DESC:FIELD OF INVENTION
The current invention relates to the field of aging related degenerative changes in the brain, and the method of inducing autophagy and/or mitophagy by peptides called Kisspeptins to help prevent and/or cure these neurodegenerative changes in the human brain.
BACKGROUND
Aging has been associated with decline in the quality and activity of mitochondria; learning and memory. Many age related neurodegenerative diseases, such as Alzheimer’s , Parkinson’s, Huntington’s and amyotrophic lateral sclerosis, share a common cellular and molecular pathogenetic mechanism involving aberrant misfolded protein aggregation and deposition. Autophagy is a major route for degradation of aggregated cellular proteins and dysfunctional organelles.
Moreover, mitochondrial function defined by quality, quantity, dynamics and homeostasis; and regulated by mitophagy and mitochondrial biogenesis is an important metric of human aging and disease. Therefore, it has been proposed in several studies that therapeutic interventions that can improve mitochondrial function can have profound impact on human health and longevity.

The elimination of abnormal and dysfunctional cellular constituents through autophagy is an essential prerequisite for nerve cells to maintain their homeostasis and proper function. Several studies have shown that age-related decrease in autophagy can impede neuronal homeostasis and, thus lead to the progression of neurodegenerative disorders due to the accumulation of toxic protein aggregates in neurons. Thus, it has been suggested that upregulation of autophagy may be crucial for development of therapeutic interventions for neurodegenerative diseases
Autophagy is an evolutionary conserved intracellular catabolic process to eliminate protein aggregates that can eliminate, damaged organelles such as endoplasmic reticulum, and cellular pathogens. Several studies have shown that autophagy is not restricted only to stressed cells to conserve resources for survival, but also plays a central role in the maintenance of cellular homeostasis in normal cells. Defects in autophagy pathway are associated with various myopathies that include cancer, aging, infections and neurodegenerative diseases like Parkinson’s and Alzheimer’s (Menzies FM et al Neuron 93:1015-1034.). Some studies have also demonstrated a strong nexus between a decrease in autophagic activity, aging and longevity (Cuervo AM et al (2005) Autophagy 1:131-40). Hence, modulation of the autophagy pathway may have a profound effect on health and longevity and most importantly it provides an avenue for therapeutic intervention.

Kisspeptin (Kp) belongs to a family of metastasis suppressors that are known to regulate functions like infertility, reproduction and metabolism. The current invention discloses method of inducing autophagy and/or mitophagy by kisspeptins, to protect neuronal cell health, and decrease neuronal cell death. This method can serve as a therapeutic intervention for preventing/ slowing down and/or curing hippocampus associated impairments such as memory loss, cognitive aging and other diseases linked to mitochondrial dysfunction.
SUMMARY
The current invention relates to the field of aging related degenerative changes in the brain, and discloses method of inducing autophagy and/or mitophagy by kisspeptins, to protect neuronal cell health, and decrease neuronal cell death.
One embodiment of the current invention is a method of inducing autophagy and mitophagy in mammalian neuronal cells, the method comprising the steps of increasing the expression of an amino acid sequence in the cells, wherein the amino acid sequence has at least 95% sequence identity to seq id no:2, 3, 4, 5 or 6.
In one embodiment, SEQ ID NO: 2 is encoded by a nucleic acid sequence which has at least 95% sequence identity to SEQ ID NO:1.
In one embodiment, the increase in autophagy and/or mitophagy in the method disclosed herein is not induced constitutively. In one embodiment, the increase in autophagy and/or mitophagy is induced in a pulsatile manner. In one embodiment, the increase in autophagy and/or mitophagy is induced once in ten days.
In one embodiment, the mammalian neuronal cells are human neuronal cells.
In one embodiment, the method of inducing autophagy and mitophagy in mammalian neuronal cells, the method comprising the steps of increasing the expression of an amino acid sequence in the cells, wherein the amino acid sequence has at least 99% sequence identity to seq id no: 6
In one embodiment the increase in autophagy and/or mitophagy ameliorates neuronal cell death associated with neuropathological disorders in brain cells.
In one embodiment, the neuropathological disorder is Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS).
In one embodiment, in the method disclosed herein, the amino acid sequence induces mitophagy by activating the GPR54 receptor.
In one embodiment, the increase in autophagy and/or mitophagy causes decrease in hippocampus associated brain impairments such as memory loss, cognitive aging.

