Populations of small extracellular vesicles for use in the treatment or prevention of ischemia

Abstract

The present invention provides a population of small extracellular vesicles (sEVs) comprising at least one polynucleotide encoding a dominant negative AMP-activated protein kinase alpha 2 (AMPKα2-DN) mutant protein. Uses in medicine, particularly in the treatment or prevention of ischemia are also provided.

Description

Cerebral ischemia produces a lack of oxygen (hypoxia) and a decrease in nutrients, in addition to the release of neurotransmitters such as glutamate at toxic concentrations. This lack of nutrients and oxygen produces a negative energy balance (increased AMP:ATP ratio), leading to a quick activation of AMPK (in a matter of minutes / hours after the ischemic insult), mitochondrial diffusion, and cell death. However, despite this evidence, the exact role and regulation of AMPK are unclear. Some studies based in AMPKal show its neuroprotective effect. However, this effect can vary significantly based on factors such as the type of tissue, the severity of stress, and the metabolic state of the cells involved. Notably, these reports primarily investigated the AMPKal subunit rather than the AMPKa2 subunit, which is highly expressed in cortical neurons. Because of this, the authors of the present invention decided to investigate the impact of AMPK inhibition by nicotine and by sEVs harbouring AMPKa2-DN isoforms in the context of ischemic stroke. First, the inventors tested the role of nicotine after cerebral ischemia. Fig. 2 shows that nicotine treatment reduces brain ischemic injury and improves neurological recovery after cerebral ischemia. Fig. 3 to 6 revealed that this effect of nicotine is mediated by inhibition of AMPK signalling, indicating that the activation of AMPK reverses the beneficious action of nicotine on cerebral ischemia, and consequently that AMPK is mediating the neuroprotective effect of nicotine on ischemic brain injured areas. Next, the inventors generated a population of small extracellular vesicles (sEVs) comprising a gene encoding for the AMPKa2-DN mutant protein of SEQ ID NO: 2. The expression of said AMPKa2-DN mutant protein was driven the CMV promoter of SEQ ID NO: 4. The sEVs also expressed the fusion protein Lamp2b-RVG of SEQ ID NO: 5 to provide the sEVs with tropisms to cerebral tissues. Fig. 7-20 demonstrate that CMV-AMPKa2-DN EVs treatment improves cognitive impairment promoted by cerebral ischemia, indicating that systemic treatment with CMV-AMPKa2-DN EVs inhibits the activation of AMPK in areas of ischemic brain tissue, reducing the affected area, improving the underlying cognitive impairment after ischemic, and reducing apoptotic processes derived from the injury. In view of the above results, the present invention provides a AMPKa2-DN mutant protein. Said AMPKa2-DN mutant protein is defined in the first aspect of the invention and preferably comprises or consists of SEQ ID NO: 2. Said AMPKa2-DN mutant protein is preferably used in the treatment or prevention of ischemia or ischemia-reperfusion, more preferably ischemic stroke or cerebral ischemia. Although the AMPKa2-DN mutant protein defined herein, particularly in the first aspect of the invention, can be vehiculized in any carrier, it is preferred that the gene that encodes for it is comprised in sEVs, preferably exosomes, as will be also defined in the first aspect of the invention. Thus, in a first aspect, the present invention relates to a population of small extracellular vesicles (sEVs), also called herein the “sEVs of the invention”, comprising at least one polynucleotide comprising a gene encoding a dominant negative AMP-activated protein kinase alpha 2 (AMPKa2-DN) mutant protein. Preferably, the sEVs are exosomes, also called herein the “exosomes of the invention”. As used herein, “AMPKa2” refers to the isoform 2 of the subunit a of AMPK protein, preferably the AMPK protein from mus musculus. The term “dominant negative AMPKa2 mutant protein” (AMPKa2-DN) refers to an inactive isoform of the AMPKa2. Preferably, the inactive isoform of AMPKa2 is a AMPKa2 protein modified in its active site with at least the point mutation D157A, wherein the amino acid position (157) is expressed with respect to the wild-type AMPKa2 sequence, preferably the wild-type mus musculus AMPKa2 sequence of SEQ ID NO: 1 or SEQ ID NO: 8. However, although the mus musculus sequence is the preferred sequence, the sequence of the AMPKa2-DN protein can be the sequenceof a homologous sequence of the Mus musculus AMPKa2 