Phospholipid compositions comprising EPA and / or DHA for ameliorating neurodegenerative and age-related neurodegenerative disorders

Abstract

The present invention relates to a composition comprising a phospholipid of marine origin, the present invention relates to a composition for improving at least one aging marker selected from the group consisting of genomic instability, telomere shortening, epigenetic alterations, loss of protein homeostasis, dysnutrition induction, mitochondrial dysfunction, cellular senescence, stem cell depletion, and dysnutrition induction and intercellular communication.

Description

Technical Field

[0001] The present invention relates to a composition comprising marine-derived phospholipids for improving at least one aging hallmark selected from the group consisting of genomic instability, telomere shortening, epigenetic alterations, loss of proteostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and dysregulated nutrient sensing and intercellular communication. Background Art

[0002] The United Nations estimates that by 2050, 1 in 6 people will be aged over 65 (16%), up from 1 in 11 (9%) in 2019. However, the increase in life expectancy has not been accompanied by an equal increase in healthy lifespan. As ageing is one of the main risk factors for most chronic diseases, the prevalence of age-related diseases rises with increasing life expectancy, representing a socioeconomic problem for developed societies. It is believed that the prevalence of multimorbidity in the new 65-74 year old population will increase from 45.7% in 2015 to 52.8% in 2035 (Andrew Kingston, 2018). Therefore, unless combined with a longer healthy lifespan, increased lifespan may translate into more years of pain and suffering as we spend more years unable to carry out normal daily activities autonomously. A recent pilot study of objective population-wide NHS data showed that currently in developed Western societies such as the UK, women can expect to live 29 years and men 23 years in poor health (Garth, 2020).

[0003] Increasing age also tends to be accompanied by a stronger desire to live in good health for as long as possible. Therefore, it is expected that as the elderly population grows, they may shift their spending toward supplements that protect cognition, maintain vitality, and slow physical decline, creating demand for longevity supplements with proven effects.

[0004] Longevity supplements are different from other "general purpose supplements" in that they do not simply provide the body with ingredients that simply prevent a deficiency (such as, for example, a vitamin or mineral deficiency); longevity supplements are able to provide the body with ingredients that can act on pathways that change the rate at which we age.

[0005] Aging is a biological process characterized by a progressive loss of vitality and increasing frailty. For humans, aging can be viewed as a continuous decline in health and loss of intrinsic function. A specific effect of normal aging in humans is the loss of neurons, known as normal age-related neurodegeneration. Since aging is also a major risk factor for many major age-related diseases, especially for most neurodegenerative diseases (NDDs), it is widely believed that slowing aging may reduce the incidence of age-related diseases. In fact, there is growing evidence that interfering with the conserved processes that lead to aging can extend healthy lifespan.

[0006] The dramatic increase in these NDDs worldwide is about to create a huge healthcare challenge. Currently, medications to treat such NDDs are limited to curing symptoms in a short period of time. There is an urgent and unmet need to find a new class of medications and supplements that can slow down the aging process and the progression of the accompanying diseases. It is believed that slowing down the aging process may delay the onset and slow progression of major NDDs.

[0007] Many cellular processes contribute to aging, and these are called “hallmarks of aging.”

[0008] It would be beneficial if anti-aging treatments could have an effect on one or more of the hallmarks of aging. Summary of the invention

[0009] Thus, in some preferred embodiments, the present invention relates to marine-derived phospholipid compositions for use in treatment (prophylactic or therapeutic) and methods for ameliorating one or more symptoms and / or signs of aging.

[0010] In some preferred embodiments, the present invention provides a method for improving at least one marker of aging in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising phospholipids with bound eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA), wherein the marker of aging is selected from the group consisting of genomic instability, telomere loss, epigenetic alterations, loss of proteostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication.

[0011] In some preferred embodiments, the present invention provides a method for improving dopaminergic neuron survival in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a composition comprising a phospholipid with bound eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA). In some preferred embodiments, administration of an effective amount of a composition comprising a phospholipid with bound eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA) improves the clearance of alpha-synuclein aggregates in the subject.

[0012] In some preferred embodiments, the present invention provides the use of a composition comprising phospholipids with bound EPA and / or DHA for improving aging markers selected from the group consisting of genomic instability, telomere loss, epigenetic changes, loss of protein homeostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. In some preferred embodiments, the present invention provides the use of a composition comprising phospholipids with bound EPA and / or DHA for treating (preventatively or therapeutically) a disease or condition associated with a marker of aging. In another embodiment, administration of the composition is sufficient to improve at least one marker of aging or for improving the survival rate of dopaminergic neurons.

[0013] The present invention also provides the use of a composition comprising phospholipids with bound EPA and / or DHA in the manufacture of a medicament: (i) for improving a marker of aging selected from the group consisting of genomic instability, telomere loss, epigenetic changes, loss of protein homeostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication, or for improving dopaminergic neuron survival; or (ii) for treating (preventatively or therapeutically) a disease or condition associated with a marker of aging or dopaminergic neuron degeneration. Administration of the medicament may be sufficient to improve at least one marker of aging.

[0014] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is characterized by comprising 5 to 80 g / 100 g, 5 to 60 g / 100 g, 5 to 40 g / 100 g, or 10 to 40 g / 100 g EPA and / or DHA as determined by gas chromatography fatty acid methyl ester (GC-FAME) analysis. In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is characterized by comprising 10 to 70%, 10 to 50%, or 10 to 40% EPA and / or DHA w / w, wherein w / w is determined by measuring the total weight of EPA and / or DHA residues in the composition and dividing the total weight of EPA and / or DHA residues by the total weight of the composition.

[0015] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is characterized by having a ratio of EPA to DHA of 1:1 to 5:1, and preferably 1:1 to 3:1. In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is characterized by having a ratio of DHA to EPA of 1:1 to 5:1, and preferably 1:1 to 3:1.

[0016] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a total phospholipid content of 10 to 90% w / w, 10 to 80% w / w, 20 to 80% w / w, or 20 to 60% w / w, wherein w / w is determined by measuring the total weight of phospholipids in the composition by 31P-NMR and dividing the total weight of phospholipids by the total weight of the composition.

[0017] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a choline content of 2 to 20% w / w, 2 to 15% w / w, or 2 to 10% w / w, wherein w / w is determined by measuring the total weight of choline in the composition by 31P-NMR and dividing the total weight of phospholipids by the total weight of the composition.

[0018] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is further characterized as comprising astaxanthin esters in an amount of 50 to 1000 mg / kg, 50 to 500 mg / kg, or 50 to 300 mg / kg, or 100 to 300 mg / kg.

[0019] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a TMAO content expressed as mgN / 100g of 1 to 30, 1 to 20 or 1 to 10 mgN / 100g.

[0020] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is further characterized by a peroxide value of less than 10, 5, or 2.

[0021] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is further characterized as having an acid number of less than 50, 40, 30, or 20.

[0022] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a Na+ content of less than 1.000%, 0.500%, 0.300% or 0.200% w / w of the composition, wherein w / w is determined by measuring the total weight of Na+ in the composition and dividing the total weight of Na+ by the total weight of the composition.

[0023] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a phosphatidylcholine (PC) content of 20 to 70%, 20 to 60%, or 20 to 50% w / w, wherein w / w is determined by measuring the total weight of PC molecules with two fatty acids attached in the composition and dividing the total weight of PC molecules with two fatty acids attached by the total weight of the composition.

[0024] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a lysophosphatidylcholine (LPC) content of 10 to 70%, 10 to 60% or 10 to 50% w / w, wherein w / w is determined by measuring the total weight of LPC molecules with one fatty acid attached in the composition and dividing the total weight of LPC molecules with one fatty acid attached by the total weight of the composition.

[0025] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is prepared from a natural source. In some preferred embodiments, the natural source is selected from the group consisting of krill, fish or fish by-products, Daphnia, shrimp, molluscs, cephalopods, and algae. In some preferred embodiments, the natural source is krill. In some preferred embodiments, the fish or fish by-product is derived from herring, salmon, anchovies, or mackerel.

[0026] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is an oil.

[0027] In some preferred embodiments, the compositions comprising phospholipids with bound EPA and / or DHA are encapsulated.

[0028] In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is encapsulated in a gel capsule.

[0029] In some preferred embodiments, the compositions comprising phospholipids with bound EPA and / or DHA comprise antioxidants and / or flavoring agents that are not naturally present in the source of the phospholipid composition.

[0030] In some preferred embodiments, the compositions comprising phospholipids with bound EPA and / or DHA are provided as a medical food.

[0031] In some preferred embodiments, the composition is provided to a subject in need thereof. In some preferred embodiments, the subject in need thereof suffers from or is at risk of suffering from a disease or condition selected from the group consisting of age-related neurodegeneration or disorder, Alzheimer's disease, Parkinson's disease, executive function decline, age-related cognitive decline, cognitive disease, disorder or injury, and aging associated with age-related pathology selected from the group consisting of atherosclerosis, cardiovascular dysfunction (including cardiovascular problems caused by certain genotoxic chemotherapy), tumor progression, loss of hematopoietic and skeletal muscle stem cell function, non-alcoholic fatty liver disease, pulmonary fibrosis, osteoarthritis and osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 a-1f. Krill oil promotes healthy dopaminergic neuron aging. (a) Representative images of the head region of PD animals at day 1, 3, and 6 of adulthood, scale bar, 20 μm. (b) Survival of anterior CEP and ADE DA neurons in PD worms co-expressing human α-synuclein (α-syn) in response to krill oil during aging (n = 35 worms per experiment; three independent experiments, sem; ***p < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test). (c) Representative aggregation of α-SYN in body wall muscles of day 1 and day 6 animals, scale bar, 20 μm. (d) Quantification of the number of aggregates in day 1 and day 6 animals in response to krill oil (n = 15 worms, sem; NS and ***p < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test). (E) Bar graphs represent basal slowing responses in wild-type and PD animals at day 6 of adulthood. Body bending was measured every 20 s on NGM plates with and without bacteria (n = 30; error bars, sem; ***p < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test. (B) Bar graphs represent locomotor activity in wild-type and PD animals at day 6 of adulthood. Activity was scored for 60 min (n = 50 animals per experiment; error bars, sem; NS and *p < 0.05; one-way ANOVA followed by Bonferroni multiple comparison test.

[0033] Figure 2a-2e. Krill oil delays senescence through p21 and TGFβ. (ab) Representative images of β-gal staining of the head region of wild-type and PD animals treated with and without krill oil at 9 days of adulthood. The bar scatter plots represent the percentage of worms with positive senescence markers in three independent experiments (n=50-100 individuals, bars indicate mean, error bars, sem, ***p<0.001; one-way ANOVA followed by Bonferroni multiple comparison test). (cd) Images and quantifications represent senescence in the BJ cell line in response to krill oil using β-gal staining (three independent experiments, error bars, sem; *p<0.05; one-way ANOVA followed by Bonferroni multiple comparison test). (e) Relative p21 and TGFβ mRNA levels in BJ cells of passages 10 and 21 treated with krill oil (100 μg / ml, 6 days) measured by qPCR. Data represent mean ± sd, n=3. *P≤0.05, **P≤0.01, ****P≤0.0001 (two-tailed Student t test).

[0034] Figure 3 a-3e. Krill oil improves mitochondrial health. (ab) Images and quantification of 8-Oxo (dG) staining in 6-day-old PD animals in response to krill oil (n=10 individuals, error bars, sem; ****p<0.0001; one-way ANOVA followed by Bonferroni multiple comparison test). (c) Oxygen consumption rate in wild-type and PD animals on days 1 and 6 in response to krill oil (n=50 individuals, two independent experiments, error bars, sem; NS and *p<0.05, **p<0.01; one-way ANOVA followed by Bonferroni multiple comparison test). (de) Images of BJ fibroblasts at passage 16 (p16) without and with krill oil and quantification of mitochondrial membrane potential measured using TMRE staining (n=6 independent experiments, error bars, sem; **p<0.01; one-way ANOVA followed by Bonferroni multiple comparison test).

[0035] Figure 4a-4c. Krill oil alters gene regulation. (a) In this PCA plot, all time points are depicted in the same plot for the BY273 strain in control only and in krill oil treated animals. The difference between conditions is very low in day 1 animals, while the difference between day 3 and day 6 animals is almost 22%. Clearly, krill oil alters gene regulation with age. (b) Representation of differentially expressed genes (DEGs) in PD animals with and without krill oil treatment in day 1, day 3, and day 6 animals. (c) Volcano plot showing significant up- and down-regulated genes in PD animals treated with krill oil at three different time points, day 1, day 3, and day 6.

[0036] Figure 5 a-5c. Krill oil stimulated gene clusters. (a) Clusters 6, 7, 8, and 12 are upregulated in PD animals treated with krill oil at three different time points: day 1, day 3, and day 6. (b) GO, KEGG, and HP of cluster 8. (c) GO, KEGG, and HP of cluster 12.

[0037] Figure 6 a-6b. Krill oil promotes DA neuron aging via cnnm-3, pbo-2, and rim-1 in PD animals. (ab) Images and scatter plots represent the intensity of dopaminergic neurons after depletion of cnnm-3, pbo-2, and rim-1 in PD animals (n = 14 individuals, scale bar, 20 μm error bar, sem; ***p < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test).

[0038] Figure 7 a-7c. WGCNA analysis correlates positive and negative correlations between gene expression and krill oil-dependent parameters. (a) Heatmap shows the correlation values ​​between all discovered modules (gene sets) and the defined parameters (krill oil treatment, DA neuron survival, OCR, and locomotion). The correlation ranges from +1 (perfect positive correlation) to -1 (perfect negative correlation). (bc) Represents the gprofiler plots used to study each module. (b) GO, KEGG, HP for green modules. (c) GO, KEGG, HP for blue modules.

[0039] Figure 8a-8c. Krill oil up-regulates CNNM3, PLCB4 and RIMS1 in human BJ cell line. (ac) Relative mRNA levels of differentially expressed genes (DEGs) in BJ cells treated with krill oil (100 μg / ml) for different times (1, 3 and 6 days) measured by qPCR. Data represent mean ± sd, n = 3. *P ≤ 0.05, **P ≤ 0.01, **P ≤ 0.001, ****P ≤ 0.0001 (two-tailed Student's t-test).