BRIEF DESCRIPTION OF DRAWINGS AND SEQUENCES:
Fig. 1 shows the effect of human Kp-10 polypeptide (SEQ ID NO:6) on autophagy flux by determining LC3-II, in the presence or absence of Peptide 234 (150 nM) and ChQ (100 µM).
Fig. 2 shows fluorescence emission of SKNSH cells that were transfected with mcherry-GFP-LC3 plasmids. The figure shows human Kp-10 treatment significantly increased the number of red puncta and the red to yellow signal ratio compared to control (Fig. 2Aand B).

Fig. 3 shows the association of p62 and phosphor ULK1 proteins in mitochondria upon human Kp-10 peptide treatment. It also shows dynamin-related protein 1 (DRP1) phosphorylation.
Fig. 4 shows the effect of rat Kp-10 (SEQ ID NO: 10) on autophagy in aging rat model. Fig. 4 western blotting of hippocampus tissue lysates from aged rat treated with rat Kp-10 peptide (SEQ ID NO; 10), and probed with various antibodies as shown (Fig. 4A). A marked difference in the phosphorylation profiles between the adult and old aged control samples was evident. While the adult samples are enriched in phosphorylation of CaMKKß, ULK1 and mTOR compared to old aged control samples. Aged Wistar rats treated with Kp-10 exhibited a remarkable increase in the phosphorylation level of CaMKKß, ULK1 and Drp1, the intensity of the latter two was higher than that observed in adults.
SEQ ID NO:1 is the nucleic acid sequence encoding full length human Kisspeptin (Kp-FL).
SEQ ID NO:2: is the amino acid sequence of full length human Kisspeptin (Kp-FL).
SEQ ID NO:3: is the amino acid sequence of human Kisspeptin -54, (Kp-54).
SEQ ID NO:4: is the amino acid sequence of human Kisspeptin-14 (Kp-14).
SEQ ID NO:5: is the amino acid sequence of human Kisspeptin -13, (Kp-13).
SEQ ID NO:6: is the amino acid sequence of human Kisspeptin-10 (Kp-10).
SEQ ID NO:7 is the nucleic acid sequence encoding full length rat kisspeptin
SEQ ID NO:8 is the amino acid sequence of full length rat Kisspeptin (ratKp-FL).
SEQ ID NO:9 is the amino acid sequence of 54-aa long rat Kisspeptin (Kp-54).
SEQ ID NO:10 is the amino acid sequence of 10-aa long rat Kisspeptin (Kp-10).

DETAILED DESCRIPTION
Many neurodegenerative diseases that are aging related, such as Alzheimer’s , Parkinson’s, Huntington’s and amyotrophic lateral sclerosis, share a common cellular and molecular pathogenic mechanism involving aberrant misfolded protein aggregation and deposition. Autophagy is a major route for degradation of aggregated cellular proteins and dysfunctional organelles.

The elimination of abnormal and dysfunctional cellular constituents through autophagy is an essential prerequisite for nerve cells to maintain their homeostasis and proper functionality. Several studies have shown that age-related decrease in autophagy can impede neuronal homeostasis and, thus lead to the progression of neurodegenerative disorders due to the accumulation of toxic protein aggregates in neurons.
Some studies have suggested that upregulation of autophagy may be crucial for development of therapeutic interventions for neurodegenerative diseases
As a consequence, therapeutic interventions that can improve mitochondrial function can have profound impact on human health and longevity.
Definitions:
“Autophagy” as used herein is defined as a process of lysosome-dependent intracellular degradation that participates in the liberation of resources including amino acids and energy to maintain homoeostasis. Autophagy facilitates degradation of unnecessary or dysfunctional cytoplasmic components and organelles in the lysosome. Depending on the substrate subjected to selective degradation, autophagy can, among other types, be categorized in ER-phagy (endoplasmic reticulum), mitophagy (mitochondria), ribophagy (ribosomes), lysophagy (lysosomes) and pexophagy (peroxisomes). The autophagic system is involved in bulk degradation of primarily long-lived cytoplasmic proteins, as well as in selective degradation of cytoplasmic organelles.

Mitophagy is a specialized form of autophagy that regulates the turnover of damaged and dysfunctional mitochondria, organelles that function in producing energy for the cell in the form of ATP and regulating energy homeostasis. Mitophagy is essential for the maintenance of mitochondrial quality in neuron, and compromised autophagy/mitophagy is known to cause neurodegenerative diseases (Fivenson E.M. (2017) Neurochemistry International Volume 109 (Pages 202-209), October issue).