protein corresponding to the species where the treatment is applied, such as human homologous of AMPKa2 protein, or humanised or a codon-optimized AMPKa2 protein for treatment in humans. Please note that here and thorough the whole document, the positions or locations of the amino acid residues of a protein or a polypeptide sequence are numbered sequentially starting from the first amino acid residue which would then be located at position 1. For example, a protein of 137 amino acids will have those residues numbered 1 (first amino acid residue) until 137 (the last amino acid residue). Preferably, the first position or position number 1 corresponds to the first amino acid residue located at the 5-prime (5') end of the polypeptide chain that has a nitrogen atom or a free amino group. Thus, the numbering preferably starts from the first amino acid residue at the N terminal or 5’ end of the protein or polypeptide and ends at the 3’ end or C terminal end of the protein or polypeptide. Preferably, the positions of the amino acid residues are numbered using the amino acid sequence of the translated mature protein. It is also noted that the amino acid numbering, and therefore the number of the amino acid substitution that results in AMPKa2-DN mutant protein as defined herein, may change within AMPKa2 proteins from different species. A single nucleotide substitution that results in a codon change encoding a different amino acid is called “missense point mutation” (called “mutation” or “substitution” thereafter). In the context of the present invention, missense point mutations or substitutions are noted following the structure “A1-number-A2”, wherein “A1” is the amino acid residue that is replaced, “number” is the amino acid position within the sequence where A1 is, and “A2” is the amino acid that is used instead of “A1”. For example, a substitution D157A in SEQ ID NO:1, as used herein, means that the amino acid aspartic acid in position number 157 in SEQ ID NO: 1 is substituted by an alanine. In an embodiment, the AMPKa2-DN mutant protein encoded by the at least one polynucleotide comprised in the sEVs or exosomes comprises, consists, or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 1, with the proviso that the amino acid residue in position number 157 in SEQ ID NO: 1, which corresponds with an aspartic acid, is substituted by an amino acid residue that is not aspartic acid. Preferably, the aspartic acid residue in position number 157 in SEQ ID NO: 1 is substituted by an alanine (mutation D157A). Preferably, the AMPKa2-DN mutant protein comprises a sequence that is at least 85%, 90%, preferably 95% identical to SEQ ID NO: 1, with the proviso that the amino acid residue in position number 157 in SEQ ID NO: 1, which corresponds with an aspartic acid, is substituted by an amino acid residue that is not aspartic acid, preferably alanine. Preferably, the AMPKa2-DN mutant protein is SEQ ID NO: 1, with the proviso that the amino acid residue in position number 157 in SEQ ID NO: 1, which corresponds with an aspartic acid, is substituted by an amino acid residue that is not aspartic acid, preferably alanine. In an embodiment, the AMPKa2-DN mutant protein encoded by the at least one polynucleotide comprised in the sEVs or exosomes comprises, consists, or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 2, with the proviso that the amino acid residue in position number 169 in SEQ ID NO: 2 is not an aspartic acid. In an embodiment, the AMPKa2-DN mutant protein encoded by the at least one polynucleotide comprised in the sEVs or exosomes comprises, consists, or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 2, with the proviso that the amino acid residue in position number 169 in SEQ ID NO: 2 corresponds to amino acid alanine (169A). In an embodiment, the AMPKa2-DN mutant protein encoded by the at least one polynucleotide comprised in the sEVs or exosomes comprises, consists, or consists essentially of an amino acid sequence with at least 85%, 90%, preferably 95%, sequence identity with SEQ ID NO: 2, with the proviso that the amino acid residue in position number 169 in SEQ ID NO: 2 is not an aspartic acid. Preferably, the AMPKa2-DN mutant protein comprises or consists of SEQ ID NO: 2. In an embodiment, the gene encoding for the AMPKa2-DN mutant protein comprises, consists, or consists essentially of polynucleotide sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 3. Preferably, the gene encoding for the AMPKa2-DN mutant protein comprises or consists of SEQ ID NO: 3. In an embodiment, the