[0040] Fig. 9 a-9d. Krill oil protects dopaminergic neurons via oxidative stress modulators. (a) Representative images of the head region of 6-day-old PD animals under control and krill oil-treated conditions after knockdown of jnk-1, skn-1, hmg-5, and lmd-3. Scale bar, 20 μm. (b) Scatter plots represent GFP intensity of CEP dopaminergic neurons in 6-day-old PD worms after knockdown of jnk-1, skn-1, hmg-5, and lmd-3 (n = 20 individuals; error bars, sem; ***p < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test). (c) Pharyngeal pumping frequency (Hz) monitored in wild-type and PD animals was quantified using a nematrixscreenchip-based assay. The bar graphs represent the frequency of pharyngeal pumping in animals at day 3 of adulthood (n = 15-20 individuals, error bars, sem NS and **P ≤ 0.001, ****p < 0.0001; one-way ANOVA followed by Bonferroni multiple comparison test). (d) The learning index was calculated from the positive correlation with butanone in wild-type and PD animals with and without krill oil at day 6 of adulthood (n = 150 individuals, two independent experiments, error bars, sem; NS, *p < 0.05; one-way ANOVA followed by Bonferroni multiple comparison test).

[0041] Fig.10 Krill oil, such as Superba Krill Oil, improves aging by promoting genome stability, mitochondrial health, and inhibits senescence and loss of proteostasis. This graphic representation uses C. elegans and human BJ fibroblasts as models to demonstrate the benefits of krill oil and the associated pathways that lead to protection of DA neurons.

[0042] definition

[0043] Throughout this disclosure, relevant terms should be understood consistently with their typical meanings as established in the relevant fields, ie, medicinal chemistry, medicine, biology, biochemistry, and physiology.

[0044] The term "EPA" means eicosapentaenoic acid.

[0045] The term "DHA" refers to docosahexaenoic acid.

[0046] EPA and DHA as used herein in connection with the compositions of the present invention refer to fatty acid chains that may be bound to a lipid backbone such as a phospholipid, lysophospholipid, triglyceride, diglyceride, monoglyceride or any other lipid backbone, or they may be present in the composition as free fatty acids or ethyl esters.

[0047] The term "signature of aging" (used synonymously with "age-related markers" and "signatures associated with aging") is a term well known in the art. In certain embodiments, the term refers to cellular processes that lead to aging, including but not limited to genomic instability, epigenetic changes, mitochondrial dysfunction, loss of protein homeostasis, senescence, telomere shortening, dysregulated nutrient sensing (metabolic changes) and dysregulated intercellular communication and stem cell exhaustion. These markers are known and reviewed in depth by C. Lopez Otin, et al. 2013. Excerpts of Lopez Otin, 2013 are given below.

[0048] As used herein, the term "amelioration" when used in reference to a hallmark of aging includes, but is not limited to: (i) slowing, stopping, reversing, or preventing the progression of a hallmark, (ii) slowing, stopping, reversing, or preventing the progression of a symptom of a hallmark, (iii) preventing or reducing the likelihood that a hallmark will recur, and / or (iv) preventing or reducing the likelihood that a symptom of a hallmark will recur. In one embodiment, treating a subject with a hallmark of aging means (i) reversing the progression of a hallmark, ideally to the extent of eliminating the hallmark, and / or (ii) reversing the progression of a symptom of a hallmark, ideally to the extent of eliminating the symptom.

[0049] The term "total phospholipids" is used herein to describe the total content of phospholipids including lysophospholipids in the composition. "Phospholipids" used herein refers to an organic compound having two fatty acid moieties connected to the sn-1 and sn-2 positions of glycerol, and comprising a head group (head group) connected to the sn-3 position of glycerol by a phosphate residue. Exemplary head groups include choline, ethanolamine, serine and inositol. Phospholipids include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol and phosphatidic acid. The fatty acid moiety is a part of the fatty acid molecule that is combined (e.g., by an ester or ether bond) at sn-1 or sn-2. When the fatty acid moiety is a fatty acyl group, the fatty chain of the fatty acyl group is connected by an ester bond, and when the fatty acid moiety is a fatty chain of a fatty acid, the fatty chain is connected by an ether bond. Therefore, when a specific fatty acid is mentioned in relation to the phospholipids of the present invention (e.g., EPA or DHA), it should be regarded as a reference to the relevant fatty acyl group or its fatty chain. In krill oil, a major amount of the total amount of EPA and / or DHA is bound to phospholipids, in particular a major amount of EPA and / or DHA is bound to phosphatidylcholine.

[0050] The term "total phosphatidylcholine" is used herein to describe the total content of phosphatidylcholine (including lysophosphatidylcholine (LPC)) in a composition.

[0051] The term "total LPC" is used herein to describe the total content of lysophosphatidylcholine in a composition.

[0052] The term "pharmaceutically acceptable excipient" refers to a substance other than the components of the LPC composition mentioned in the claims and which is usually used with oily drugs. Such excipients include, but are not limited to, trioleylglycerol, soybean oil, safflower oil, sesame oil, castor oil, coconut oil, triglycerides, tributyglycerol, tricaproylglycerol, tricaprylylglycerol, vitamin E, antioxidants, α-tocopherol, ascorbic acid, deferoxamine mesylate, thioglycolic acid, emulsifiers, lecithin, polysorbate 80, methylcellulose, gelatin, serum albumin, sorbitan lauraute, sorbitan oleate, sorbitan trioleate, polyethylene glycol (PEG), PEG 400, polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), poloxamer, glycerol, sorbitol, xylitol, pH adjusters; sodium hydroxide, antimicrobial agent EDTA, sodium benzoate, benzyl alcohol and proteins such as albumin. A pharmaceutically acceptable excipient must be acceptable in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0053] The term "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable salts derived from a wide variety of organic and inorganic counterions well known in the art, and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium and tetraalkylammonium salts, and, when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, methanesulfonate, acetate, maleate and oxalate. Suitable salts include those described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of pharmaceutical salts properties, Selection, and Use; 2002.

[0054] The term "prophylaxis" refers to measures taken to prevent, rather than treat, a disease or condition.

[0055] The term "therapeutically effective amount" is a term well known in the art. In certain embodiments, the term refers to the amount of the composition disclosed herein that produces some desired effect at a reasonable benefit / risk ratio applicable to medical treatment. In certain embodiments, the term refers to the amount necessary or sufficient to eliminate, alleviate or relieve medical symptoms over a period of time. The effective amount may vary due to factors such as the disease or condition being treated, the specific composition being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can determine the effective amount of a specific composition empirically without excessive experimentation.

[0056] The term "treatment" is well known in the art and includes preventing a disease, illness or condition from occurring in a subject who may be susceptible to the disease, illness and / or condition but has not yet been diagnosed with the disease, illness and / or condition; inhibiting the disease, illness or condition, such as hindering its progress; and alleviating the disease, illness or condition, such as causing the disease, illness and / or condition to regress. Treating a disease or condition may include affecting the underlying pathophysiology of the disease or condition, thereby bringing an overall benefit to the patient (such as inhibiting the disease, illness or condition, such as preventing its progress; and alleviating the disease, illness or condition, such as causing the regression of the disease, illness and / or condition). Treating a disease or condition may include ameliorating at least one or more than one symptom of a particular disease or condition, e.g., an overall improvement of symptoms, even if the underlying pathophysiology is not affected, such as treating dry eye selected from inflammation of the eye, corneal nerve abnormalities, and ocular surface abrasions; or a neurodegenerative disease selected from age-related macular degeneration, diabetic retinopathy, non-proliferative retinopathy, proliferative retinopathy, diabetic macular edema, retinitis pigmentosa, central vein occlusion, and glaucoma; as well as other related diseases or any other medical condition, as is known in the art, and includes administering a composition that reduces the frequency of symptoms of the medical condition or delays the onset of the symptoms in a subject relative to a subject not receiving the composition.

[0057] Unless otherwise defined herein, all technical and scientific terms used have the same meanings as commonly understood by those skilled in the fields of medicine, pharmacology, medicinal chemistry, biology, biochemistry, and physiology.

[0058] All methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described herein. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail.

[0059] Where numerical limitations or ranges are stated herein, the endpoints are included. Furthermore, all values ​​and sub-ranges within the numerical limitations or ranges are specifically included as if expressly written.

[0060] Headings are for organizational purposes and should not be construed as limiting the subject matter herein. DETAILED DESCRIPTION

[0061] Marine oils have been extensively studied for their health-promoting properties. Oil from the Antarctic krill species Euphausia Superba is rich in long-chain omega-3 fatty acids (EPA and DHA), phospholipids, choline, and astaxanthin. Both EPA / DHA and astaxanthin have antioxidant and anti-inflammatory properties. 4,5In krill oil, a large amount of EPA / DHA is bound to phospholipids (PL), especially phosphatidylcholine (PC), which makes the fatty acids more stable and ensures effective absorption in tissues. 6,7 . It has also been shown that one PC, lysophosphatidylcholine (LPC), is essential for the transport of long-chain omega-3 fatty acids across the blood-brain barrier (Nguyen et al, Nature, volume 509, p. 503-506 (2014)) and therefore the absorption of EPA and DHA in the brain. Therefore, krill oil-derived phospholipids may be a first-choice nutritional supplement for promoting health, including brain health. In support of this, short-term supplementation experiments have shown that PC- and LPC-DHA / EPA have anti-inflammatory and antioxidant properties, with the potential to improve motor function and some cognitive traits in mice 8,9 A study that fed mice a diet rich in LPC from krill oil for 12 months also showed improvements in memory 10 .

[0062] However, studies evaluating the overall potential of krill-derived phospholipids as nutraceuticals targeting the aging process are lacking.

[0063] Signs of aging

[0064] Many cellular processes contribute to aging. These processes are often referred to as the “hallmarks of aging” and include genomic instability, epigenetic alterations, mitochondrial dysfunction, loss of proteostasis, senescence, telomere shortening, dysregulated nutrient sensing (altered metabolism) and intercellular communication, and stem cell exhaustion. 1-3 .

[0065] Hallmarks of aging are types of biochemical changes that occur in all organisms that experience biological aging and lead to a gradual loss of physiological integrity, impaired function, and ultimately death. An effective anti-aging supplement should be able to reduce at least one of the hallmarks of aging. The nine hallmarks of aging are divided into the following three categories:

[0066] Main signs (cause of injury):

[0067] Genome instability

[0068] Telomere shortening

[0069] Epigenetic changes

[0070] Loss of protein homeostasis

[0071] Antagonistic markers (response to injury):

[0072] Nutritional sensing disorders

[0073] Mitochondrial dysfunction

[0074] Cellular senescence

[0075] Integrative markers (culprits of the phenotype):

[0076] Stem cell exhaustion

[0077] Altered intercellular communication

[0078] Primary markers are the primary cause of cellular damage. Antagonistic markers are antagonistic or compensatory responses to the manifestation of primary markers. Integrative markers are the functional consequence of the first two groups of markers, leading to further functional deterioration associated with aging.

[0079] A review based on Lopez-Otin, 2013 is provided:

[0080] Genome instability

[0081] Genomic instability is caused by defects in certain processes that control how cells divide. The accumulation of genetic damage throughout life is a common feature of aging, and increased accumulation of DNA damage has also been linked to premature aging diseases.

[0082] Epigenetic changes

[0083] A heritable change that does not affect the DNA sequence but results in changes in gene expression. Examples include promoter methylation and histone modifications. Also called epigenetic variation and epimutation. There is multiple evidence that aging is accompanied by epigenetic changes and that epigenetic perturbations can induce premature aging syndromes in model organisms. For example, studies on SIRT6 have shown that it is an epigenetically linked enzyme whose loss of function shortens lifespan, while its gain of function extends lifespan in mice.

[0084] Mitochondrial dysfunction

[0085] Mitochondrial dysfunction occurs when mitochondria are unable to function properly. This can be due to other diseases, conditions or age. Most aging-related diseases, especially neurodegenerative diseases, are linked to mitochondria, including Alzheimer's disease and Parkinson's disease.

[0086] Loss of protein homeostasis

[0087] Loss of proteostasis, which can be described as a malfunction of the cell's protein-building machinery and the accumulation of misfolded proteins, is one of the root causes of age-related diseases, including Alzheimer's disease. Proteostasis involves mechanisms for the stabilization of correctly folded proteins, most notably the heat shock protein family, and for their degradation by the proteasome or lysosome.

[0088] Senescence

[0089] Cellular senescence can be defined as a stable arrest of the cell cycle, which can be triggered in normal cells in response to various intrinsic and extrinsic stimuli and developmental signals. Senescent cells remain viable, have altered metabolic activity and undergo significant changes in gene expression, and form a complex senescence-associated secretory phenotype. Cellular senescence impairs tissue repair and regeneration, leading to aging, and the number of senescent cells increases with age. Removal of senescent cells can alleviate age-related tissue dysfunction and extend healthy lifespan. However, the main purpose of senescence is to prevent the reproduction of damaged cells and trigger their death through the immune system. Therefore, senescence can serve as an effective anti-tumor mechanism by preventing the proliferation of potential cancer cells. This is a cellular program that is like a double-edged sword, with both benefits and disadvantages for the health of the organism. However, as a cellular checkpoint, it requires an effective cell replacement system involving the clearance of senescent cells and the mobilization of progenitor cells to re-establish the cell population. In aging organisms, this turnover system may become inefficient or may exhaust the regenerative capacity of progenitor cells, ultimately leading to the accumulation of senescent cells, which may exacerbate damage and lead to aging.

[0090] Telomere shortening

[0091] Telomere shortening is the result of problems with end replication and occurs in somatic cells without telomerase activity. Telomere shortening is one of the more widely accepted theories of aging in the scientific community.

[0092] Dysregulated nutrient sensing (metabolism changes)

[0093] Nutrient sensing pathways are often dysregulated in human metabolic diseases. Metabolic activity stresses our cells. Excessive activity as well as changes in nutrient availability and composition can cause cells to age more rapidly. Consistent with the relevance of dysregulated nutrient sensing as a hallmark of aging, dietary restriction (DR) has been shown to extend lifespan or healthspan in all eukaryotic species studied. Currently available evidence supports the view that anabolic signaling accelerates aging, whereas reduced nutrient signaling extends lifespan.