Many factors such as mutations, accumulation of oxidation species, activity of different complexes, determine mitochondrial health and/or quality. Ageing is associated with progressive accumulation of mutations/deletions in nuclear and mitochondrial genomes and is becoming increasingly clear that increased mtDNA mutagenesis can lead to premature ageing.
“Autophagy flux” as defined herein is a measure of autophagic degradation activity. Any method may be utilized to assess autophagic flux. The higher (or lower) the degradation activity, the higher (or lower) is the respective rate of degradation of the proteins/ organelles. Examples of systems that can be utilized to assess autophagic degradation activity include, but are not limited to, any protein assay techniques including, but not limited to, western blot analysis for specific key proteins, transmission electron microscopy, and fluorescence microscopy, or a combination of these.

Mitochondrial biogenesis and mitophagy dictate the supply of energy to meet the physiological demand. Mitochondria are the sites of adenosine triphosphate (ATP) production via oxidative phosphorylation (OXPHOS) taking place in the electron transport chain (ETC). ETC consists of multi-subunit protein complexes embedded in the inner membrane. Electrons are transferred through complexes I–IV to the final electron acceptor oxygen. In the final step, the terminal enzyme of the respiratory chain, cytochrome c oxidase (complex IV) catalyses the complete reduction of molecular O2 to water.
Mitochondrial activity, quality and quantity determine the state of the cell’s health. is a mitochondrial quality control mechanism that eliminates defective mitochondria to maintain a healthy mitochondrial network, thus contributing to cell viability and whole organism health span. Evidence shows that complex I activity is most severely compromised amongst all the mitochondrial enzymes in aging and age-associated neurodegenerative diseases Hence, the activity of complex I and the level of ATP reflect on the activities of the mitochondrial enzymes and mitochondrial bioenergetics respectively.
Mitochondria are powerhouse organelles for a cell, with myriad crucial roles in apoptosis, necrosis, autophagy, stress regulation, production of lipids and carbohydrates, Ca2+ storage and innate immunity. Mitochondria are exposed to damages caused by intra- and extra-mitochondrial events, such as mutated mitochondrial proteins and reactive oxygen species (ROS). Mitochondrial quality at the organelle level is sustained through synthesis of new mitochondria, fusion and fission, and elimination of the damaged ones. Mitochondrial quality control systems actively function in physiological and pathological conditions, to protect the mitochondria from stress and damage at the protein and organelle level. Damage beyond the protein level activates a bigger response that includes, mitochondrial fission and degradation, also known as mitophagy. Autophagy is regulated by multiple signalling cascades that are modulated by intracellular or extracellular stimuli. Target of Rapamycin (TOR) is considered a master regulator of autophagy and is highly conserved. Inhibition of TOR by lack of nitrogen or rapamycin stimulates autophagy. The mammalian homologs of the classic yeast autophagy associated gene Atg1, ULK1 (unc-51 like kinase) and ULK2 act downstream of TOR. Unlike TOR, AMP activated protein kinase (AMPK) is a positive regulator of autophagy and it inhibits TOR, more specifically TORC1. Nutrient starvation, energy depletion, hypoxia and increase in cytosolic Ca2+ cause phosphorylation of AMPK and thereby its activation. Increase in Ca2+levelsactivates AMPK through Ca2+/Calmodulin dependent kinase kinase beta (CaMKKß).
Kisspeptin (Kp) polypeptides belong to a family of metastasis suppressors that are known to regulate functions like infertility, reproduction and metabolism. Kisspeptin is a family of peptides derived from the KISS1/kiss1 gene with structural similarity, forming from differential proteolysis of a common precursor, prepro-kisspeptin. Kisspeptin peptides are classified as an RF amide peptide family i.e., neuroactive peptides with characteristic Arg-Phe-NH2 motif . The most abundant Kisspeptin in the human circulation is kisspeptin-54 (SEQ ID NO:3), which can be further cleaved to 14, 13, and 10 amino acid peptides (SEQ ID NOS: 4, 5 and 6 respectively).
Kisspeptin (Kp) is a G protein coupled receptor ligand that was initially identified as a metastasis suppressor. Kp’s are encoded by KISS1/Kiss1 gene and initially translated as long Kp precursors. Upon differential proteolytic cleavage of the Kp precursors, shorter, mature peptides are formed.

As used herein the term “Kisspeptin” refers to any one, more than one, or all of the Kisspeptin polypeptide sequences which are referred to herein as Kisspeptin-FL(Kp-FL), Kisspeptin-54 (Kp-54), Kisspeptin -14 (Kp-14) , Kisspeptin-13 (Kp-13) and Kisspeptin-10 (Kp-10). It refers to kisspeptins from any species, including humans and rats.