gene encoding for the AMPKa2-DN protein is under the control of a promoter, that is also comprised in the polynucleotide. A coding sequence and a gene expression control sequence or promoter are said to be operably linked when they are linked in such a way as to place the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence. For example, the gene encoding the AMPKa2-DN may be operably linked to the CMV promoter so that the expression levels of the AMPKa2-DN are regulated by the CMV promoter. In an embodiment, the promoter is a ubiquitous or a tissue specific promoter. As used herein, a “tissue specific” promoter is a promoter that is only active in specific types of cells / tissues, preferably specific mammal tissues, most preferably specific human tissues. As used herein, a ubiquitous promoter is a promoter that is active in many or in any different tissue(s), preferably mammal tissues, most preferably human tissues. Usually, “many” in this context may mean more than 5 or at least 6, 10, 15, 20 or in 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 different tissues. In a preferred embodiment, the promoter used to regulate the expression of the AMPKa2-DN protein is ubiquitous and / or constitutive. In a more preferred embodiment, said promoter is ubiquitous and constitutive. A “constitutive” promoter is a promoter that is active under most physiological and developmental conditions. Preferably, the promoter is selected from the list consisting of cytomegalovirus promoter (CMV), SV40, CMV, UBC, EF1A, PGK and CAGG. Preferably, the ubiquitous and constitutive promoter that drives the expression of AMPKa2-DN protein in the sEVs or exosomes of the invention is the CMV promoter. In an embodiment, the CMV promoter comprises, consists, or consists essentially of a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 4. Preferably, the CMV promoter comprises or consists of SEQ ID NO: 4. In another embodiment, the promoter used to regulate the expression of the AMPKa2-DN protein is ubiquitous and / or inducible. An “inducible” promoter is a promoter that is preferably regulated depending on physiological or developmental conditions, preferably in physiological or developmental conditions of hypoxia. In a more preferred embodiment, said promoter is ubiquitous and inducible in hypoxia conditions. Preferably, the ubiquitous and inducible promoter that drives the expression of AMPKa2-DN protein in the sEVs or exosomes of the invention, under hypoxic conditions, is heat-shock protein 70 (Hsp70) promoter, also known as Hspala or Hsp68. Hsp70 is a transcription factor, which expression was found to be increased under hypoxia. Preferably, the promoter that drives the expression of AMPKa2-DN protein in the sEVs or exosomes of the invention is the heat-shock protein 70 (Hsp70) promoter. In an embodiment, the Hsp70 promoter comprises, consists, or consists essentially of a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 9. Preferably, the Hsp70 promoter comprises or consists of SEQ ID NO: 9. Additionally, the Hypoxia-Inducible Factor 1 (HIF-1) is a crucial transcription factor involved in the cellular response to hypoxia. In normal concentrations, HIF-1 is rapidly degraded, however, in hypoxic conditions its structure is stabilized, binding to specific regions of certain promoters called hypoxia-responsive elements (HRE), such as its own or VEGF among others to increase your expression. The consensus sequence for HRE is 5'-ACGTGNNNNACGTG-3' (SEQ ID NO: 7), which can be added to the 5’ end of the promoter (e.g., CMV or Hsp70 promoter) to enhance the expression under hypoxic conditions. Preferably, the promoter that drives the expression of AMPKa2-DN protein in the sEVs or exosomes of the invention is an CMV promoter comprising the HRE sequence in its 5’ end. Preferably, the promoter that drives the expression of AMPKa2-DN protein in the sEVs or exosomes of the invention is an Hsp70 promoter comprising the HRE sequence in its 5’ end. Hence, the population of sEVs or exosomes provided herein comprise or are loaded with at least one polynucleotide comprising a gene encoding an AMPKa2-DN mutant protein. Preferably, each of the sEVs of the population is loaded with at least one polynucleotide comprising a gene encoding an AMPKa2-DN mutant protein, wherein said gene may be operably linked and under the control of a CMV promoter. Preferably, at least one polynucleotide is DNA, more preferably a plasmid. Thus, designing a plasmid encoding for AMPKa2-DN protein under the control of CMV promoter (CMV-AMPKa2-DN