immunocytochemistry.
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).
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-SP-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.
Imaging and Analyses
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 μm 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).
All data are presented as mean±SEM. 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.
Cell Culture
The human neuroblastoma cell line, SH-SY5Y (ECACC, UK) was maintained in Dulbecco's Modified Eagle Medium supplemented with glucose (4500 μg/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 2×105 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 μM 6-hydroxydopamine; 10 μM CuCl2; 1 mM Aβ1-42.
Thioflavin S Staining
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.
Determination of Cell Viability
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 μl 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.
Mitored Analysis of Mitochondrial Morphology
Cells were treated with pre-warmed cell culture medium containing 1 μM 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.
Results
Leptin (116-130) Facilitates NMDA Receptor-Dependent Hippocampal Synaptic Plasticity
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;
Leptin (116-130) Induces Synaptic Plasticity at Adult Hippocampal CA1 Synapses
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 188±13% 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 140±13% of baseline; n=5; P<0.05;
Leptin (116-130), But Not Leptin (22-56) Enhances the Surface Expression of GluA1
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 184±7%; (n=36; P<0.001;
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 144±9% of control (n=48; P<0.001) and it also enhanced the degree of colocalization between surface GluA1 and synaptophysin immunostaining from 43±5.4% to 62±4.4% (n=36; P<0.05;
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 148±8% 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 (140±7.2%; n=36; P<0.001;
Leptin (116-130), But Not Leptin (22-56) Reverses Aβ1-42 Inhibition of Hippocampal Synaptic Plasticity
Several studies indicate that soluble Aβ oligomers impair activity-dependent synaptic plasticity, as exposure to Aβ 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 Aβ1-42 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 Aβ1-42 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 127±3.4% of baseline (n=8; P<0.01). Similarly in slices treated with the inactive peptide Aβ42-1 (1 tiM; 40 min), an enhancement of synaptic transmission (132±8.8% of base-line) was induced (n=5; P<0.05;
Leptin (116-130) Reverses Aβ1-42-Induced LTD
It is known that oligomeric Aβ 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 Aβ1-42 (Doherty et al. 2013). Thus we assessed if either of the leptin fragments mirror leptin action. Initially we verified that leptin prevented Aβ1-42-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 (94±5.6% of baseline; n=5; P>0.05), whereas robust LTD (73±3.8% of baseline; n=5; P<0.001) was induced in Aβ1-42-treated slices (
Leptin (116-130) Prevents Aβ-Induced Internalization of GluA1
Previous studies indicate that Aβ 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 Aβ1-42 (500 nM; 20 min) significantly attenuated (to 70±2% of control) GluA1 surface expression compared with control (Aβ42-1-treated) hippocampal neurons (n=48; P<0.001;
We have demonstrated previously that leptin attenuates cortical neuronal death triggered by Aβ1-42 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 μM CuCl2 or 10 μM Aβ1-42. 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 CuCl2-treated cells, 10 nM leptin reduced LDH release by 39.5±2.73% compared with CuCl2 alone (n=5; P<0.001;
In parallel studies a crystal violet assay was used to verify these findings by assessing cell number. In CuCl2-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 16.7±3.4% increase in cell number and this increased to 43.4±9.2% 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 27.8±10.6% and 39.9±13.5%, respectively (n=5;
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 Aβ1-42. In contrast to leptin (116-130), treatment with leptin (22-56) had no effect on the viability of cells exposed to Aβ1-42 (41.2±5.8% survival following Aβ1-42 treatment and 48.6±9.3% in Aβ1-42 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.
The Neuroprotective Effects of Leptin (116-130) Involve Activation of STAT3 and PI3-Kinase-Dependent Signalling Pathways
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 Aβ1-42-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 18.3±3.2% increase in LDH release in leptin (116-130) and Aβ1-42-treated cultures was observed, which is similar to the 26.7±4.4% increase observed with Aβ1-42 alone (n=5; P>0.5;
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;
Leptin (116-130) Enhances Episodic-Like Memory
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 (
Leptin (116-121) and Leptin (117-122) Facilitate Hippocampal LTP.
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 (117±10.8% of baseline; n=5; p<0.05;
Leptin (116-121) and Leptin (117-122) Increase the Surface Expression of GluA1 in Hippocampal Neurons.