[0094] Furthermore, pharmacological manipulations that mimic states of limited nutrient availability, such as rapamycin, have been shown to extend lifespan in mice.

[0095] Altered intercellular communication

[0096] Aging involves changes at the level of intercellular communication, where the changes can be endocrine, neuroendocrine, or neuronal. One of the most striking changes in cell signaling biomarkers is "inflammaging," the development of chronic, low-grade inflammation throughout the body with age. The normal role of inflammation is to recruit the body's immune system and repair mechanisms to specific damaged areas as long as the insult and threat exist. The continued presence of systemic inflammatory markers can exhaust the immune system and damage healthy tissue. It has been shown that neurohormonal signaling (e.g., renin-angiotensin, adrenergic, insulin-IGF1 signaling) tends to become dysregulated in aging as the inflammatory response increases, immune surveillance of pathogens and precancerous cells decreases, and the composition of the peri- and extracellular environments changes, affecting the mechanical and functional properties of all tissues.

[0097] Stem cell exhaustion

[0098] Stem cells are undifferentiated or partially differentiated cells that can proliferate indefinitely. In the first few days after fertilization, the embryo consists almost entirely of stem cells. As the fetus grows, the cells multiply, differentiate and exert their appropriate functions in the organism. In adults, stem cells are mainly located in areas that are gradually depleted (intestines, lungs, mucous membranes, skin) or require constant replenishment (red blood cells, immune cells, sperm cells, hair follicles).

[0099] The loss of regenerative capacity is one of the most obvious consequences of aging. This is mainly because the proportion of stem cells and their division rate gradually decrease over time. It has been found that stem cell regeneration can reverse some of the effects of aging at the organismal level.

[0100] Models for studying aging

[0101] The nematode Caenorhabditis elegans (C. elegans) is a nematode worm that is much simpler anatomically than humans, however, does share many similarities at the molecular level, making it a good candidate for a model organism. C. elegans is considered a good organism to study aging because most pathways that influence the aging process are conserved between nematodes and humans. 11 Importantly, C. elegans also enables us to study the mechanisms of how the aging process leads to the development of age-related diseases.

[0102] Effects of krill-derived phospholipid compositions

[0103] Previous studies using krill oil as a supplement have shown improvements in movement abnormalities and cognitive deficits in a mouse model of MS (multiple sclerosis). 10 In addition, LPC-bound EPA / DHA has been shown to effectively enhance DHA availability in the brain and improve brain function6 , making it a unique nutritional supplement for treating AD brain 9 In addition, phospholipid-bound DHA and EPA have been shown to improve damage caused by Aβ amyloid aggregation through the anti-inflammatory and antioxidant properties of krill oil. 8 In the MPTP-induced PD mouse model, astaxanthin protected dopaminergic neurons in the nigrostriatal circuit in young mice but not in aged animals 40 However, to date, there is no evidence that krill-derived products can influence the cellular processes that lead to aging, or that such products have the potential to preventively or therapeutically treat age-related damage and disease.

[0104] In Example 1, Caenorhabditis elegans was used to explore whether a krill-derived composition comprising phospholipid-bound EPA and DHA could promote healthy aging. To evaluate the potential to mitigate age-related damage and disease, Parkinson's disease (PD) was used as a model, as aging is a major risk factor for this devastating neurodegenerative disease (NDD). 12-15 These transgenic strains facilitate the visualization of age-related degeneration of dopaminergic neurons and accumulation of α-SYN aggregates in body muscle; two of the most prominent pathological hallmarks of PD.

[0105] The results of Example 1 show that krill-derived phospholipid compositions counteract the main drivers of aging, including oxidative stress, proteotoxic stress, mitochondrial dysfunction, senescence, and genomic instability, in C. elegans and human fibroblasts. In the C. elegans model of Parkinson's disease, krill-derived phospholipid compositions protect dopaminergic neurons from aging-related degeneration, reduce α-synuclein aggregation, and improve dopamine-dependent behavior and cognition. Mechanistically, krill-derived phospholipid compositions increase neuronal resilience through temporal transcriptome reorganization, thereby promoting resistance to oxidative stress and inflammation through healthy lifespan regulatory transcription factors such as SNK-1. However, the phospholipid compositions also promote DA neuron survival by regulating synaptic transmission and neuronal function through PBO-2 and RIM-1. Overall, krill-derived phospholipid compositions rewire global gene expression programs and promote healthy aging by eliminating multiple aging hallmarks.

[0106] The results showed that krill-derived phospholipid composition could effectively protect dopaminergic neurons and reduce α-synuclein aggregation in aged PD worms ( Figure 1ad). This data directly correlates with clusters 4, 6, 7, 8, and 12 derived from RNAseq analysis, which represent the majority of upregulated genes in PD animals treated with the krill-derived composition ( Figure 5 ) and the green module from WGCNA ( Figure 7 ). GO enriched in cluster C5 is associated with cellular component morphogenesis and metalloendopeptidase activity. A recent study demonstrated that these metallopeptidases play a role in age-related muscle degeneration. 41 This suggests the possibility that krill-derived phospholipid composition could maintain mobility and BSR in aged PD animals by upregulating metallopeptidase activity.

[0107] It has been reported that the mechanism of aging may be affected by sex. In men, mitochondrial gene expression decreases with age. 42 The elevated expression of GO in the C10 cluster associated with sex determination and reproductive organ development in response to krill-derived phospholipid composition can be attributed to improved mitochondrial health. Figure 3 ), and increased the expression of mitochondrial encoded genes ctc-1 and nduo-2. Notably, HMG-5, a master regulator of mitochondrial transcription, was also involved in the induced neuroprotection of DA neurons. This clearly suggests that krill-derived phospholipid composition improves mitochondrial health and promotes DA neuron protection by improving mitochondrial transcriptional activation. Mitochondria also play an important role in regulating oxidative stress. Using a reverse genetics approach, it was demonstrated that the protective effect of DA neurons in response to krill-derived phospholipid composition depends on the oxidative stress regulators skn-1 and lmd-3 ( Fig. 9 a and 9b).

[0108] The results showed that the krill-derived PL composition tested in Example 1 improved dopamine-dependent behaviors, such as BSR and pharyngeal pumping ( Figure 1 e and Fig. 9 c). Interestingly, GO for synaptic organization was found in the 122-gene cluster C12 ( Figure 5 a and 5c). Therefore, the tested composition can improve pharyngeal pumping and motor activity in PD strains due to better synaptic organization. In addition, the C. elegans PD model further confirmed that neuroprotection of DA neurons was significantly dependent on cnnm-3, pbo-2 and rim-1. This confirms that the tested PL composition improved dopamine receptor signaling pathways, positive regulation of acetylcholine secretion, cholinergic synaptic transmission signaling and transmembrane transporter activity through vertebrate CNNM family homologs.

[0109] The downregulation of some GOs in clusters 16, 22, 30, and 31 in response to the tested PL compositions is interesting; C16 represents heme and iron binding proteins. Heme and iron binding proteins are associated with the early onset of Alzheimer's disease. 43 . Consistently, C. elegans AD strains showed improved memory in first-day-old adults fed a krill-derived phospholipid composition. Similarly, overexpression of pan-neuronal Tau protein in the C. elegans AD model BR5270 strain resulted in a 60% senescence induction rate at day 5. AD animals fed a krill-derived PL composition showed a 30% reduction in positive β-gal staining. Cluster 31 predicts downregulation of GO in pantothenate biosynthesis and rod-cone atrophy (HP: human phenotype). Pantothenate kinase-associated neurodegeneration (PKAN) is exacerbated by iron accumulation in the basal ganglia, leading to clinical symptoms such as Parkinson's disease and retinal degeneration. 44 . This may suggest that neuroprotection of DA neurons in PD animals fed the krill-derived PL composition may also be affected by downregulation of the pantothenate biosynthesis pathway. The observation that the krill-derived PL composition downregulates genes associated with rod-cone atrophy suggests that the claimed composition may also be a useful supplement for the treatment of retinal degeneration. In PD animals fed the krill-derived PL composition, the expression of UDP-glucuronosyltransferase was reduced in clusters 22 and 31. In early development, UDP-glucuronosyltransferase is highly upregulated and controls hormone signaling. This may suggest that the claimed PL composition may also affect aging by regulating the expression of the liver enzyme UDP-glucuronosyltransferase 37 .

[0110] in conclusion

[0111] The study in Example 1 showed that the krill-derived phospholipid composition effectively improved dopaminergic neuron survival by improving the clearance of α-synuclein aggregates. Thus, the health benefits of the claimed phospholipid composition are demonstrated by improving mitochondrial health, slowing aging, maintaining genomic stability, and improving protein homeostasis ( Fig. 9 ). From the RNAseq data analysis, it was confirmed that the tested composition improved cholinergic, dopaminergic and transmembrane transport systems. Therefore, antioxidant and anti-inflammatory therapies such as supplementation with the phospholipid composition may become a new and feasible approach for healthy brain aging intervention. Based on the results, it was concluded that the krill-derived phospholipid composition modulated different gene expression programs in middle-aged and elderly animals, all promoting healthy aging by attenuating several aging hallmarks of proteotoxic stress and senescence, and promoting genome stability and mitochondrial health. In summary, these results demonstrated a significant protection of DA neuron survival in aged animals.

[0112] Thus, the present invention provides marine-derived phospholipid compositions and methods for improving multiple markers of aging and age-related damage. The compositions described herein are shown to counteract the major drivers of aging, such as mitochondrial dysfunction, senescence, and genomic instability. Thus, the effectiveness of the phospholipid compositions as longevity supplements is demonstrated.

[0113] In a first aspect, a phospholipid composition and a method for improving at least one marker of aging in a subject are provided, comprising administering a composition of the invention to a subject, wherein the marker of aging is selected from the group consisting of, for example, genomic instability, telomere loss, epigenetic alterations, loss of proteostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication.

[0114] In one embodiment, the method improves genomic instability. In another embodiment, the method improves telomere attrition. In another embodiment, the method improves one or more epigenetic changes. In another embodiment, the method improves loss of proteostasis. In another embodiment, the method improves nutrient sensing disorders. In another embodiment, the method improves mitochondrial dysfunction. In another embodiment, the method improves cellular aging. In another embodiment, the method improves stem cell exhaustion. In another embodiment, the method improves alterations in intercellular communication.

[0115] Phospholipid composition

[0116] The present invention provides a marine-derived phospholipid composition and its use in treatment, medicine, nutrition, medical food and functional food.

[0117] Normal intake of PL is about 2-8 grams per day, which accounts for 1-10% of dietary fat intake. Phosphatidylcholine (PC) is an important phospholipid because it contains choline, which is important for many body functions, such as nerve signaling and liver and muscle function. Choline is an essential nutrient that is critical for normal cell function and is especially important for liver health, heart health and brain development.

[0118] PLs are found in eggs, milk, meat and fish, however, common fatty acids in these PLs are palmitic, stearic, oleic and linoleic acids. Many marine-derived phospholipids are unique for their high EPA and DHA levels. In krill, up to 80% or more of the total amount of EPA and DHA can be bound to phospholipids. In addition, most of the EPA and DHA present in krill oil are bound to PC. In contrast, EPA and DHA in fish oil are mostly bound to triglycerides (TG).

[0119] The metabolism of omega-3 (EPA / DHA) is very important in assessing bioavailability and retention in the body.

[0120] In a preferred embodiment, the phospholipid composition is prepared from krill, such as Antarctic krill (Euphausia superba) or Pacific krill (Euphausia pacifica). In other preferred embodiments, the phospholipid composition is prepared from fish or fish by-products (e.g., herring, herring roe, salmon, anchovies, mackerel, and related by-products of processed fish), Daphnia, shrimp, molluscs (including cephalopods such as squid), or algae. Lipid compositions from herring, herring roe, squid, and some microalgae (such as some strains of Chlorella) show rich phospholipids, including phosphatidylcholine. Suitable krill crude phospholipid extracts, desalted krill phospholipid extracts, and methods for processing krill are disclosed in PCT / IB2016 / 000208 and PCT / IB2016 / 000326, each of which is incorporated herein by reference in its entirety.

[0121] In a preferred embodiment, the phospholipid composition of the present invention is derived from krill.

[0122] In a preferred embodiment of the present invention, the phospholipid composition includes phospholipid-bound EPA and / or phospholipid-bound DHA. The amount of phospholipid-bound EPA and / or DHA can preferably be determined by methods known in the art, including by 31P-NMR program. When determining the amount of EPA and / or DHA bound to phospholipids present in the composition and using the NMR method, the amount of the combined EPA and / or DHA can be expressed as a weight percentage relative to the total weight of the composition, i.e. w / w. Although most of the EPA and / or DHA in the expected composition will be combined with the phospholipid molecules in the composition, some EPA and / or DHA may also be combined with other molecules such as triglycerides. In some preferred embodiments, the total amount of EPA and / or DHA in the composition (for example, whether combined with phospholipids or triglycerides or present as free fatty acids) can be determined by GC-FAME and expressed as g / 100g fatty acids. Alternatively, the total amount of EPA and / or DHA in the mixed composition can also be expressed as a weight percentage based on the total weight of the composition, i.e. w / w.

[0123] Thus, in some preferred embodiments, at least 50% of the EPA and / or DHA present in the composition comprising phospholipid-bound EPA and / or DHA, on a molar basis, is bound to phospholipid molecules (including lyso forms). In some preferred embodiments, at least 60% of the EPA and / or DHA present in the composition comprising phospholipid-bound EPA and / or DHA, on a molar basis, is bound to phospholipid molecules (including lyso forms). In some preferred embodiments, at least 70% of the EPA and / or DHA present in the composition comprising phospholipid-bound EPA and / or DHA, on a molar basis, is bound to phospholipid molecules (including lyso forms). In some preferred embodiments, at least 80% of the EPA and / or DHA present in the composition comprising phospholipid-bound EPA and / or DHA, on a molar basis, is bound to phospholipid molecules (including lyso forms). In some preferred embodiments, at least 90% of the EPA and / or DHA present in the composition comprising phospholipid-bound EPA and / or DHA, on a molar basis, is bound to phospholipid molecules (including lyso forms). In these embodiments, the amount of phospholipid-bound EPA and / or DHA relative to other EPA- and / or DHA-containing molecular species is preferably determined by 31 P-NMR.