plasmid) will allow the AMPKa2-DN to be expressed in most, if not all tissues. Once the sEVs or exosomes are fused with the target cells, the AMPKa2-DN mutant protein would be expressed in the target cell. The techniques used to load sEVs with various cargo include free-thaw cycles to fuse sEVs and liposomes, sonication, extrusion, permeabilization with saponin, and electroporation. Several commercial kits are available for loading nucleic acids into sEVs and the skilled person would be familiar with them. Preferably, the at least one polynucleotide, preferably a plasmid, is mainly located in the core of the sEVs or exosomes. The sEVs or exosomes of the invention may further comprise or be loaded with, preferably mainly at their outer membrane, at least a fusion protein. Said fusion protein, also named Lamp2b-RVG protein, comprises or consists of the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b). Preferably, said fusion protein comprises, consists or consists essentially of an amino acid sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 5. Preferably, the fusion protein comprises or consists of SEQ ID NO: 5. By “fusion protein” is referred herein as to a protein formed by at least two domains, wherein the at least two domains have been joined, one after the other, so that they are synthetized or translated as a single unit, and thus the two domains of the fusion protein are part of a single polypeptide. In this particular case, the domains comprised or consisting of the fusion protein are the RVG peptide and the Lamp2b protein. In an embodiment, the domains of the Lamp2b-RVG fusion protein may be linked by a linker peptide. “Linker peptide” as used herein is a short peptide sequence that is located between the domains of a fusion protein. Linker peptides are placed to provide the two domains comprised in the fusion protein with movement flexibility. In the context of the present invention, the linker peptide has at least one amino acid residue, preferably at least two consecutive amino acid residues, optionally 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 20 more amino acid residues. The linker peptide includes flexible linkers, rigid linkers, and in vivo cleavable linkers. Therefore, it is preferred that the sEVs or exosomes of the invention comprise at least one polynucleotide encoding an AMPKa2-DN protein and are produced to have in their outer membrane at least one fusion protein, said fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b). For that purpose, a plasmid comprising the Lamp2b-RVG gene can be transfected in exosome generating-cells, preferably in exosome generating-immature dendritic cells, to transiently express the Lamp2b-RVG fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, wherein, preferably, such fusion protein comprises an amino acid sequence with at least 95% sequence identity with SEQ ID NO 5. This way, when the sEVs or exosomes are produced, they will carry the Lamp2b-RVG fusion protein, preferably in their surface. Preferably, the sEVs or exosomes of the invention comprise at least one polynucleotide comprising a gene encoding an AMPKa2-DN mutant protein operably linked and under the control of a ubiquitous promoter, preferably CMV, and comprise in their outer membrane at least one fusion protein comprising the RVG peptide fused to Lamp2b. Preferably, the sEVs or exosomes of the invention comprise at least one polynucleotide comprising a gene encoding an AMPKa2-DN mutant protein operably linked and under the control of the HSp70 promoter, and comprise in their outer membrane at least one fusion protein comprising the RVG peptide fused to Lamp2b. Preferably, the sEVs or exosomes of the invention comprise at least one polynucleotide encoding AMPKa2-DN mutant protein operably linked and under the control of a tissue-specific promoter and comprise in their outer membrane at least one fusion protein comprising the RVG peptide fused to Lamp2b. In an embodiment, the polynucleotide comprised in the sEVs of the invention, preferably in the exosomes of the invention, comprises, consists, or consists essentially of a sequence with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with SEQ ID NO: 6. Preferably, the polynucleotide comprises or consists of SEQ ID NO: 6. In an embodiment, the sEVs or exosomes further comprise markers derived from the cells used to produce them (hereinafter referred to as producer cell). Preferably, the producer cells are immune cells, more preferably antigen presenting cells, and even more preferably dendritic