In humans, the pre-pro Kp-FL (SEQ ID NO: 2; Kisspeptin-FL) is comprised of 145 amino acids that harbours a putative 19 amino acid signal sequence at the N terminal, two dibasic cleavage sites at 57th and 68th amino acid positions and a third cleavage site at 121-124 amino acid positions in the C terminal region. SEQ ID NO:2 is coded by the cDNA sequence of full length Kisspeptin given in SEQ ID NO: 1. Kisspeptin-FL has an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2.
The pre-pro Kp-FL (SEQ ID NO:2) undergoes proteolytic cleavage in vivo to give rise to a 54-mer peptide called Kp-54 (SEQ ID NO:3) . Kp-54 has also been described as a metastin as it is able to inhibit tumour metastasis. Kisspeptin-54 has an amino acid sequence with at least 95% sequence identity to SEQ ID NO:3.
Kisspeptins 14 and 13 are also proteolytic forms of Kisspeptin. Kisspeptin-14 has an amino acid sequence with at least 95% sequence identity to SEQ ID NO:4. Kisspeptin-13 has an amino acid sequence with at least 95% sequence identity to SEQ ID NO:5.
Kp-10 (SEQ ID NO:6) is a derivative of Kp-54 that is a more mature and active form, efficient in activating the GPR54 (G-protein couple receptor-54) signalling pathways (Pinilla L, et al (2012) Physiol Rev 92:1235-316.). The binding of Kp-10 (SEQ ID NO:6) to GPR54 triggers and activates a whole repertoire of signalling events that include phospholipase C (PLC), protein kinase C (PKC), intracellular calcium (Ca2+) mobilization, mitogen-activated protein kinase (MAPK) and phosphotidylinositol 3-kinase (PI3K/Akt) pathways. Kisspeptin-10 has an amino acid sequence with at least 95% sequence identity to SEQ ID NO:6.
“Kisspeptin” or Kp is well known to stimulate GnRH secretion and play a vital role in fertility and reproduction by regulating the hypothalamic-pituitary-gonadal (HPG) axis. Though Kp has been shown to be expressed in hippocampus, its role in autophagy has not been clearly delineated (14, 16-19).
As used herein, the term “cognitive ageing” refers to the decline in cognitive processing that occurs as people get older. Age-related impairments in reasoning, memory and processing speed can arise during adulthood and progress into the elder years.
Embodiments:
The current invention relates to the field of aging related degenerative changes in the brain, and the method of inducing autophagy and/or mitopahgy in neuronal cells by peptides called Kisspeptins. One embodiment of the current invention is a method of inducing autophagy and mitophagy in mammalian neuronal cells, the method comprising the steps of increasing the expression of an amino acid sequence in the cells, wherein the amino acid sequence has at least 95% sequence identity to seq id no:2, 3, 4, 5 or 6.
In one embodiment, SEQ ID NO: 2 is encoded by a nucleic acid sequence which has at least 95% sequence identity to SEQ ID NO:1.
In one embodiment, the increase in autophagy and/or mitophagy in the method disclosed herein is not induced constitutively. In one embodiment, the increase in autophagy and/or mitophagy is induced in a pulsatile manner.
In one embodiment, the increase in autophagy and/or mitophagy is induced in a pulsatile manner by inducing the increase once in 10 days.
In one embodiment, the mammalian neuronal cells are human neuronal cells.
In one embodiment, the method of inducing autophagy and mitophagy in mammalian neuronal cells, the method comprising the steps of increasing the expression of an amino acid sequence in the cells, wherein the amino acid sequence has at least 99% sequence identity to seq id no: 6
In one embodiment the increase in autophagy and/or mitophagy ameliorates signals associated with neuropathological disorders in brain cells.
In one embodiment, the neuropathological disorder is Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS).
In one embodiment, in the method disclosed herein, the amino acid sequence induces mitophagy by activating the GPR54 receptor.
In one embodiment, the increase in autophagy and/or mitophagy causes decrease in hippocampus associated brain impairments such as memory loss, cognitive aging.