[0124] In some preferred embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA can be further characterized by the total amount of EPA and / or DHA in the composition, which is expressed as grams of EPA and / or DHA per 100 grams of fatty acids determined by GC-FAME. Therefore, in some preferred embodiments, the amount of EPA and / or DHA in the composition is 5 to 80g / 100g fatty acids. In some preferred embodiments, the amount of EPA and / or DHA in the composition is 5 to 60g / 100g fatty acids. In some preferred embodiments, the amount of EPA and / or DHA is 5 to 50g / 100g fatty acids. In some preferred embodiments, the amount of EPA and / or DHA combined in the composition is 5 to 40g / 100g fatty acids. In some preferred embodiments, the amount of EPA and / or DHA in the composition is 10 to 40g / 100g fatty acids. In some preferred embodiments, the amount of EPA and / or DHA in the composition is 10 to 30g / 100g fatty acids. In some preferred embodiments, the amount of EPA and / or DHA in the composition is 15 to 40 g / 100 g fatty acids. In some preferred embodiments, the amount of EPA and / or DHA combined in the composition is 15 to 30 g / 100 g fatty acids.

[0125] In further preferred embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA can be further characterized by the total amount of EPA and / or DHA in the composition, the total amount of EPA and / or DHA in the composition being expressed as the weight percentage of EPA and / or DHA relative to the total weight of the composition. Thus, in some preferred embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA comprises 10% to 70% EPA and / or DHA w / w (i.e., grams of bound or free EPA and / or DHA per total weight of the composition). In some preferred embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA comprises 10% to 50% EPA and / or DHA w / w. In some preferred embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA comprises 10% to 40% EPA and / or DHA w / w. In some preferred embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA comprises 20% to 60% EPA and / or DHA w / w. In some preferred embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA comprises 20% to 50% EPA and / or DHA w / w. In some preferred embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA comprises 20% to 40% EPA and / or DHA w / w.

[0126] In further preferred embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA may be further characterized by the ratio of EPA and / or DHA present in the composition. Thus, in some preferred embodiments, the ratio of EPA to DHA in the composition is from 1:1 to 5:1. In some preferred embodiments, the ratio of EPA to DHA in the composition is from 1:1 to 3:1. In some preferred embodiments, the ratio of EPA to DHA in the composition is from 1:1 to 2.5:1. In some preferred embodiments, the ratio of DHA to EPA in the composition is from 1:1 to 5:1. In some preferred embodiments, the ratio of DHA to EPA in the composition is from 1:1 to 3:1. In some preferred embodiments, the ratio of DHA to EPA in the composition is from 1:1 to 2.5:1.

[0127] In a further preferred embodiment, the phospholipid composition comprising EPA and / or DHA bound to phospholipids can be further characterized by the total amount of phospholipids in the composition. In some preferred embodiments, the amount of total phospholipids (including lysozyme) is determined by 31P-NMR to provide a total amount (total weight of weight / composition) based on w / w. In some preferred embodiments, the composition comprises 10% to 90% total phospholipids (w / w). In some preferred embodiments, the composition comprises 10% to 80% total phospholipids (w / w). In some preferred embodiments, the composition comprises 10% to 70% total phospholipids (w / w). In some preferred embodiments, the composition comprises 10% to 60% total phospholipids (w / w). In some preferred embodiments, the composition comprises 20% to 90% total phospholipids (w / w). In some preferred embodiments, the composition comprises 20% to 80% total phospholipids (w / w). In some preferred embodiments, the composition comprises 20% to 70% total phospholipids (w / w). In some preferred embodiments, the composition comprises 20% to 60% total phospholipids (w / w). In some preferred embodiments, the composition comprises 30% to 90% total phospholipids (w / w). In some preferred embodiments, the composition comprises 30% to 80% total phospholipids (w / w). In some preferred embodiments, the composition comprises 30% to 70% total phospholipids (w / w). In some preferred embodiments, the composition comprises 30% to 60% total phospholipids (w / w).

[0128] In some embodiments, the phospholipid composition comprising phospholipid-bound EPA and / or DHA can be further characterized by the total amount of other lipids in the composition. In some embodiments, the composition comprises about 26% w / w to 46% w / w triglycerides, more preferably about 31% w / w to about 41% w / w triglycerides, and most preferably about 36% w / w triglycerides, wherein w / w refers to the weight of triglycerides as a percentage of the total weight of the phospholipid composition. In some embodiments, the phospholipid composition preferably comprises about 1% w / w to 7% w / w diglycerides, more preferably about 1.5% w / w to about 4.5% w / w diglycerides, and most preferably about 3% w / w diglycerides, wherein w / w refers to the weight of diglycerides as a percentage of the total weight of the phospholipid composition. In some further embodiments, the phospholipid composition preferably comprises about 2% w / w to 12% w / w free fatty acids, more preferably about 4.5% w / w to about 9.5% w / w free fatty acids, and most preferably about 7% w / w free fatty acids, wherein w / w refers to the weight of free fatty acids as a percentage of the total weight of the phospholipid composition. In some embodiments, the phospholipid composition preferably comprises about 1% w / w to 7% w / w cholesterol, more preferably about 1.5% w / w to about 4.5% w / w cholesterol, and most preferably about 3% w / w cholesterol, wherein w / w refers to the weight of cholesterol as a percentage of the weight of the phospholipid composition. In some embodiments, the krill phospholipid composition preferably comprises about 0.1% w / w to 2% w / w cholesterol esters, more preferably about 0.2% w / w to about 1.0% w / w cholesterol esters, and most preferably about 0.5% w / w cholesterol esters, wherein w / w refers to the weight of cholesterol esters as a percentage of the weight of the phospholipid composition. In one embodiment, the phospholipid composition comprises a major amount of phosphatidylcholine (PC) compared to the amount of lysophosphatidylcholine (LPC). Therefore, in some preferred embodiments, the composition can be further characterized by the ratio of PC to LPC. In some preferred embodiments, the ratio of PC to LPC in the composition is 2:1 to 50:1. In some preferred embodiments, the ratio of PC to LPC in the composition is 5:1 to 50:1. In some preferred embodiments, the ratio of PC to LPC in the composition is 5:1 to 20:1.

[0129] In embodiments where the composition comprises a major amount of PC, the composition may be further characterized based on the total amount of PC in the composition on a w / w basis, which may preferably be determined by 31P-NMR. In some preferred embodiments, the composition comprises 20% to 70% PC (w / w). In some preferred embodiments, the composition comprises 20% to 60% PC (w / w). In some preferred embodiments, the composition comprises 20% to 50% PC (w / w). In some preferred embodiments, the composition comprises 30% to 70% PC (w / w). In some preferred embodiments, the composition comprises 30% to 60% PC (w / w). In some preferred embodiments, the composition comprises 30% to 50% PC (w / w).

[0130] In another embodiment, the phospholipid composition comprises a major amount of LPC compounds compared to the amount of PC. The LPC composition can be prepared by enzymatic treatment of a phospholipid extract. PCT / IB2018 / 001588 discloses a method for processing phospholipids into lysophospholipids.

[0131] Thus, in some preferred embodiments, the composition can be further characterized by the ratio of LPC to PC. In some preferred embodiments, the ratio of LPC to PC in the composition is from 1:1 to 20:1. In some preferred embodiments, the ratio of LPC to PC in the composition is from 1:1 to 10:1. In some preferred embodiments, the ratio of LPC to PC in the composition is from 1:1 to 5:1. In some preferred embodiments, the ratio of LPC to PC in the composition is from 2:1 to 5:1.

[0132] In embodiments where the composition comprises a major amount of LPC, the composition may be further characterized based on the total amount of LPC in the composition on a w / w basis, which may preferably be determined by 31P-NMR. In some preferred embodiments, the composition comprises 10% to 70% LPC (w / w). In some preferred embodiments, the composition comprises 10% to 60% LPC (w / w). In some preferred embodiments, the composition comprises 10% to 50% LPC (w / w). In some preferred embodiments, the composition comprises 15% to 50% LPC (w / w). In some preferred embodiments, the composition comprises 15% to 40% LPC (w / w). In some preferred embodiments, the composition comprises 15% to 30% LPC (w / w).

[0133] Krill oil (Superba Boost) and further processed krill-derived LPC compositions (Lysoveta) are both rich in phospholipid-bound EPA and DHA, as well as astaxanthin and choline.

[0134] Therefore, in some preferred embodiments, the composition can be further characterized by the amount of choline based on w / w in the composition, which can preferably be determined by 31P-NMR. In some preferred embodiments, the composition comprises 2% to 20% choline (w / w). In some preferred embodiments, the composition comprises 2% to 15% choline (w / w). In some preferred embodiments, the composition comprises 2% to 10% choline (w / w). In some preferred embodiments, the composition comprises 5% to 20% choline (w / w). In some preferred embodiments, the composition comprises 5% to 15% choline (w / w). In some preferred embodiments, the composition comprises 5% to 10% choline (w / w).

[0135] In a further embodiment, the phospholipid composition comprising phospholipid-bound EPA and / or DHA can be further characterized by the total amount of phosphatidylcholine. In some preferred embodiments, the composition comprising phospholipids with bound EPA and / or DHA is characterized by having a phosphatidylcholine (PC) content of 20 to 70%, 20 to 60% or 20 to 50% w / w, wherein w / w is determined by dividing the total weight of PC molecules with two fatty acids attached by the total weight of the composition, and preferably can be determined by 31P-NMR. In other embodiments, the amount of PC can be expressed as the w / w of total phospholipids. In other embodiments, the amount of PC can be expressed as the w / w of total phospholipids. In these embodiments, the composition can be characterized as comprising about 75% w / w to 95% w / w phosphatidylcholine, more preferably about 80% w / w to about 90% w / w phosphatidylcholine, and most preferably about 85% to 90% w / w phosphatidylcholine, wherein w / w refers to the weight of phosphatidylcholine as a percentage of the weight of total phospholipids.

[0136] In some preferred embodiments, the composition can be further characterized by the amount of astaxanthin esters in mg / kg of the composition, preferably measured by UV spectroscopy. In some preferred embodiments, the composition comprises 50 to 1000 mg / kg of astaxanthin esters. In some preferred embodiments, the composition comprises 50 to 500 mg / kg of astaxanthin esters. In some preferred embodiments, the composition comprises 50 to 300 mg / kg of astaxanthin esters. In some preferred embodiments, the composition comprises 100 to 400 mg / kg of astaxanthin esters. In some preferred embodiments, the composition comprises 100 to 300 mg / kg of astaxanthin esters.

[0137] In some preferred embodiments, the composition can be further characterized by the amount of TMAO in the composition, which is preferably expressed in mgN / 100g. In some preferred embodiments, the amount of TMAO is 1 to 30 mgN / 100g. In some preferred embodiments, the amount of TMAO is 1 to 20 mgN / 100g. In some preferred embodiments, the amount of TMAO is 1 to 10 mgN / 100g.

[0138] In some preferred embodiments, the composition can be further characterized by its peroxide value (PV), which is preferably determined by the acetic acid-isooctane method. In some preferred embodiments, the peroxide value is less than 10. In some preferred embodiments, the peroxide value is less than 5. In some preferred embodiments, the peroxide value is less than 3. In some preferred embodiments, the peroxide value is less than 2. In some preferred embodiments, the peroxide value is less than 5.

[0139] In some preferred embodiments, the composition can be further characterized by its acid value, which is preferably determined by AOC Sd 3d-63. In some preferred embodiments, the acid value is less than 50. In some preferred embodiments, the acid value is less than 40. In some preferred embodiments, the acid value is less than 30. In some preferred embodiments, the acid value is less than 20.

[0140] In some preferred embodiments, the composition can be further prepared by Na + In some preferred embodiments, the composition comprises less than 1.000% Na + (w / w). In some preferred embodiments, the composition comprises less than 0.500% Na + (w / w). In some preferred embodiments, the composition comprises less than 0.300% Na + (w / w). In some preferred embodiments, the composition comprises less than 0.200% Na + (w / w).