cells. Preferably, the producer cells are mammalian cells. The producer cell may be a primary cell or an immortalized cell. In an embodiment, the producer cell is a cell with low immunogenicity, preferably an immature immune cell, preferably an immature antigen presenting cell, and most preferably an immature dendritic cell or monocyte. By “immature” is referred herein as a cell that is not activated or biologically active, or that does not present activation markers or molecules on the surface. An immature dendritic cell has a different morphological phenotype than the mature one. Immature dendritic cells have a round and smooth surface, while mature cells, such as mature dendritic cells, have a rough surface with multiple pseudopodia. Immature dendritic cells produce large quantities of exosomes devoid of T-cell activators such as MHC-II, CD80 and CD86. Thus, in an embodiment, the producer cell, preferably the immature dendritic cell, does not express activation markers, preferably T-cell activator markers or molecules such as major histocompatibility complex II (MHC-II), cluster of differentiation 80 (CD80) or cluster of differentiation 86 (CD86), or a combination thereof. In an embodiment, the producer cell is an immature immortalized cell, preferably an immature immortalized dendritic cell or monocyte, most preferably the JAWS II cell line from the American Type Culture Collection CRL-1194; ATCC; Manassas, VA, USA or a cell derived from said JAWS II cell line. In an embodiment, the producer cell is an immature immortalized cell, preferably an immature immortalized dendritic cell or monocyte, most preferably the JAWS II cell line from the American Type Culture Collection CRL-3612; ATCC; Manassas, VA, USA or a cell derived from said JAWS II cell line. JAWSII is a cell with a monocyte morphology that was isolated from the bone marrow of a mouse. This product is an ATCC manufactured and accessioned progeny of ATCC CRL-11904 cited in US Pat. No. 5,648,219. Also, preferably, the producer cell is a genetically modified cell that expresses the RVG-Lamp2b fusion protein, preferably in its outer membrane. In an embodiment, the producer cell is an immature antigen presenting cell characterized by having a statistically significant reduced expression of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of at least one T-cell activator molecule in comparison to the expression of said T-cell activator molecule in a mature antigen presenting cell. The percentage of reduction of said T-cell activator molecule can be measured by techniques known in the art, e.g. by flow cytometry or RT-PCR. The producer cells may be grown in a conditioned media, and the sEVs or the exosomes are produced and released to said media. In the context of the present invention, the term “conditioned media” is understood as the supernatant of cell cultures. Since the population of sEVs or exosomes is released into the extracellular media when the intracellular multivesicular bodies fuse with the plasma membrane of the producer cell, the sEVs or exosome outer membrane would be similar to the one of the producing cell. In particular embodiments, the producer cell is a mammalian cell, e.g., a human cell. In particular embodiments, the producer cell is an immature dendritic cell. Thus, in an embodiment, the population of sEVs or exosomes are positive for cellular markers, preferably cellular immature dendritic cell (iDC) markers. Further preferred, the population of sEVs or exosomes comprise one or more of the specific exosome markers selected from the groups consisting of ALIX, TSG101, CD9 or CD81, or any combination thereof. In another embodiment, the sEVs or exosomes are characterized by lacking the MHC-II, CD80, CD86 or GRP94 markers, or any combination thereof. In an embodiment, the sEVs or exosomes have an immature phenotype, so that they are low immunogenic. In some embodiments, the sEVs or exosomes are produced from autologous producer cells of the subject to be treated or were obtained from cells that were isolated from the subject to be treated. In other embodiments, the sEVs or exosomes are produced from allogeneic producer cells to the subject to be treated or were obtained from cells that were isolated from a donor other than the subject to be treated. In particular embodiments, the producer cell is an autologous mammalian cell, eg., an autologous human cell. In particular embodiments, the producer cell is an autologous immature dendritic cell. Preferably, the sEVs or exosomes are isolated. In the context of the present