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 146±9% of control (p<0.001; n=36;
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 μM Aβ1-42. 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 Aβ31-42-treated cells, 10 nM leptin and 10 nM leptin (116-130) significantly reduced LDH release by 25.3±2.5% and 24.0+2.2*% respectively compared with Aβ1-42 alone (n=11; P<0.001;
To confirm the findings of the LDH assay, cells were treated with Aβ1-42 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 Aβ1-42-treated cells, 10 nM leptin and 10 nM leptin (116-130) increased mitochondrial activity by 32.6±5.2% and 40.9+5.8% respectively compared with Aβ1-42 alone (n=11; P<0.001;
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)
Hyper-phosphorylation of tau is the underpinning mechanism of the development of neurofibrillary tangles—one of the key pathological feature of AD. Human SH-SY5Y neuronal cells were exposed to Aβ1-42 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 α-tubulin determined (
Previous studies have shown that exposure to amyloid beta (Aβ) promotes phosphorylation of tau, and that leptin protects against this aberrant action of Aβ in our models of AD [6]. Recent evidence indicates that treatment with oligomeric Aβ increases translocation of tau to synapses and this has been linked to synaptic dysfunction and ultimately cognitive impairments.
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 Aβ 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
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 Aβ increases the dendritic levels of p-tau, and in particular the synaptic levels of p-tau are increased after exposure to Aβ (
Murine Leptin116-130
Previously we have shown neuroprotection in models of Alzheimer's Disease (AD)[1].
In accordance with the existing data we have demonstrated that leptin116-130 prevents the accumulation of amyloid beta following seeding of cultures with amyloid (
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 (
In a cellular Parkinson's Disease (PD) model, leptin116-130 prevents mitochondrial swelling and clumping in response to 6-hydroxydopamine (6-OHDA;
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.
Murine Leptin Hexamers Based on Leptin116-130
The data presented above reported pro-survival effects of leptin116-121 and leptin117-122 in AD models.
Building on these findings we have evaluated the potential for these hexamer fragments to ameliorate amyloid propagation after initial seeding with 1 μM Aβ1-42. We have demonstrated that leptin116-121 and leptin117-122 prevent the accumulation of amyloid beta following seeding of cultures with amyloid (
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 (
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 (
Thus hexamer fragments of leptin116-130 mirror the effects of leptin and of leptin116-130 validating their further investigation as potential small peptide therapeutics.
Humanised Leptin Fragments Based on leptin116-130
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 (
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 (
Furthermore both hleptin117-125 and hleptin116-121 prevent neuronal loss in vitro in response to either 10 μM amyloid betal-42 (
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.
Peptide Modification
Halogenation and cyclisation: Target sequences for halogenation should ideally contain a tryptophan and there is no such residue in murine leptin116-130. Therefore, this work is focused on the human sequences, and to date 3 sequences (hleptin117-125, hleptin116-121, and hleptin117-122) 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.
Discussion
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 Aβ 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 Aβ 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 Aβ 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.
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.
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.
It is known that Aβ inhibits the induction of hippocampal LTP (Shankar et al. 2008), and this detrimental effect of Aβ1-42 is reversed by leptin (Doherty et al. 2013). Similarly, leptin (116-130) reversed the acute effects of Aβ1-42 in this study as synaptic plasticity was readily induced in hippocampal slices exposed to leptin (116-130) and Aβ1-42. Contrastingly, application of leptin (22-56) failed to prevent the detrimental effects of Aβ1-42 as no increase in synaptic strength was induced after exposure to Aβ1-42 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.
Several studies indicate that Aβ1-42 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 Aβ1-42-treated slices. Similarly, leptin (116-130), but not leptin (22-56) attenuated the effects of Aβ1-42 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 Aβ1-42-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 Aβ1-42 on hippocampal synaptic function.
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 Aβ1-42 or Cu2+. 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.
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.
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 Aβ-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 Aβ on hippocampal-dependent learning and memory.
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 Aβ1-42 on hippocampal synaptic function and neuronal viability in various cellular models of amyloid toxicity.
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.
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.
In accordance with the data above, both leptin (116-121) and leptin (117-122) attenuated Aβ1-42-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 Aβ1-42. Neither leptin (124-129) nor leptin (125-130) had any significant effect.
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.
All references cited herein are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2018/052441 | 8/30/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62553050 | Aug 2017 | US |