[0141] In some preferred embodiments, the composition is characterized by:

[0142] i) contains 10 to 40 g / 100 g EPA and DPA combined as determined by gas chromatography fatty acid methyl ester (GC-FAME) analysis;

[0143] ii) having a ratio of EPA to DHA of 1:1 to 3:1;

[0144] iii) having a total phospholipid content of 20 to 60% w / w, wherein w / w is determined by measuring the total weight of phospholipids in the composition by 31P-NMR and dividing the total weight of phospholipids by the total weight of the composition;

[0145] iv) having a choline content of 2 to 10% w / w, wherein w / w is determined by measuring the total weight of choline in the composition by 31P-NMR and dividing the total weight of the phospholipids by the total weight of the composition;

[0146] v) having a phosphatidylcholine (PC) content of 20 to 50% w / w, wherein w / w is determined by measuring the total weight of PC molecules with two fatty acids attached in the composition and dividing the total weight of PC molecules with two fatty acids attached by the total weight of the composition, optionally wherein the ratio of PC to LPC in the composition is from 2:1 to 50:1;

[0147] vi) has a lysophosphatidylcholine (LPC) content of less than 10% w / w, wherein w / w is determined by measuring the total weight of LPC molecules with one fatty acid attached in the composition and dividing the total weight of LPC molecules with one fatty acid attached by the total weight of the composition;

[0148] vii) comprising astaxanthin esters in an amount of 100 to 300 mg / kg;

[0149] viii) having a TMAO content, expressed in mgN / 100g, of 1 to 10 mgN / 100g;

[0150] ix) having a peroxide value of less than 2;

[0151] x) has an acid number of less than 30; and / or

[0152] xi) has a Na+ content of less than 0.200% w / w of the composition, wherein w / w is determined by measuring the total weight of Na+ in the composition and dividing the total weight of Na+ by the total weight of the composition;

[0153] In some preferred embodiments, the composition is characterized by:

[0154] i) contains 10 to 40 g / 100 g EPA and DPA combined as determined by gas chromatography fatty acid methyl ester (GC-FAME) analysis;

[0155] ii) having a ratio of EPA to DHA of 1:1 to 3:1;

[0156] iii) having a total phospholipid content of 20 to 60% w / w, wherein w / w is determined by measuring the total weight of phospholipids in the composition by 31P-NMR and dividing the total weight of phospholipids by the total weight of the composition;

[0157] iv) having a choline content of 2 to 10% w / w, wherein w / w is determined by measuring the total weight of choline in the composition by 31P-NMR and dividing the total weight of the phospholipids by the total weight of the composition;

[0158] v) has a phosphatidylcholine (PC) content of less than 20% w / w, wherein w / w is determined by measuring the total weight of PC molecules with two fatty acids attached in the composition and dividing the total weight of PC molecules with two fatty acids attached by the total weight of the composition;

[0159] vi) having a lysophosphatidylcholine (LPC) content of 20 to 50% w / w, wherein w / w is determined by measuring the total weight of LPC molecules with one fatty acid attached in the composition and dividing the total weight of LPC molecules with one fatty acid attached by the total weight of the composition, optionally wherein the ratio of LPC to PC in the composition is from 1:1 to 20:1;

[0160] vii) comprising astaxanthin esters in an amount of 100 to 300 mg / kg;

[0161] viii) having a TMAO content, expressed in mgN / 100g, of 1 to 10 mgN / 100g;

[0162] ix) having a peroxide value of less than 2;

[0163] x) has an acid number of less than 30; and / or

[0164] xi) has a Na+ content of less than 0.200% w / w of the composition, wherein w / w is determined by measuring the total weight of Na+ in the composition and dividing the total weight of Na+ by the total weight of the composition;

[0165] In some preferred embodiments, the composition is characterized by any one of i) to xi) above. In some preferred embodiments, the composition is characterized by any two of i) to xi) above. In some preferred embodiments, the composition is characterized by any three of i) to xi) above. In some preferred embodiments, the composition is characterized by any four of i) to xi) above. In some preferred embodiments, the composition is characterized by any five of i) to xi) above. In some preferred embodiments, the composition is characterized by any six of i) to xi) above. In some preferred embodiments, the composition is characterized by any seven of i) to xi) above. In some preferred embodiments, the composition is characterized by any eight of i) to xi) above. In some preferred embodiments, the composition is characterized by any nine of i) to xi) above. In some preferred embodiments, the composition is characterized by any ten of i) to xi) above. In some preferred embodiments, the composition is characterized by all of i) to xi) above.

[0166] In some preferred embodiments, the composition is characterized by i) and ii) above. In some preferred embodiments, the composition is characterized by i) and iii) above. In some preferred embodiments, the composition is characterized by i) and iv) above. In some preferred embodiments, the composition is characterized by i) and v) above. In some preferred embodiments, the composition is characterized by i) and vi) above. In some preferred embodiments, the composition is characterized by i) and vii) above. In some preferred embodiments, the composition is characterized by i) and viii) above. In some preferred embodiments, the composition is characterized by i) and ix) above. In some preferred embodiments, the composition is characterized by i) and x) above. In some preferred embodiments, the composition is characterized by i) and xi) above.

[0167] In some preferred embodiments, the composition is characterized by ii) and iii) above. In some preferred embodiments, the composition is characterized by ii) and iv) above. In some preferred embodiments, the composition is characterized by ii) and v) above. In some preferred embodiments, the composition is characterized by ii) and vi) above. In some preferred embodiments, the composition is characterized by ii) and vii) above. In some preferred embodiments, the composition is characterized by ii) and viii) above. In some preferred embodiments, the composition is characterized by ii) and ix) above. In some preferred embodiments, the composition is characterized by ii) and x) above. In some preferred embodiments, the composition is characterized by ii) and xi) above.

[0168] In some preferred embodiments, the composition is characterized by iii) and iv). In some preferred embodiments, the composition is characterized by iii) and v). In some preferred embodiments, the composition is characterized by iii) and vi). In some preferred embodiments, the composition is characterized by iii) and vii). In some preferred embodiments, the composition is characterized by iii) and vii). In some preferred embodiments, the composition is characterized by iii) and viii). In some preferred embodiments, the composition is characterized by iii) and ix). In some preferred embodiments, the composition is characterized by iii) and x). In some preferred embodiments, the composition is characterized by iii) and xi).

[0169] In some preferred embodiments, the composition is characterized by iv) and v). In some preferred embodiments, the composition is characterized by iv) and vi). In some preferred embodiments, the composition is characterized by iv) and vii). In some preferred embodiments, the composition is characterized by iv) and viii). In some preferred embodiments, the composition is characterized by iv) and viii). In some preferred embodiments, the composition is characterized by iv) and ix). In some preferred embodiments, the composition is characterized by iv) and x). In some preferred embodiments, the composition is characterized by iv) and vi).

[0170] In some preferred embodiments, the composition is characterized by v) and vi). In some preferred embodiments, the composition is characterized by v) and vii). In some preferred embodiments, the composition is characterized by v) and viii). In some preferred embodiments, the composition is characterized by v) and viii). In some preferred embodiments, the composition is characterized by v) and ix). In some preferred embodiments, the composition is characterized by v) and x). In some preferred embodiments, the composition is characterized by v) and xi).

[0171] In some preferred embodiments, the composition is characterized by vi) and vii) above. In some preferred embodiments, the composition is characterized by vi) and viii) above. In some preferred embodiments, the composition is characterized by vi) and ix) above. In some preferred embodiments, the composition is characterized by vi) and x) above. In some preferred embodiments, the composition is characterized by vi) and xi) above.

[0172] In some preferred embodiments, the composition is characterized by vii) and viii) above. In some preferred embodiments, the composition is characterized by vii) and ix) above. In some preferred embodiments, the composition is characterized by vii) and x) above. In some preferred embodiments, the composition is characterized by vii) and xi) above.

[0173] In some preferred embodiments, the composition is characterized by the above viii) and ix). In some preferred embodiments, the composition is characterized by the above viii) and x). In some preferred embodiments, the composition is characterized by the above viii) and xi).

[0174] In some preferred embodiments, the composition is characterized by ix) and x) above. In some preferred embodiments, the composition is characterized by ix) and xi) above.

[0175] In some preferred embodiments, the composition is characterized by x) and xi) above.

[0176] Table 1: Lipid composition and fatty acid profiles in two different PL compositions from krill

[0177]

[0178]

[0179]

[0180]

[0181] NMR method according to USP / FCC

[0182] Therapeutic applications

[0183] 1. Protects against neurodegeneration in the brain

[0184] The finding that krill-derived phospholipid compositions attenuated several aging hallmarks in both C. elegans and human fibroblasts supports the idea that consuming krill-derived phospholipid compositions prevents fundamental aging mechanisms in neural tissue. Thus, krill-derived phospholipid compositions have demonstrated neuroprotective benefits, which have important implications for brain health and the treatment of age-related neurodegeneration and neurodegenerative diseases (NDDs) in the brain.

[0185] Although systemic diseases are the most detrimental to human health and well-being, brain decline is the defining factor in the gradual loss of life essence leading to death. Aging, which underlies neurodegeneration and dementia, affects every organ in the body and appears to be encoded in part by blood-based signatures. In fact, factors in circulation have been shown to regulate aging and rejuvenate many organs, including the brain. Because brain tissue is composed primarily of post-mitotic cells, it is particularly sensitive to the effects of aging.

[0186] Studies have shown that the brain's uptake of EPA and DHA is greater when the EPA and DHA in the blood are in the form of LPC-EPA and LPC-DHA. Multiple studies have shown that the LPC-EPA / DHA form preferentially crosses the blood-brain barrier, which is supported by the discovery of a specific transporter for LPC-EPA / DHA called Mfsd2a (major facilitator superfamily domain-containing protein 2A) (Nguyen et al 2014). This has important implications for brain treatments.

[0187] Based on the documented effects, the present invention is a method of improving at least one marker of aging in a subject comprising administering to a subject in need thereof an effective amount of a phospholipid-bound EPA and / or DHA compound or a phospholipid composition as described above.

[0188] The present invention may also be defined as a phospholipid-bound EPA and / or DHA compound or a phospholipid composition as described above for use in improving at least one marker of aging or for preventing and / or treating age-related neurodegenerative diseases.

[0189] In some embodiments of the present invention, an effective amount of a phospholipid-bound EPA / DHA compound or the above-described phospholipid composition is administered to a subject in need thereof.

[0190] - to treat, prevent or ameliorate normal age-related neurodegeneration and neurodegenerative disorders, or

[0191] - To treat or prevent Alzheimer's disease or Parkinson's disease

[0192] - To treat, prevent or ameliorate executive function decline or age-related cognitive decline, or

[0193] - to treat, prevent or improve cognition and / or cognitive diseases, disorders or damage (memory, attention, learning (deficits)). In a particularly preferred embodiment, the composition for use in the method of treating or for treating neural tissue in the brain (including neurodegeneration, cognitive diseases, disorders or damage or decreased executive function) comprises phospholipid-bound EPA and / or DHA, wherein a major amount of EPA and / or DHA is bound to an LPC compound compared to the amount of EPA and / or DHA bound to PC.

[0194] 2. Protects against age-related cellular damage

[0195] The phospholipid composition derived from krill reduces multiple aging signs such as mitochondrial dysfunction, aging and genomic instability in Caenorhabditis elegans and human fibroblasts. This finding supports that the phospholipid composition derived from krill also prevents the basic aging mechanism in tissues other than neural tissue. To support this, it has been demonstrated in different organs that supplementation with the phospholipid composition derived from krill is effective. For example, in healthy elderly people, after 6 months of dietary supplementation with the phospholipid composition derived from krill, it was found that muscle size and function increased significantly (Alkhedhairi, SAA et al, Clinical Nutrition 41 (2022) 1228-1235). Studies have also shown that the phospholipids derived from krill fed to mice inhibit the inflammatory signaling pathways in adipose tissue and liver induced by a high-fat diet (Gart et al, Nutrients 2021, 13, 2836).

[0196] Cellular senescence is considered the most important age-related phenotypic change relevant to the concept of tissue aging. 45 Historically, senescence has been viewed as an irreversible cell cycle arrest mechanism that protects against cancer, but discoveries have expanded its known roles to complex biological processes such as development, tissue repair, aging, and age-related disorders. The transition from transient to persistent cell cycle arrest during senescence appears to be unplanned and stochastic in nature, likely involving the combined effects of different senescence-inducing stressors acting simultaneously on cells.

[0197] Currently, many studies are devoted to elucidating the specific molecular pathways of aging in different cells, tissues or organs, and it has been found that senescent cells are the driving factors of a large number of age-related pathologies. 46 In addition to Alzheimer's and Parkinson's diseases, these pathologies include atherosclerosis, cardiovascular dysfunction (including those caused by certain genotoxic chemotherapy), tumor progression, loss of hematopoietic and skeletal muscle stem cell function, nonalcoholic fatty liver disease, pulmonary fibrosis, osteoarthritis, and osteoporosis. In the liver, the biliary and vascular components have emerged as important determinants of some forms of liver disease. 47 Multiple findings in preclinical animal models and human liver specimens support the presence of specific hallmarks of aging in diseases involving these liver regions.

[0198] In some embodiments of the present invention, an effective amount of a phospholipid-bound EPA / DHA compound or the above-described phospholipid composition is administered to a subject in need thereof.

[0199] - To treat, prevent or ameliorate senescence associated with age-related pathologies selected from the group consisting of atherosclerosis, cardiovascular dysfunction (including cardiovascular problems caused by certain genotoxic chemotherapy), tumor progression, loss of hematopoietic and skeletal muscle stem cell function, non-alcoholic fatty liver disease, pulmonary fibrosis, osteoarthritis and osteoporosis.

[0200] In a particularly preferred embodiment, the composition for treatment or for use in a therapeutic method to prevent or ameliorate aging associated with age-related pathologies comprises phospholipid-bound EPA and / or DHA, wherein a major amount of EPA and / or DHA is bound to a PC compound compared to the amount of EPA and / or DHA bound to LPC.

[0201] It has been previously shown that mitochondria play an important role in the retina, one of the most bioenergetic organs in the human body. 48 The eye is exposed to visible light and has extensive antioxidant protection mechanisms. Mitochondrial function is impaired in aged retinal pigment epithelial cells and reactive oxygen species production is increased. Age-related mitochondrial dysfunction has been shown to lead to increased oxidative damage, which, combined with impaired repair mechanisms, results in retinal dysfunction and retinal cell loss, leading to visual impairment. This has been linked to age-related blindness such as age-related macular degeneration. Mitochondrial dysfunction has also been suggested to play a role in diabetic retinopathy and glaucoma.

[0202] The transporter Mfsd2 involved in LPC-mediated absorption of EPA and DHA has a wide tissue distribution and is expressed in multiple organs such as brain, retina, liver, kidney, intestinal mucosa, lung, breast, ovary, uterus, prostate and testis.

[0203] In another particularly preferred embodiment, the composition for use in a method of treating or ameliorating at least one marker of aging in a tissue selected from the group consisting of brain, retina, liver, kidney, intestinal mucosa, lung, breast, ovary, uterus, prostate and testis comprises phospholipid-bound EPA and / or DHA, wherein a major amount of the EPA and / or DHA is bound to an LPC compound compared to the amount of EPA and / or DHA bound to PC.

[0204] Example

[0205] Embodiment 1:

[0206] The purpose of this study was to determine whether a marine-derived phospholipid composition could affect signs of aging. A krill-derived phospholipid composition (Superba Boost) rich in phosphatidylcholine was selected as a model substance for this study. The composition of SuperbaBoost is listed in Table 1.