In one embodiment, the current invention encompasses the use of Kisspeptins described herein, to stimulate autophagy in neuronal cells. In one embodiment, Kisspeptins can be used to stimulate autophagy in neuronal cells, wherein the Kisspeptin is selected from the group consisting of Kisspeptin-FL, Kisspeptin-54, Kisspeptin -14, Kisspeptin-13 and Kisspeptin-10 (Kp-10). In one embodiment, Kisspeptins can be used to stimulate autophagy in neuronal cells, wherein the Kisspeptin sequence is selected from the group consisting of SEQ ID NO: 2, 3, 4, 5 and 6. In one embodiment, the Kisspeptin sequences given in SEQ ID NO: 2, 3, 4, 5 and 6 can be used to stimulate autophagy in human neuronal cells.
In one embodiment, any one or more than one of the Kisspeptin sequences given in SEQ ID NO: 2, 3, 4, 5 and 6 can be used to stimulate autophagy in human cells. In one embodiment, a kisspeptin sequence can be used to stimulate mitophagy in human cells, wherein the Kisspeptin sequence is selected from the group consisting of SEQ ID NO: 2, 3, 4, 5 and 6. In one embodiment, a kisspeptin sequence can be used to stimulate mitophagy in human neuronal cells, wherein the Kisspeptin sequence is selected from the group consisting of SEQ ID NO: 2, 3, 4, 5 and 6. In one embodiment, the current invention encompasses the use of Kisspeptin-FL, Kisspeptin-54, Kisspeptin -14, Kisspeptin-13 and Kisspeptin-10 (Kp-10) in protecting mitochondrial heath in neuronal cells.
In one embodiment, the current invention encompasses the use of Kisspeptin-FL, Kisspeptin-54, Kisspeptin -14, Kisspeptin-13 and Kisspeptin-10 (Kp-10) in protecting mitochondrial health in hippocampal cells. In one embodiment, kisspeptin sequence selected from the group consisting of Kisspeptin-FL, Kisspeptin-54, Kisspeptin -14, Kisspeptin-13 and Kisspeptin-10 (Kp-10) can be used as a therapeutic intervention to hippocampus associated impairments such as memory loss, cognitive aging and other diseases linked to mitochondrial dysfuntion.
In one embodiment, the current invention encompasses the use of a kisspeptin polypeptide, wherein the kisspeptin polypeptide is Kisspeptin-10 (Kp-10) in protecting mitochondrial heath in hippocampal cells. In one embodiment, Kisspeptin-10 (Kp-10) is used as a therapeutic intervention to hippocampus associated impairments such as memory loss, cognitive aging and other diseases linked to mitochondrial dysfuntion.
In one embodiment, the current invention encompasses the use of Kisspeptin-10 (Kp-10, SEQ ID NO:6), to stimulate autophagy in neuronal cells.
In one embodiment, the current invention encompasses the use of Kisspeptin-FL, Kisspeptin-54, Kisspeptin -14, Kisspeptin-13 and Kisspeptin-10 (Kp-10), to stimulate mitophagy in neuronal cells.
In one embodiment, the current invention encompasses the use of Kisspeptin-10 (Kp-10) in protecting mitochondrial heath in neuronal cells.
In one embodiment, the current invention encompasses the use of Kisspeptins in protecting mitochondrial heath in hippocampal cells, wherein the Kisspeptin is selected from the group consisting of Kisspeptin-FL, Kisspeptin-54, Kisspeptin -14, Kisspeptin-13 and Kisspeptin-10 . In one embodiment, Kisspeptin, Kisspeptin-54, Kisspeptin -14, Kisspeptin-13 or Kisspeptin-10 (Kp-10) can be used as a therapeutic intervention to hippocampus associated impairments such as memory loss, cognitive aging and other diseases linked to mitochondrial dysfuntion. Examples of such neurodegenerative disorders include, but are not limited to, Parkinson’s disease, Mitochondrial Encephalopathy, Lactic acidosis, and Stroke-like episodes.
In one embodiment, the current invention encompasses the use of Kisspeptin-10 (Kp-10) in protecting mitochondrial heath in hippocampal cells. In one embodiment, Kp-10 can be used as a therapeutic intervention to hippocampus associated impairments such as memory loss, cognitive aging and other diseases linked to mitochondrial dysfunction. In one embodiment, Kp-10 has an amino acid sequence with at least 95% sequence identity to the sequence given in SEQ ID NO: 6.
In one embodiment, the current invention encompasses the use of Kisspeptin-10 (Kp-10) in enhancing autophagy in neuronal cells. In one embodiment, Kisspeptin polypeptide induced autophagy enhances mitochondrial complex I activity and ATP levels in brain cells. In one embodiment, Kisspeptin polypeptide induced autophagy enhances mitochondrial complex I activity and ATP levels in hippocampal cells.