invention, the term "isolated" indicates that the sEVs or exosomes or population thereof are not within the environment or cell culture where they were produced. The term isolated also encompasses a population of sEVs or exosomes that have been removed from the environment or ell culture, e.g., from the supernatant or conditioning media, from which they originated. In an embodiment, the population of sEVs or exosomes is a substantially pure population, and the sEVs or exosomes or population thereof has been substantially separated from surrounding environment or cell culture. In some embodiments, the population is substantially pure or enriched in sEVs or exosomes and comprises at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% of sEVs or exosomes. In an embodiment, the population is pure in sEVs or exosomes. They are released by many cell types as a means of communicating with other cells and thus cell content may be potentially removed from the population. The cargo of extracellular vesicles includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. Preferably, the population according to the first aspect or any of its embodiments comprises a substantially pure population of immature dendritic cells (iDC)-derived sEVs or exosomes, wherein the iDC-derived sEVs or exosomes, preferably each of the iDC-derived sEVs or exosomes, comprise at least one polynucleotide comprising a gene encoding an AMPKa2-DN mutant protein operably linked and under the control of a CMV promoter, and wherein the iDC-derived sEVs or iDC-derived exosomes have in their outer membrane at least one fusion protein comprising the RVG peptide fused to Lamp2b. In an embodiment, the sEVs or exosomes are spherical or round-shaped. Further, the sEVs or exosomes may have a size of greater than 2 nm. The sEVs or exosomes may have a size of greater than 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm or 160 nm. The exosome may have a size of substantially 160 nm or greater. The sEVs or exosomes may have a range of size distribution, such as between 30 nm to 50 nm, 30 nm to 100 nm, preferably 30 nm to 150 nm or 30 nm to 200 nm. The size distribution may be determined by various means. In principle, the size may be determined by size fractionation and filtration through a membrane with the relevant size cut-off. The sEVs or exosomes size may then be determined by tracking segregation of component proteins with SDS-PAGE or by a biological assay. The size may also be determined by electron microscopy or by Nanoparticle tracking analysis (NTA). As discussed above, the results provide evidence that the population of sEVs or exosomes described herein is able to decrease or reduce the activation of AMPKa2 in the target cell / tissue. In view of this, in a preferred embodiment, the population of sEVs or exosomes according to the first aspect or any of its embodiments is capable, preferably when administered systemically, of significantly decreasing the activation levels of AMPKa2 in the target cells in comparison to the activation levels of AMPKa2 in untreated cells or reference cells, preferably neurons. Thus, as a result of AMPKa2 inhibition, the exogenous AMPKa2-DN isoform lacks kinase activity and therefore the AMPK heterotrimer (alpha, beta, gamma; preferably alpha2 in this case) cannot be properly regulated and exert its phosphorylation effect on downstream targets. By “AMPKa2 inhibition” is referred herein as a decrease in AMPKa2 activation levels. Preferably, the sEVs or exosomes of the invention are capable of reducing, preferably statistically significant reducing, the activation levels of AMPKa2 in the target cell, preferably in neurons. By “statistically significant” or “significant” is referred herein as the determination by an analyst that the results in the data are not explainable by chance alone. Statistical hypothesis testing is the method by which the skilled person makes this determination. This test provides a p-value, which is the probability of observing results as extreme as those in the data, assuming the results are truly due to chance alone. A p-value of 0.1 or lower (preferably 0.05, 0.01, 0.001 or lower) is considered herein to be statistically significant. For example, the reduction in the activation levels of AMPKa2 protein is a statistically significant reduction or decrease when a statistical test is performed to compare sEVs- or exosomes-treated target cells with sEVs- or exosomes-untreated target or reference cells, and wherein the resulting p-value of said statistical test is of 0.1 or lower, preferably 