[0207] Materials and methods

[0208] C. elegans strains and culture conditions

[0209] Animals were grown on nematode growth medium (NGM) plates with Escherichia coli strain OP50 at 20°C using standard procedures (Brenner, 1974). Adult animals were bleached to obtain a synchronized population. For all aging-related studies, L4 hermaphrodites were grown from day 1 to day 6 in NGM plates containing krill oil (0.5 μl / ml). Aged populations of worms were maintained in NGM plates without FdUrd. Instead, animals were washed daily with M9 buffer after the adult day 2 stage and filtered through a nylon mesh filter (Catalog #NY4104700) until the desired age was reached. Day 1 of adulthood was defined as 24 hours after the L4 stage. The following nematode strains were used in this study: N2: wild type; BY273 Is[p dat- 1GFP; p dat-1 α-syn] was used to monitor dopaminergic neurons. The HLN107 strain was constructed by crossing TU3401:sid-1(pk3321)V;uls69 V with BY273 to perform RNAi in dopaminergic neurons. unc-54 α-syn::GFP is used to study α-synuclein aggregation. To study Alzheimer's disease (AD) using Caenorhabditis elegans; strain BR5270 [P rab-3 ::F3(delta)K280] and strain CK12[aex-3::tau4R1N(P301L)+myo-2p::gfp] were used for the Tau model; strain CL2355[P snb-1 ::Aβ 1-42 ] for Aβ model; strain UM0001[P snb-1 ::Aβ 1-42 ;P rab-3 ::F3(delta)K280] was used for the Tau;Aβ model. In the following, strains N2 and BY273 will be referred to as wild-type (WT) and PD animals, respectively. Strains BR5270, CK12, CL2355, and UM0001 will be referred to as AD animals.

[0210] Cell lines

[0211] The human fibroblast-like cell line BJ was grown in Dulbecco's modified Eagle's medium GlutaMAX (DMEM, Life Technologies) supplemented with 10% (vol / vol) fetal bovine serum (FBS, Lonza) and 1x penicillin-streptomycin (Life Technologies). The cells were grown at 37°C and 5% CO2.

[0212] RNA isolation and qPCR

[0213] Total RNA was isolated using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) on the QuantStudio 7Flex detection system (Applied Biosystems). Each sample was analyzed in triplicate.

[0214] Chemicals and antibodies

[0215] The senescence marker X-gal was purchased from Sigma (B4252-250MG Oslo, Norway). Serotonin hydrochloride was purchased from Sigma (Oslo, Norway). The following commercially available antibodies were used: monoclonal 8-oxo-dG (4354-MC-050 Trevigen). Secondary antibodies were AlexaFluor 555-conjugated anti-rabbit and anti-mouse (Invitrogen). For RNA isolation, Direct-zol RNA Miniprep Kits (#R2050) from Zymo-Research were used. The senescence marker SPiDER-β-gal for fibroblasts was purchased from Dojindo (SG02-10). TMRE (mitochondrial membrane potential assay kit) was purchased from Abcam (ab113852). The nuclear staining dye Hoechst was purchased from Thermo Scientific (Hoechst 33342).

[0216] Degeneration of dopaminergic neurons

[0217] BY273Is[p dat-1 GFP; p dat-1 Degeneration of dopamine neurons in α-syn 16,17BY273 animals were exposed to vehicle and krill oil (0.5 μl / ml medium) at the L4 larval stage. In vivo imaging of dopaminergic neurons was performed on day 1, 3, and 6 old animals. At this stage, 15-20 worms were mounted on agar-pad glass slides with 2 mM levamisole and glass coverslips. Dopaminergic (DA) neurons were imaged under a Zeiss LSM780 confocal microscope equipped with a x20 objective.

[0218] Immunohistochemistry

[0219] For immunostaining, adult worms were washed twice with milliQ water. Washed worms were placed on poly-L-lysine coated slides (Thermo Scientific) and frozen on dry ice using coverslips. The primary antibody 8-oxo-dG was used at a dilution of 1:200. The secondary antibody Alexa Fluor 555 conjugated anti-mouse was used at a dilution of 1:1500. Prolong Gold with DAPI was used for mounting (Invitrogen, P36931). Slides were imaged on a Zeiss LSM780 confocal microscope equipped with a x63 plan-Apochromat 1.4NA objective.

[0220] Basic slowed reaction

[0221] The basal slowing reaction was performed as described 16 Well-fed synchronized day 6-old worms were tested. NGM plates with and without OP50 were used to count body bends every 20 s. Movement rates were calculated 5 min after transfer to avoid overstimulation. The assay was performed blindfolded with three independent replicates.

[0222] Pharyngeal pumping

[0223] We used the microfluidics-based ScreenChip system from Invivo Biosystem to measure the pharyngeal pumping rate of each individual worm. 16 . For this assay, worms were age synchronized. Worms were first washed twice with M9 buffer, and then incubated with 10mM serotonin prepared in M9 buffer for 30 minutes at room temperature. At the same time, the screenchip fluidics system was prepared according to the user guide. Worms were loaded into 1.5ml Eppendorf tubes with 1ml M9 buffer, and vacuum was drawn into the screening chip using a vacuum pump. Pumping frequency was measured by EPG recording of 3-day-old worms using screen-chip 40. Each recording lasted 1 to 2 minutes, with 15-20 worms per genotype.

[0224] Chemotaxis-related learning

[0225] Short-term associated memory training (STAM) test 16,18 The assay is based on the ability of C. elegans to learn and remember the positive association between food and the weak chemical attractant butanone. Briefly, age-synchronized worms maintained at 20°C were divided into initial and training groups. Each group had 200 to 300 worms per condition. The training group was washed three times with M9 and starved in M9 for one hour before training. After two hours of conditioning, the worms were trained to actively associate food with butanone and were then placed on seeded NGM plates for 120 minutes. STAM was measured after a two-hour gap in (interval) training. Worms were tested for chemotaxis to 10% butanone before (initial) or after (trained) two hours of conditioning. The chemotaxis index was calculated as follows: (CI) = (n 丁酮 -n 乙醇 ) / (total-n 起始 The learning index was calculated by subtracting the initial CI from the CI after conditioning (CI 丁酮 -CI 初始 Memory assays in AD animals were performed as described elsewhere. 19 .

[0226] Oxygen consumption rate

[0227] Oxygen consumption rate was measured using a DW1 / AD clark polarographic oxygen sensor (Hansatech Instruments, model: Oxygraph Plus System). The protocol was performed exactly as described without modification. 20 .

[0228] senescence

[0229] Senescence assay was performed as described 21 The animals were thawed at room temperature for 10 minutes and stained with a freshly prepared X-gal staining solution containing 1 mg / ml X-gal (stock solution 20 mg / ml in DMSO), 40 mM citric acid / sodium phosphate pH 6.5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide, 150 mM NaCl and 2 mM MgCl2, and incubated overnight at 37 ° C in the dark. The stained animals were washed with 1 x PBS and sealed on a glass slide with a cover glass. The animals were imaged under a bright field microscope with a x20 objective.

[0230] Senescence assay was performed on BJ fibroblasts using the Spider-β-Gal marker kit purchased from Dojindo. 50,000 (late passage) fibroblasts were seeded on a 35 mm imaging dish (from IBIDI) with a polymer coverslip bottom, and the cells were grown overnight at 37° C. in a 5% CO2 incubator. 24 hours after seeding, the cells were treated with 100 μg / mL krill oil and grown in a 5% CO2 incubator at 37° C. for 6 days. Senescence assay was performed on live cells on day 7 according to the manufacturer's protocol. In brief, the cells were washed once with PBS, then treated with 1 mL of Bafilomycin working solution and incubated in a 5% CO2 incubator at 37° C. for 1 hour. After 1 hour, 1 mL of SPiDER-β-Gal working solution containing 2 μg / mL Hoechst was added to the cells, and they were incubated again in a 5% CO2 incubator for 30 minutes. After incubation, cells were washed with PBS and then imaged under a Zeiss LSM780 confocal microscope at 63X using appropriate filters.

[0231] TMRE staining

[0232] Mitochondrial membrane potential was measured using the TMRE (tetramethylrhodamine, ethyl ester) mitochondrial membrane potential assay kit (Cat. No. ab113852; Abcam). A total of 50,000 BJ fibroblasts (late passage) were plated onto a 35 mm imaging dish (from IBIDI) with a polymer coverslip bottom. 24 hours after inoculation, cells were treated with 100 μg / mL krill oil and allowed to grow for another 5 days at 37°C in a 5% CO2 incubator. On day 6, TMRE assays were performed according to the manufacturer's protocol. In brief, cells were stained with a final concentration of 200 nM TMRE and incubated at 37°C in the dark for 30 min. After incubation, cells were washed again with PBS and live cell imaging was performed at 63X magnification on a Zeiss LSM780 confocal microscope using appropriate filters.

[0233] Mitochondrial copy number and gene expression analysis

[0234] Mitochondrial DNA (mtDNA) copy number was quantified using droplet digital PCR (ddPCR). Briefly, age-synchronized wild-type and PD animals were individually picked in lysis buffer (TE low containing 1 mg / ml proteinase K and 0.01 mg / ml RNase). Lysis was performed at 65°C for 1 h, followed by 95°C for 15 min. From here on, the method was exactly as described in 16 Execute as described in .

[0235] For mitochondrial gene expression analysis, transcriptional activation of ctc-1 and nduo-2 was measured in age-synchronized wild-type and PD animals. Individual worms were collected in 1 ml PBS and immediately snap-frozen in dry ice. This protocol was performed exactly as described without modification. 16 .

[0236] RNA Isolation for RNAseq

[0237] Age-synchronized day 1, 3, 6 wild-type and PD animals were treated or not with krill oil. These animals were harvested and RNA isolation was performed on the same day. Briefly, animals were washed x2 with sterile milliQ water and collected in a 1.5 ml Eppendorf. The worm pellet was distributed in 600 μl of TRI reagent or Trizol and transferred to a tube with sterile beads to homogenize the animals. From here on, RNA isolation of tissues was performed as described in the Direct-zol RNA Miniprep Kits protocol section. The isolated RNA was dissolved in 10 μl of nuclease-free water and the quality was analyzed in a bioanalyzer.

[0238] RNA sequencing

[0239] Sequencing libraries were prepared from 200 ng of total RNA using the Nugen Universal Plus Total RNA-Seq Library Preparation Kit (Tecan) with a custom AnyDeplete design for C. elegans. Final libraries were pooled (10 samples / run) and paired-end sequenced (2x71 bp) on a NextSeq 550 sequencer using the NextSeq High Output Kit. Raw sequencing data were demultiplexed using BCL Convert (Illumina) and sequenced using kallisto 22 Sequencing reads were aligned to the C. elegans WBcel235 cDNA and quantified using umi-tools 23 Remove UMI duplicates. Use samtools 24 The depulicated bam files were converted to fastq, and then the abundance of transcripts was quantified using kallisto.

[0240] RNA-seq data analysis

[0241] All analyses were done using R version 4.1.0 on a x86_64-pc-linux-gnu (64-bit) platform, running the Pop!_OS operating system, version 21.04. The computer used for the analyses had 32Gb of RAM and 4 8th generation i5 cores, threaded twice each, resulting in 8 available CPUs. The DESeq2R package was used 25 Version 1.32.0 was used for differential gene expression. The count files were divided into their respective groups and attributed to defined conditions, such as 'control' and 'experimental'. The counts of the individual conditions were merged into a data frame, from which genes with no counts for each sample were removed. A sample definition file containing the sample names and their associated conditions was also created, which contained relevant information about the samples in the merged count files. The values ​​were then normalized using the DESeq2 normalization method. The standard DESeq2 pipeline was used to obtain differential gene expression results. The merged count files were first used to create a DESeqDataSet-class by calling the DESeqDataSetFromMatrix function, and the sample definition file was used as input, while the condition was used as the design. The design always used 'experimental' and compared it to 'control'. The size factors of the DESeqDataSet object were estimated using the median ratio method using the estimateSizeFactors function. The DESeq2 function was called, which performed dispersion estimation, followed by negative binomial GLM fitting and wal statistics to obtain differential gene expression results. Significant differentially expressed genes were selected based on an adjusted p-value (false discovery rate) lower than 0.05 and a log2 fold change value greater than 1 or less than -1.

[0242] Using the ClusterProfiler package 26 (version 4.0.0) performed gene set enrichment analysis (GSEA). The analysis was run against the biological process (BP) ontology. The AnnotationDbi package (version 1.54.1) was used to load information related to the organism under study. Using this package, the background of the GSEA was set using the org.Hs.eg.db library (version 3.13.0). GSEA was performed on differential gene expression analysis, where every gene in the differential expression results, regardless of significance, was entered for the analysis. The p-value and false discovery rate (FDR) cutoff were set to 0.05. Semantic distances were added to the GSEA results using the pairwise_termsim function in the enrichplot package (version 1.12.1). The ‘Wang’ method was used. 27 Compute the added similarity matrix.

[0243] WGCNA Package 28(version 1.70-3) is used to obtain weighted correlation associations between gene modules and behavioral parameters. WGCNA receives RNAseq count data. A sample file was manually prepared for the WGCNA package that details which behavioral values ​​are associated with the input samples. The pipeline first removes any samples with too many missing values ​​based on the number of input samples and values. The samples are then clustered and their efficacy is evaluated. 28 The power value used is the first value above the 0.8 threshold, which is 4. The block module function is used to calculate the gene modules and obtain the correlation values ​​with clinical parameters. Each gene module is generated by gprofiler 29 Run to extract the potential biological relevance of each gene module.

[0244] Biological age prediction was performed using fastq files as raw data as input in the preprocessing specified in the BitAge pipeline. First, the data was processed using fastp (version 0.23.2) with parameters -gxq 30-e30-w 8. Second, the reads were aligned using the STAR aligner with parameters --quantMode GeneCounts --runThreadN 8, --outSAMtype BAM Unsorted --sjdbOverhang 69, and again the parameters suggested by the STAR manual in the ENCODE options as described above. To predict biological age, we took the raw counts from the STAR aligner output and calculated counts per million (CPM) using the edgeR library (version 3.38.1) as required by the BitAge method. We then calculated the predicted biological age using the elastic network coefficients and code provided in the BitAge materials.