In one embodiment, the current invention encompasses the use of a Kisspeptin to have a therapeutic application in the treatment cognitive impairment, wherein the kisspeptin is selected from the group consisting of Kisspeptin, Kisspeptin-54, Kisspeptin -14, Kisspeptin-13 and Kisspeptin-10 (Kp-10).
In one embodiment, Kp-10 induced autophagy enhances mitochondrial complex I activity and ATP levels in brain cells. In one embodiment, Kp-10 induced autophagy enhances mitochondrial complex I activity and ATP levels in hippocampal cells.
In one embodiment, the current invention encompasses the use of Kisspeptin-10 (Kp-10; SEQ ID NO:6), to have a therapeutic application in the treatment cognitive impairment.
In one embodiment, the current invention encompasses the use of one or more than one Kisspeptin polypeptide, to ameliorate the symptoms associated with neurodegenerative diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS).
In one embodiment, the current invention encompasses the use of Kisspeptin-10 (Kp-10), a member of the family of peptides derived from kiss1 gene, to ameliorate the symptoms associated with neurodegenerative diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). In one embodiment, the therapeutic intervention can be done by injecting any one or more than one of the Kisspeptin polypeptides into the subject. In one embodiment, the therapeutic intervention can be done by gene therapy. In one embodiment, the therapeutic intervention can be done by gene therapy, and gene therapy can be done by using adenoviral vectors.
In one embodiment, the therapeutic intervention can be done by gene therapy, and gene therapy can be done by using adeno-associated virus (AAV) vector-mediated overexpression of human kisspeptin polypeptide (Menzies et al (2017) Neuron Volume 93, ISSUE 5, P1015-1034, March 08).
In one embodiment, the autophagy upregulation using Kisspeptins as a therapeutic approach is not done by constitutive activation of the pathway, but is done by a pulsatile strategy (periodic activation of autophagy). In one embodiment, the autophagy upregulation is done in a periodic manner over a long time period.
In one embodiment, the current invention encompasses the use of one or more than one Kisspeptins in inducing mitophagy, and increasing the quality of mitochondria in the hippocampus of aged mammals.
In one embodiment, the current invention encompasses the use of Kp-10 in inducing mitophagy, and increasing the quality of mitochondria in the hippocampus of aged mammals.
In one embodiment, the current invention encompasses the use of Kisspeptins to treat abnormal mitochondrial homeostasis. The Kisspeptin is selected from the group consisting of Kp-FL, Kp-54, Kp-14, Kp-13 and Kp10.
In one embodiment, Kp induced autophagy enhances mitochondrial complex I activity and ATP levels in hippocampal cells.
In one embodiment, any one or more than one of the Kisspeptins induces mitophagy via CaMKKß, AMPK, and ULK1 signalling independent of mTOR (mammalian Target of Rapamycin). In one embodiment, Kisspeptin treatment increases autophagy flux via the Ca+2/Calmodulin pathway autonomously without affecting mTOR, wherein the Kisspeptin is selected from the group consisting of Kp-FL, Kp-54, Kp-14, Kp-13 and Kp10.
In one embodiment, Kp-10 induced autophagy enhances mitochondrial complex I activity and ATP levels in hippocampal cells. In one embodiment, Kp-10 induces mitophagy via CaMKKß, AMPK, and ULK1 signalling independent of mTOR (mammalian Target of Rapamycin). In one embodiment, Kp treatment increases autophagy flux via the Ca+2/Calmodulin pathway autonomously without affecting mTOR.
In one embodiment, treatment with any one, or more than one of the Kisspeptins disclosed herein induces significant increase in the phosphorylated forms of CaMKKß, AMPK and ULK1. In one embodiment, treatment with Kp-10 induces significant increase in the phosphorylated forms of CaMKKß, AMPK and ULK1.
In one embodiment, treatment with any one or more than one of the Kisspeptins disclosed herein induces mitophagy by increasing recruitment of p62 protein to mitochondria.
In one embodiment, treatment with any one or more than one of the Kisspeptins disclosed herein induces mitophagy by increasing recruitment of phosphor ULK1 protein to mitochondria.
In one embodiment, treatment with any one or more than one of the Kisspeptins disclosed herein induces mitophagy by increasing levels of ubiquitinylated proteins in mitochondria.
In one embodiment, treatment with any one or more than one of the Kisspeptins disclosed herein induces mitophagy by increasing phosphor Drp1 level in cells.
In one embodiment, treatment with Kp-10 induces mitophagy by increasing recruitment of p62 protein to mitochondria.