0.05, 0.01, 0.001 or lower. By “decrease” or “reduction” is referred herein as a reduction in at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in comparison to a reference cell or value or reference tissue. By “reference cell or tissue” is referred herein to a cell or tissue not treated with the sEVs of exosomes described herein. Preferably, said cell or tissue is a mammalian cell e.g., a human cell, preferably a neuron or a cell or tissue from the nervous system. The reference cell may also be a single cell or a population of cells derived from the same subject or from a population of subjects. By “reference value” is referred herein to the average known and correct measurement of a parameter, for instance activity levels of AMPKa2 protein in untreated cells or in reference cells. Reference values are an average of repeated measurements from more accurate measuring equipment. A skilled artisan would know how to obtain said reference value. The levels of AMPKa2 enzymatic activity may be expressed or measured by an “absolute” quantification or by a “relative” or comparative quantification and they can be calculated by a skilled person using suitable techniques known in the art. For example western blot, Immunochestry, Immunofluorescence, qPCR, etc. The population may be part of a composition comprising emulsions containing sEVs or exosomes. Such emulsions may comprise oil-in-water emulsions. Thus, the first aspect of the invention also includes compositions comprising the sEVs or exosomes of the invention, preferably together with one or more pharmaceutically acceptable carriers. The compositions, preferably emulsions, may be made by means known in the art. They may be made from freshly prepared sEVs or exosomes, or lyophilised sEVs or exosomes or sEVs or exosomes stored in oil. Emulsions are heterogeneous systems composed of at least two immiscible liquids, for example water and oil, one of which is usually uniformly dispersed as fine droplets throughout the other liquid phase by a mechanical agitation process. The composition may further comprise at least one emulsifier. An emulsifier (also known as an “emulgent”) is a substance that stabilizes an emulsion by increasing its kinetic stability. The carriers are biologically acceptable without eliciting an adverse reaction (e.g. immune response) when administered to the host. Suitable pharmaceutically acceptable carriers are well known in the art and vary with the desired form and mode of administration of the pharmaceutical formulation. For example, they may include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, lubricants and the like. Typically, the carrier is a solid, a liquid or a vaporizable carrier, or a combination thereof. Each carrier should be “acceptable” in the sense of being compatible with the other ingredients in the formulation and not injurious to the subject. The sEVs or exosomes may be conveniently stored as a suspension or dispersion in oil. For this purpose, freshly prepared sEVs or exosomes or lyophilised exosomes may be suspended or dispersed in oil. The oil may comprise any suitable oil, such as a plant oil. The sEVs or exosomes may be suspended or dispersed in for example olive oil, palm oil, soy oil or coconut oil. The sEVs or exosomes may be suspended or dispersed in the oil in any suitable proportion. The sEVs or exosomes suspension or dispersion in oil may be stored, for example at room temperature, prior to use. The sEVs or exosomes suspension or dispersion in oil may be such that the exosome exhibits at least one biological activity of an sEVs or exosomes following a period of storage or after a period of storage. The biological activity may comprise a therapeutic activity, such as correcting, reducing, ameliorating, treating, preventing ischemia, as will be described below. The biological activity may be derived from the presence of a dominant negative AMPKa2 protein (AMPKa2-DN), which in turn decreases AMPK activity and pACC phosphorylation. The biological activity may be derived from the reduced levels of phosphorylation of acetyl-CoA carboxylase alpha (pACCa). Method for producing the population or the composition or pharmaceutical composition according to the present invention are known in the art, and they optionally comprise the step of specifically enriching for substantially pure populations of sEVs or exosomes. For this, generally any suitable method for purifying and / or enriching can be used, such as methods comprising magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA), and / or