[0245] result

[0246] Krill oil protects dopaminergic neurons from age-related degeneration and improves dopamine-dependent behaviors

[0247] To determine whether growth in the presence of krill oil protects DA neurons from degeneration over time, we used the Caenorhabditis elegans Parkinson's disease (PD) model, a humanized strain in which overexpression of α-synuclein under the control of the dat-1 promoter exacerbates age-dependent degeneration of dopaminergic (DA) neurons. 12,16 As previously reported, we found that when assessed by DA neuron GFP fluorescence intensity ( Figure 1 a) and scoring of DA neuron numbers by differential interference contrast (DIC) microscopy ( Figure 1b), the number of DA neurons was significantly reduced in 6-day-old animals compared to young adults (days 1 and 3). Growth in krill oil-enriched medium resulted in significant protection of DA neurons from age-related degeneration ( Figure 1 a and 1b). As expected, aging reduced the proportion of DA neurons in day 6 adults to 60% compared to 90% in day 1 adults when grown in standard medium. In contrast, there was no reduction in the proportion of DA neurons that survived from day 3 to day 6 in krill oil-treated animals ( Figure 1 a and 1b). In addition, the proportion of surviving DA neurons was slightly increased in day 1 animals, suggesting a general protection against α-synuclein-mediated proteotoxicity.

[0248] To test whether krill oil inhibits α-synuclein aggregate formation, a key pathogenic hallmark of Parkinson's disease, we used a reporter strain that expresses α-synuclein coupled to GFP in body wall muscle cells. 16 While day 1 young animals showed no signs of alpha-synuclein aggregation, day 6 old animals had clear alpha-synuclein aggregates, with an average of approximately 50 aggregates per animal. Interestingly, krill oil treated animals showed a significant reduction in alpha-synuclein aggregation, with an average of approximately 20 aggregates ( Figure 1 c and 1d).

[0249] Dopamine-regulated behavioral features are evident in the Caenorhabditis elegans PD model 30 Many research groups, including ours, have demonstrated that DA neuron-dependent behaviors, such as basal slowed response (BSR) and motor performance, deteriorate with age. 16 Therefore, we wanted to determine whether the increased survival of DA neurons would lead to the preservation of DA neuron function, and we examined the BSR in wild-type and day 6 PD animals. Interestingly, animals fed krill oil had significantly higher BSR than untreated animals ( Figure 1 e). Further supporting the DA-dependent improvement in behavioral performance, the mean activity score for 6-day-old animals was shown to be 400 / hour, compared to only 340 / hour for untreated PD animals. There was no change in the response of wild-type animals to krill oil ( Figure 1 f). This clearly showed that aged PD animals fed krill oil showed improved motor activity compared to untreated animals. These findings suggest that krill oil protects DA neurons from degeneration, inhibits α-synuclein aggregate formation, and improves dopamine-dependent behaviors.

[0250] Krill oil slows aging in Caenorhabditis elegans and human fibroblasts

[0251] Senescence is a hallmark of aging, and there is evidence that senescence-like atrophy may be responsible for age-dependent loss of germline gonadal cells and self-destruction of intestinal biomass in Caenorhabditis elegans 31-33 In 9-day-old animals, 45% of wild-type animals and 80% of PD animals showed positive β-gal staining. When these animals were fed krill oil, wild-type and PD animals showed a 35% and 60% decrease in positive β-gal staining, respectively ( Figure 2 a and 2b).

[0252] Since it is unclear whether C. elegans undergoes a process similar to the senescence-associated secretory phenotype, which is an effector of senescence-driven aging in human tissues, we used human BJ fibroblasts and treated these cells with krill oil to support our data obtained in worms. Late-passage BJ fibroblasts, which are widely accepted as a model for tracking aging, showed a 1.75-fold decrease in β-gal-positive cells compared to untreated cells ( Figure 2 c and 2d). The reduction of senescence in late passage BJ cells was accompanied by a decrease in the expression of senescence markers p21 and TGFβ ( Figure 2 e). Taken together, these data indicate that krill oil inhibits senescence in both C. elegans and human fibroblasts.

[0253] Krill Oil Improves Mitochondrial Health

[0254] Oxidative stress is a fundamental feature of aging and a causative factor in Parkinson's disease 1,16 Reactive oxygen species (ROS) generated as a result of mitochondrial metabolic activity are an important source of oxidative stress in brain cells. ROS can cause oxidative DNA damage in both nuclear and mitochondrial DNA. 34 The oxidation of guanine to 8-oxoG is a biomarker of nucleic acid oxidation and, as previously demonstrated 16 PD animals accumulate 8-oxoG ( Figure 3 a and 3b). Since DHA and astaxanthin are components of krill oil with antioxidant properties, we were interested in understanding how 8-oxoG levels were affected. Notably, day 6 PD animals supplemented with krill oil showed a significant six-fold decrease in 8-oxoG levels compared to untreated animals, indicating attenuated oxidative stress.

[0255] Since mitochondrial dysfunction is a major source of oxidative stress, we tested whether krill oil could prevent age-related decline in mitochondrial function by monitoring oxygen consumption rate (OCR). 20Krill oil suppressed the age-related decline in OCR in PD animals, with the decline in OCR only evident in 6-day-old animals, compared to untreated animals where the decline in OCR was evident as early as day 3 ( Figure 3 c). This cannot be attributed to an increase in mitochondrial copy number, which remained unchanged in response to krill oil in both wild-type and PD 6-day-old animals (data not shown). This clearly indicates that krill oil promotes mitochondrial health in PD animals.

[0256] To test whether krill oil also promotes mitochondrial health in human cells, we measured mitochondrial membrane potential using tetramethylrhodamine ethyl ester (TMRE), a dye that enters mitochondria based on mitochondrial membrane potential and therefore specifically stains healthy mitochondria. The fluorescence intensity of TMRE was significantly reduced in late passage human BJ fibroblasts. Notably, these cells showed a significant 1.2-fold increase in TMRE fluorescence intensity ( Figure 3 de). Taken together, these data strongly suggest that krill oil can enhance mitochondrial health in both C. elegans and human fibroblasts.

[0257] Krill oil modulates gene expression profiles with age

[0258] We have shown above that krill oil has broad effects on many phenotypic hallmarks of aging. To obtain an unbiased view of the pathways activated by krill oil from a life course perspective, we used RNA sequencing. Data were collected from wild type and PD animals at 1, 3, and 6 days of age. Principal component analysis (PCA) plots showed that the experimental groups were well separated in terms of age (day 1, 3, and 6) and treatment ( Figure 4 a; supplementary data not shown). In PD animals, differences between treatment groups were quite low in day 1 animals, compared to 22% in day 3 and day 6 animals ( Figure 4 a). The technical quality of the RNAseq analysis was confirmed in a PCA plot by representing clusters of three biological replicates with and without krill oil treatment in wild-type and PD animals. For the wild-type strain, the PC2 differences were 17%, 10%, and 8% between each biological replicate on days 1, 3, and 6, respectively. While the condition differences PC1 were 57%, 73%, and 81% on days 1, 3, and 6, respectively (data not shown). Similarly, in PD animals, the PC2 differences were 14%, 5%, and 18% between each biological replicate on days 1, 3, and 6, respectively. As expected, the condition differences were quite large, at 64%, 87%, and 71% in day 1, day 3, and day 6 animals, respectively (data not shown).

[0259] Differential gene expression analysis showed that the number of differentially expressed genes (DEGs) increased with age in animals treated with krill oil on days 1, 3, and 6. The volcano plot showed that the number of upregulated DEGs increased with age, from 356 genes on day 1, 965 genes on day 3, to 1939 genes on day 6. Interestingly, day 6 PD animals showed a greater number of upregulated DEGs compared to the age-matched wild-type population ( Figure 4 b). The largest number of down-regulated genes was found on day 3 ( Figure 4 c). Thus, krill oil has a greater effect on gene expression in older animals than in younger animals. In summary, the study found that krill oil induces temporal transcriptome rewiring, thereby reducing biological age estimates and therefore promoting healthy aging. Biological age estimates were calculated using the BiT age calculator (Meyer and Schumacher. Aging Cell 20, e13320 https: / / doiorg.ezproxy.uio.no / 10.1111 / acel.13320(2021).).

[0260] Cluster analysis

[0261] To identify groups of genes that respond similarly to krill oil, we used gene cluster analysis. DEGs with an absolute log2 fold change greater than one were included in the analysis. Multiple clusters containing genes that were temporally co-regulated in response to krill oil were identified ( Figure 5 a, Supplementary data not shown). For example, clusters 4, 6, and 7, containing 231, 47, and 61 genes, respectively, were upregulated in animals treated with krill oil from day 1 to day 6. Gene ontology (GO) enriched in C4 was associated with neural projection morphogenesis; C6 was associated with neurogenesis, stratum corneum structure, and metalloendopeptidase activity; and C7 was associated with cell differentiation, stratum corneum development, and regulation of collagen structural components ( Figure 5 a, supplementary data not shown). The upregulation of genes involved in neurogenesis and neuronal projection in PD animals treated with krill oil is of great interest given the improved survival of dopaminergic neurons ( Figure 1 a and b). In addition, genes related to stratum corneum development were associated with intact and compact stratum corneum in 9-day-old PD animals fed krill oil at the phenotypic level, compared to wrinkled stratum corneum in untreated animals, as shown by transmission electron microscopy (TEM) (data not shown). Several clusters, such as C5, C8, C9, C10, and C12, contained genes that were highly upregulated at day 3 but decreased at day 6 in krill oil-treated PD animals ( Figure 5a, supplementary data not shown), although the expression on day 6 was significantly higher than that on day 1. Cluster C8 had the largest number of genes (527), including GOs involved in cuticle structure, cytoskeletal protein binding activity, neuronal projection, presynaptic membrane, axon and synapse, MAP kinase and calcium signaling pathways, and neurotransmission of cholinergic neurons ( Figure 5 b). Again, these GOs are consistent with the preservation of DA neurons and intact stratum corneum in PD animals fed krill oil ( Figure 1 a and 1b; supplementary data not shown). MAP kinases are known to regulate oxidative stress regulators 35,36 and senescence, which corresponds to the enriched GO in cluster 8 ( Figure 5 Cluster C9 contained 194 genes involved in cell-cell, gap junction, and cytoskeletal protein binding (data not shown). Cluster C10 contained 116 genes involved in male sex determination, genital development, reproductive system development, and glycerophospholipid metabolism (data not shown). Cluster 12 represented 122 upregulated genes annotated with GO cell signaling, neuronal projection, synaptic organization, and oropharyngeal dysphagia ( Figure 5 a and 5c). Interestingly, genes in this cluster were downregulated in untreated PD animals, suggesting that krill oil can counteract age-related changes in gene expression.

[0262] The genes downregulated in response to krill oil were present in clusters C16, C22, C30, and C31 (data not shown). These clusters contained 218, 193, 41, and 73 genes, respectively. Cluster C16 contained genes regulating cell development, oxidoreductase activity, heme binding proteins, and iron ion binding proteins. UDP-glucuronosyltransferase is highly upregulated in early development and controls hormone signaling. 37 The expression of UDP-glucuronyltransferases in clusters 22 and 31 was decreased in krill oil-fed PD animals. Clusters C30 and C31 predicted that krill oil inhibits multiple biosynthetic processes, such as pantothenate biosynthesis, during development and adulthood.

[0263] To test whether these changes in gene expression indeed mitigate DA neuron degeneration in aged animals, we scored dopaminergic neuron survival after RNAi-mediated knockdown ( Figure 6 a and 6b). The neuroprotective effects of krill oil in PD strains were abolished following depletion of cnnm-3, pbo-2, and rim-1 (human homologs of CNNM3, PLCB4, and RIMS1, respectively), whereas no effects were observed in the wild-type background. This therefore supports that krill oil induces global changes in gene expression in multiple pathways that promote DA neuron survival ( Figure 6 a and 6b).

[0264] WGCNA Clustering

[0265] To further define which components of the global transcriptional reprogramming were associated with changes in phenotypic endpoints, we used WGCNA analysis. This approach first clusters genes into 'modules', each of which is color-coded. Therefore, different colors represent groups of genes with similar trajectories using the WGCNA clustering method. Modules were then associated with defined clinical parameters. We used three endpoints: survival fraction of DA neurons at all time points (day 1, day 3, and day 6); oxygen consumption rate and locomotor capacity. We also added krill oil treatment as a parameter to examine whether it was associated with the presence or absence of krill oil. This pipeline provides an unbiased analysis and treats each input with equal weight at all three time points, whether control or treated PD animals, and finds correlations between genes and clinical parameters, but not necessarily biological events, such as the presence or absence of krill oil.

[0266] The correlation values ​​between all identified modules and defined parameters are displayed as a heat map ( Figure 7 a). Correlation ranges from +1 (perfect positive correlation) to -1 (perfect negative correlation). A positive correlation means that an increase in gene expression leads to an increase in the value of that parameter. A negative correlation indicates an inverse reaction.

[0267] The green module is strongly negatively correlated with the survival rate of DA neurons ( Figure 7 a and 7b), indicating that when DA neurons have high survival, less genes are expressed in this group and that they are downregulated in the presence of krill oil. Similarly, the blue module showed a strong positive correlation with krill oil animals and motility ( Figure 7 ac), indicating that the genes in this group are highly expressed and associated with intact athletic performance in the presence of krill oil. gprofiler shows GO related to the green and blue modules ( Figure 7 b and 7c). The sky-blue modules showed strong correlation with neuronal survival, but many of them have not been functionally characterized, and gprofiler gave only three hits, which did not predict any relevant pathways. The trajectory shows that the genes in the green module peaked at day 3 in the control group, while their expression was very stable in krill ( Figure 7 b) For genes in the blue modules, trajectories are defined as those with a dramatic increase in expression in aged animals ( Figure 7 c). In conclusion, we conclude that krill oil modulates separate gene expression programs in middle-aged and aged animals. Interestingly, changes in gene expression programs in middle-aged adults also promote healthy aging by attenuating multiple hallmarks of aging, thereby significantly protecting DA neuron survival in aged animals.

[0268] Validated in human BJ cell line

[0269] To test whether the signature of gene expression changes is conserved in human cells, we measured temporal changes in expression of human orthologs of selected upregulated genes from C12 (BRSK1 and KIRREL1), C4 (PRPS1), and C8 (CNNM3, PDE3A, PLCB4, RIMS1) in krill oil-treated human fibroblasts. Human BJ cell lines were cultured for 10 passages prior to krill oil treatment. Gene expression was measured by q-RT PCR after 1, 3, and 6 days of treatment; in krill oil-treated BJ fibroblasts, CNNM3 and PLCB4 mRNA expression was significantly increased at day 1 and day 6, respectively ( Figure 8 a and 8b). RIMS1 mRNA, encoding the RIMS1 protein that regulates neurotransmitter release, was upregulated in BJ cells after 3 days of krill oil exposure, but not after 6 days, a temporal regulation also observed for the C. elegans ortholog in C8, which was upregulated in middle-aged but not in aged animals ( Figure 8 c and Figure 5 a) Thus, although cell culture cannot capture aging itself, the dynamic changes in gene expression observed in C. elegans are largely echoed in human cells.