In one embodiment, treatment with Kp-10 induces mitophagy by increasing recruitment of phosphor ULK1 protein to mitochondria.
In one embodiment, treatment with Kp-10 induces mitophagy by increasing levels of ubiquitinylated proteins in mitochondria.
In one embodiment, treatment with Kp-10 induces mitophagy by increasing phosphor Drp1 level in cells.
Autophagy is known to be regulated by multiple signalling cascades that are modulated by intracellular or extracellular stimuli. Target of Rapamycin (TOR) is considered a master regulator of autophagy and is highly conserved. Inhibition of TOR by lack of nitrogen or rapamycin stimulates autophagy. The mammalian homologs of the classic yeast autophagy associated gene Atg1, ULK1 (unc-51 like kinase) and ULK2 act downstream of TOR and it has been reported that blocking ULK1 or ULK2 inhibits autophagy. Unlike TOR, AMP activated protein kinase (AMPK) is a positive regulator of autophagy and it inhibits TOR, more specifically TORC1. Nutrient starvation, energy depletion, hypoxia and increase in cytosolic Ca2+ cause phosphorylation of AMPK and thereby its activation. Increase in Ca2+levels activate AMPK through Ca2+/Calmodulin dependent kinase kinase beta (CaMKKß). Intriguingly, several lines of evidence have implicated Kp-10 for the intracellular rise in calcium levels.

Examples
Example 1 : Kp-10 enhances autophagy flux
One of the hallmarks of autophagy activation is the conversion of LC3-1 to LC3-II through lipid modification. The ratio of LC3-II/LC3-I (Microtubule-associated proteins 1A/1B light chain 3B) is monitored in presence or absence of inhibitors of specific steps of the autophagy pathway in order to assess the autophagy flux as LC3-II is degraded in the lysosomes. We initially monitored effect of human Kp-10 peptide (SEQ ID NO:6) (peptides were synthesized by Biotech Desk INDIA ) on the autophagy flux in SKNSH cells (Neuroblastoma cell line ( neuronal cells) Bought from NCCS PUNE India), the presence or absence of chloroquine (ChQ), which is a lysosomal inhibitor, and Pep234,which is a known kisspeptin anatagonist) (Roseweir, AK et. al. J Neurosci. 2009 Mar 25;29(12):3920-9.) an antagonist of Kp. 100nM Kp-10 was ascertained to be the optimum human Kp-10 (SEQ ID NO:6) concentration for use in the autophagy flux assay (FIG. 1).
Cells were treated with 100nM human Kp-10 (SEQ ID NO:6) for 60 min in the presence or absence of Peptide 234 (150 nM) and ChQ (100 µM) (FIG. 1). Cells were lysed, resolved on SDS-PAGE and western transferred and the blots probed with anti-LC3. As shown (Fig. 1A), we found that in the presence of Kp-10, there was reduced amount of LC3-II. This could be attributed to either diminished formation of autophagosomes or enhanced autolysosome degradation of LC3-II. In the presence of human Kp-10 (SEQ ID NO: 6) and ChQ, we found more accumulation of LC3-II indicating that the reduced level of LC3-II observed in presence of human Kp-10 alone was because of a rapid turnover of LC3-II and increased autophagic flux. This was further corroborated by the increase in LC3-II in presence of Pep234 (compare lanes 5 and 6 to lane 3 in Fig. 1A(i)), an antagonist of Kp-10.
Determination of ‘LC3-II net flux’ provides insights into the actual autophagic flux. To do this, we initially quantitated the amount of LC3-II visualized on the western as shown (Fig. 1A(ii)). Amongst all the lanes, the lane with human Kp-10 and ChQ had the highest amount of LC3-II. Next, we subtracted the LC3-II amount from samples without ChQ from the corresponding samples with ChQ as described in the methods (Fig. 1A(iii)). It was seen that Rat Kp-10 treatment doubled the LC3-II net flux compared to control by augmenting the autophagic flux and autolysosomal degradation (Fig. 1A(iii). This increase was abolished in the presence of Pep234 underscoring the specificity of Kp-10 action.
Example 2: Role of Kp-10 in autophagic flux, using fluorescence studies
To further confirm the role of human Kp-10 (SEQ ID NO:6) in stimulating autophagic flux, we studied the fluorescence emission of SKNSH cells that were transfected with mcherry-GFP-LC3 plasmids. Incorporation of mcherry-GFP-LC3 within autophagosome enables SKNSH cells to emit both red and green fluorescence signals that results in a yellow colour upon image overlay (FIG. 2). However, when an autophagosome with mcherry-GFP-LC3 fuses with a lysosome, the acidic environment present in the lysosome quenches the GFP signal, and only red signal is emitted. Importantly, the amount of red signal is directly proportional to the rate of autophagy flux. This assay showed that human Kp-10 treatment significantly increased the number of red puncta and the red to yellow signal ratio compared to control (Fig. 2A and B). Taken together, the aforementioned results clearly showed that Kp-10 stimulates autophagy flux.