[0270] Oxidative stress regulator-dependent protection of dopaminergic neurons in response to krill oil

[0271] Because krill oil contains compounds with antioxidant and anti-inflammatory properties, we studied factors commonly associated with promoting healthy aging. 16 The authors investigated whether the oxidative stress regulators of PD were required for DA neuron protection. Similarly, RNAi was used to downregulate the central oxidative stress regulators JNK-1, SKN-1, and LMD-3. Wild-type animals showed no significant differences in DA neuron survival at day 6 after downregulating the jnk-1, skn-1, and lmd-3 genes. In contrast, after depletion of skn-1 and lmd-3, the protective effect of DA neurons in PD animals was abolished. However, jnk-1 knockdown had no effect ( Fig. 9 a and 9b). This clearly demonstrates that krill oil-mediated DA neuroprotection is dependent on core oxidative stress regulators that are important for regulating pathways that promote healthy aging. Intact dopaminergic neurons regulate pharyngeal pumping 38 .

[0272] We observed that animals fed krill oil showed improved pharyngeal pumping in PD animals, a generally accepted indicator of healthspan in C. elegans ( Fig. 9 c) In addition, dopamine deficiency impairs chemosensory learning by affecting olfactory responses 39Consistently, PD animals fed krill oil showed significant improvements in chemotaxis-related learning abilities ( Fig. 9 d). However, wild-type animals showed no significant benefits on pharyngeal pumping or cognitive improvements in response to krill oil ( Fig. 9 c and 9d). In conclusion, we can conclude that krill oil does improve dopamine-dependent behavior and cognition in C. elegans.

[0273] Fig.10 A schematic diagram of the effects of krill oil on aging hallmarks in C. elegans is provided.

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[0322] All publications and patents mentioned in the above specification are incorporated herein by reference, as if clearly set forth herein. Those skilled in the art will appreciate that various modifications and variations of the methods and systems of the present invention may be made without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be overly limited to these specific embodiments. In fact, it is apparent to those skilled in the relevant art that various modifications may be made to the described modes of implementing the present invention, and these modifications are intended to fall within the scope of the following claims.

Claims

1. A method for improving at least one marker of aging in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a composition comprising a phospholipid having bound eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA), wherein the marker of aging is selected from the group consisting of genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication.

2. A method for improving dopaminergic neuron survival in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a composition comprising a phospholipid having bound eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).

3. The method according to claim 1, wherein: Administration of an effective amount of a composition comprising a phospholipid with bound eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA) improves clearance of α-synuclein aggregates in the subject.

4. The method according to any one of claims 1 to 3, wherein: The composition comprising phospholipids with bound EPA and / or DHA is characterized in that it comprises 5 to 80 g / 100 g, 5 to 60 g / 100 g, 5 to 40 g / 100 g, 8 to 40 g, or 10 to 40 g / 100 g EPA and / or DHA as determined by gas chromatography fatty acid methyl ester (GC-FAME) analysis.

5. The method according to any one of claims 1 to 3, wherein: The composition comprising phospholipids with bound EPA and / or DHA is characterized in comprising 10 to 70%, 10 to 50% or 10 to 40% EPA and / or DHA w / w, wherein w / w is determined by measuring the total weight of EPA and / or DHA residues in the composition and dividing the total weight of EPA and / or DHA residues by the total weight of the composition.

6. The method according to any one of claims 1 to 5, wherein: The composition comprising phospholipids with bound EPA and / or DHA is characterized by having a ratio of EPA to DHA of 1:1 to 5:1, and preferably 1:1 to 3:

1.

7. The method according to any one of claims 1 to 5, wherein: The composition comprising phospholipids with bound EPA and / or DHA is characterized by having a ratio of DHA to EPA of 1:1 to 5:1, and preferably of 1:1 to 3:

1.

8. The method according to any one of claims 1 to 5, wherein: The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a total phospholipid content of 10 to 90% w / w, 10 to 80% w / w, 20 to 80% w / w, or 20 to 60% w / w, wherein w / w is determined by measuring the total weight of phospholipids in the composition by 31P-NMR and dividing the total weight of the phospholipids by the total weight of the composition.

9. The method according to any one of claims 1 to 8, wherein: The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a choline content of 2 to 20% w / w, 2 to 15% w / w, or 2 to 10% w / w, wherein w / w is determined by measuring the total weight of choline in the composition by 31P-NMR and dividing the total weight of the phospholipids by the total weight of the composition.

10. The method according to any one of claims 1 to 9, wherein: The composition comprising phospholipids with bound EPA and / or DHA may also be characterized as comprising astaxanthin esters in an amount of 50 to 1000 mg / kg, 50 to 500 mg / kg, or 50 to 300 mg / kg, or 100 to 300 mg / kg.

11. The method according to any one of claims 1 to 10, wherein: The composition comprising phospholipids with bound EPA and / or DHA is also characterized in having a TMAO content expressed in mgN / 100g of 1 to 30, 1 to 20 or 1 to 10 mgN / 100g.

12. The method according to any one of claims 1 to 11, wherein: The composition comprising phospholipids with bound EPA and / or DHA is further characterized by a peroxide value of less than 10, 5 or 2.

13. The method according to any one of claims 1 to 12, wherein: The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having an acid number of less than 50, 40, 30 or 20.

14. The method according to any one of claims 1 to 13, wherein: The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a Na+ content of less than 1.000%, 0.500%, 0.300% or 0.200% w / w of the composition, wherein w / w is determined by measuring the total weight of Na+ in the composition and dividing the total weight of Na+ by the total weight of the composition.

15. The method according to any one of claims 1 to 14, wherein: The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a phosphatidylcholine (PC) content of 20 to 70%, 20 to 60% or 20 to 50% w / w, wherein w / w is determined by measuring the total weight of PC molecules with two fatty acids attached in the composition and dividing the total weight of PC molecules with two fatty acids attached by the total weight of the composition.

16. The method according to any one of claims 1 to 14, wherein: The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a lysophosphatidylcholine (LPC) content of 10 to 70%, 10 to 60% or 10 to 50% w / w, wherein w / w is determined by measuring the total weight of LPC molecules in the composition and dividing the total weight of LPC molecules with one fatty acid attached by the total weight of the composition.

17. The method according to any one of claims 1 to 16, wherein: The composition comprising phospholipids with bound EPA and / or DHA is prepared from natural sources.

18. The method according to claim 17, wherein: The natural source is selected from the group consisting of krill, fish or fish by-products, Daphnia, shrimp, molluscs, cephalopods and algae.

19. The method according to claim 18, wherein: The natural source is krill.

20. The method according to claim 18, wherein: The fish or fish by-product is derived from herring, salmon, anchovies or mackerel.

21. The method according to any one of claims 1 to 20, wherein: The composition comprising phospholipids with bound EPA and / or DHA is an oil.

22. The method according to any one of claims 1 to 21, wherein: The composition comprising phospholipids with bound EPA and / or DHA is encapsulated.

23. The method according to claim 22, wherein: The composition comprising phospholipids with bound EPA and / or DHA is encapsulated in a gel capsule.

24. The method according to any one of claims 1 to 23, wherein: The composition comprising phospholipids with bound EPA and / or DHA comprises antioxidants and / or flavoring agents that are not naturally present in the source of the phospholipid composition.

25. The method according to any one of claims 1 to 24, wherein: The composition comprising phospholipids with bound EPA and / or DHA is provided as a medical food.

26. The method according to any one of claims 1 to 25, wherein: The subject in need thereof has or is at risk of having a disease or condition selected from the group consisting of: age-related neurodegeneration or disorder, Alzheimer's disease, Parkinson's disease, executive function decline, age-related cognitive decline, cognitive disease, disorder or injury, and aging associated with age-related pathology selected from the group consisting of: atherosclerosis, cardiovascular dysfunction (including cardiovascular problems caused by certain genotoxic chemotherapy), tumor progression, loss of hematopoietic and skeletal muscle stem cell function, non-alcoholic fatty liver disease, pulmonary fibrosis, osteoarthritis and osteoporosis.

27. A composition comprising phospholipids with bound EPA and / or DHA for use in improving markers of aging selected from the group consisting of genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intercellular communication, or for use in improving survival of dopaminergic neurons.

28. The use according to claim 27, wherein Administration of the composition is sufficient to improve at least one hallmark of aging.

29. The use according to any one of claims 27 to 28, wherein The composition comprising phospholipids with bound EPA and / or DHA is characterized by comprising 5 to 80 g / 100 g, 5 to 60 g / 100 g, 5 to 40 g / 100 g, or 10 to 40 g / 100 g EPA and / or DHA as determined by gas chromatography fatty acid methyl ester (GC-FAME) analysis.

30. The use according to any one of claims 27 to 28, wherein The composition comprising phospholipids with bound EPA and / or DHA is characterized in comprising 10 to 70%, 10 to 50% or 10 to 40% EPA and / or DHA w / w, wherein w / w is determined by measuring the total weight of EPA and / or DHA residues in the composition and dividing the total weight of EPA and / or DHA residues by the total weight of the composition.

31. The use according to any one of claims 27 to 30, wherein The composition comprising phospholipids with bound EPA and / or DHA is characterized by having a ratio of EPA to DHA of 1:1 to 5:1, and preferably 1:1 to 3:

1.

32. The use according to any one of claims 27 to 30, wherein The composition comprising phospholipids with bound EPA and / or DHA is characterized by having a ratio of DHA to EPA of 1:1 to 5:1, and preferably of 1:1 to 3:

1.

33. The use according to any one of claims 27 to 32, wherein The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a total phospholipid content of 10 to 90% w / w, 10 to 80% w / w, 20 to 80% w / w, or 20 to 60% w / w, wherein w / w is determined by measuring the total weight of phospholipids in the composition by 31P-NMR and dividing the total weight of the phospholipids by the total weight of the composition.

34. The use according to any one of claims 27 to 33, wherein The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a choline content of 2 to 20% w / w, 2 to 15% w / w, or 2 to 10% w / w, wherein w / w is determined by measuring the total weight of choline in the composition by 31P-NMR and dividing the total weight of the phospholipids by the total weight of the composition.

35. The use according to any one of claims 27 to 34, wherein The composition comprising phospholipids with bound EPA and / or DHA may also be characterized as comprising astaxanthin esters in an amount of 50 to 1000 mg / kg, 50 to 500 mg / kg, or 50 to 300 mg / kg, or 100 to 300 mg / kg.

36. The use according to any one of claims 27 to 35, wherein The composition comprising phospholipids with bound EPA and / or DHA is also characterized in having a TMAO content expressed in mgN / 100g of 1 to 30, 1 to 20 or 1 to 10 mgN / 100g.

37. The use according to any one of claims 27 to 36, wherein The composition comprising phospholipids with bound EPA and / or DHA is further characterized by a peroxide value of less than 10, 5, or 2.

38. The use according to any one of claims 27 to 37, wherein The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having an acid number of less than 50, 40, 30 or 20.

39. The use according to any one of claims 27 to 38, wherein The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a Na+ content of less than 1.000%, 0.500%, 0.300% or 0.200% w / w of the composition, wherein w / w is determined by measuring the total weight of Na+ in the composition and dividing the total weight of Na+ by the total weight of the composition.

40. The use according to any one of claims 27 to 39, wherein The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a phosphatidylcholine (PC) content of 20 to 70%, 20 to 60% or 20 to 50% w / w, wherein w / w is determined by measuring the total weight of PC molecules with two fatty acids attached in the composition and dividing the total weight of PC molecules with two fatty acids attached by the total weight of the composition.

41. The use according to any one of claims 27 to 39, wherein The composition comprising phospholipids with bound EPA and / or DHA is further characterized as having a lysophosphatidylcholine (LPC) content of 10 to 70%, 10 to 60% or 10 to 50% w / w, wherein w / w is determined by measuring the total weight of LPC molecules with one fatty acid attached in the composition and dividing the total weight of LPC molecules with one fatty acid attached by the total weight of the composition.

42. The use according to any one of claims 27 to 41, wherein The composition comprising phospholipids with bound EPA and / or DHA is prepared from natural sources.

43. The use according to claim 42, wherein The natural source is selected from the group consisting of krill, fish or fish by-products, Daphnia, shrimp, molluscs, cephalopods and algae.

44. The use according to claim 43, wherein The natural source is krill.

45. The use according to claim 43, wherein The fish or fish by-product is derived from herring, salmon, anchovies or mackerel.

46. ​​The use according to any one of claims 27 to 45, wherein The composition comprising phospholipids with bound EPA and / or DHA is an oil.

47. The use according to any one of claims 27 to 46, wherein The composition comprising phospholipids with bound EPA and / or DHA is encapsulated.

48. The use according to claim 47, wherein The composition comprising phospholipids with bound EPA and / or DHA is encapsulated in a gel capsule.

49. The use according to any one of claims 27 to 48, wherein The composition comprising phospholipids with bound EPA and / or DHA comprises antioxidants and / or flavoring agents that are not naturally present in the source of the phospholipid composition.

50. The use according to any one of claims 27 to 49, wherein The composition comprising phospholipids with bound EPA and / or DHA is provided as a medical food.

51. The use according to any one of claims 27 to 50, wherein The composition is administered to a subject having or at risk of having a disease or condition selected from the group consisting of: age-related neurodegeneration or disorder, Alzheimer's disease, Parkinson's disease, decline in executive function, age-related cognitive decline, cognitive disease, disorder or injury, and senescence associated with age-related pathology selected from the group consisting of: atherosclerosis, cardiovascular dysfunction (including cardiovascular problems caused by certain genotoxic chemotherapy), tumor progression, loss of hematopoietic and skeletal muscle stem cell function, non-alcoholic fatty liver disease, pulmonary fibrosis, osteoarthritis and osteoporosis.

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