Example 3: Kp-10 induces mitophagy
AMPK (5' AMP-activated protein kinase) is known to promote mitochondrial fission and subsequently mitophagy. In addition, it has been shown that autophagy adopter protein, p62, is recruited to mitochondrial clusters and is essential for the aggregation and clearance of damaged mitochondria. Interestingly, p62 recruitment to mitochondria is also required for the maintenance of mitochondrial morphology and genome integrity. To check whether Kp-10 activated AMPK increases p62 recruitment along with ULK1 to mitochondria, we isolated mitochondria from SKNSH cells treated with human Kp-10 (SEQ ID NO:6). Whole cell lysate and mitochondrial fractions were separated on SDS-PAGE, western blotted and probed with phosphor-ULK1 and p62 antibodies (FIG. 3).
We found an increased association of p62 and phosphor ULK1 proteins with mitochondria though there was no significant change in the steady state levels of p62 in whole cell lysates (Fig. 3A). These studies suggested that human Kp-10 (SEQ ID NO:6) mediated phosphorylation of AMPK enhances the recruitment of p62 and thereby induces autophagy, more specifically mitophagy.
We also found that mitochondria from SKNSH cells treated with human Kp-10 harboured increased levels of ubiquitinylated proteins (Fig. 3B). Examination of phosphor Drp1 level in whole cell lysates of SKNSH cells treated with Kp in a time dependent manner (5 min to 4 h) revealed an initial spike in phosphorylation followed by a steady phosphorylation state that is more than twice that of control (Fig. 3C). Based on the aforementioned results, we concluded that Kp stimulates mitophagy.
Example 4: Kp-10 mediated autophagy process in a natural aging rat model
We administered 4 nmoles of rat Kp-10 (SEQ ID NO:10) each day to old (22-24 months) aged Wistar rats intraperitoneally for ten days as described in the methods. Animals were sacrificed at indicated time points and the hippocampus brain region was isolated from control adult (3-4 months) and old rats as well as from Kp-10 treated old rats. Tissue lysates were resolved on SDS-PAGE, western blotted and probed with various antibodies as shown (Fig. 4A). A marked difference in the phosphorylation profiles between the adult and old aged control samples was evident. While the adult samples were enriched in phosphorylation of CaMKKß, ULK1 and mTOR compared to old aged control samples, curiously, the latter had significantly higher levels of AMPK phosphorylation. Aged Wistar rats treated with Kp-10 peptide (SEQ ID NO:10) exhibited a remarkable improvement in the phosphorylation level of CaMKKß, ULK1 and Drp1, the intensity of the latter two was higher than that observed in adults. Intriguingly, there was further increase in AMPK phosphorylation while mTOR failed to get phosphorylated despite a significant increase in its protein levels. The in vivo data underscores the possibility of therapeutic intervention as rat Kp-10 (SEQ ID NO:10) treatment was able to enhance mitophagy in aged rats.

,CLAIMS:We Claim:
1. A method of inducing autophagy and mitophagy in mammalian neuronal cells, the method comprising the steps of increasing the expression of an amino acid sequence in the cells, wherein the amino acid sequence has at least 95% sequence identity to seq id no:2, 3, 4, 5 or 6.
2. The method of claim 1, wherein the increase in autophagy and/or mitophagy is not induced constitutively.
3. The method of claim 1, wherein the increase in autophagy and/or mitophagy is induced in a pulsatile manner.
4. The method of claim 1, wherein the mammalian neuronal cells are human neuronal cells.
5. The method of claim 1, wherein the amino acid sequence has at least 99% sequence identity to seq id no: 6
6. The method of claim 1, wherein the increase in autophagy and/or mitophagy ameliorates neuronal cell death associated with neuropathological disorders.
7. The method of claim 4, wherein the neuropathological disorder is Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS).
8. The method of claim 1, wherein the amino acid sequence induces mitophagy by activating the GPR54 receptor.
9. The method of claim 1, wherein the increase in autophagy and/or mitophagy causes decrease in hippocampus associated brain impairments such as memory loss, and cognitive aging.