chromatography. In an embodiment, the composition is a pharmaceutical composition. The pharmaceutical composition is preferably enriched with the population of sEVs exosomes having a size of between about 30 to 200 nm, preferably 30 to 150 nm in size. The sEVs or exosomes amount can be measured by protein amount, for example, by using a Bradford assay (BioRad) or a BCA protein assay kit (Pierce). Yet, the optimal dose will be selected according to the administration route, treatment regime and / or administration schedule, having regard to the existing toxicity and effectiveness data. Alternatively, the composition comprises the AMPKa2-DN defined in the first aspect of the invention in solution, or vehiculized in another carrier different that the sEVs defined herein. For example, in an embodiment, the composition comprises a polynucleotide encoding for the AMPKa2-DN defined herein, under the control of a promoter as defined herein, but vehiculized in a different vector other than the sEVs, such as a viral vector. In a second aspect, the present invention provides the use of the population according to the first aspect or any of its embodiments, including the composition or pharmaceutical composition, in therapy or for the generation of a medicament. The use of the second aspect also applies to the embodiments where the AMPKa2-DN defined in the first aspect of the invention is provided in a different vector other than the sEVs, such as in a viral vector. Thus, this aspect also refers to uses of the AMPKa2-DN defined in the first aspect of the invention, wherein the AMPKa2-DN is provided, either as a protein or as a polynucleotide vector encoding thereof, but without sEVs. Preferably, the use is a therapeutic and / or prophylactic treatment of ischemia or ischemiareperfusion in a subject in need thereof. The term “treatment” as used herein refers to the medical care given to a patient for a disease or injury. This term includes curing the disease but also ameliorating, mitigating, reducing or preventing the symptoms of said disease. In an embodiment, the population or composition according to the first aspect or any of its embodiments is used in the therapeutic treatment of ischemia or ischemia-reperfusion in a subject in need thereof. The term “therapeutic treatment” as used herein refers to bringing a body from a pathological state or disease back to its normal, healthy state. The term “therapeutic treatment” also refers to the mitigation, amelioration, or reduction of the harmful effect of ischemia in a subject, or in some manner reducing the symptoms associated with such effects. In an embodiment, the population or composition according to the first aspect or any of its embodiments is used in the prophylactic treatment of ischemia or ischemia-reperfusion in a subject in need thereof. The term “prophylactic treatment” as used herein refers to preventing a pathological stage derived from ischemia. In an embodiment, the population or composition according to the first aspect or any of its embodiments is used in correcting, reducing, ameliorating, treating, and / or preventing ischemia. The term “ischemia” refers to a pathological stage in which there is a period of lack of oxygen in the target cell or tissue. The period of lack of oxygen may be a few seconds or even minutes. The period of lack of oxygen may be characterized by being a period of hypoxia or anoxia. As used herein, the term “hypoxia" refers to the presence of less than normal amounts of O2 than those present in a vertebrate or in its blood, cells or tissue. As used herein, the term “anoxia" refers to the lack of O2. The ischemia may be caused by an inadequate blood flow. The ischemia may be from any cause including acute coronary syndromes (such as myocardial infarction, coronary revascularization, coronary bypass surgery and coronary angioplastyistent placement); stroke, renal, retinal, mesenteric or limb ischemia due to vascular occlusion; organ transplant surgery (for maintaining viability and function of the transplanted organ); ischemia associated with vascular surgery (including hypothermic arrest and vascular cross-clamping); ischemic reperfusion injury (such as coronary or cerebral reperfusion injury); cerebrovascular accidents (such as a thromboembolic or hemorrhagic stroke) that can lead to ischemia in the brain; operative ischemia; traumatic hemorrhage (for example, a hypovolemic stroke that can lead to CNS hypoxia or anoxia), or resuscitation injury. Preferably, the ischemia is caused by myocardial infarction or is cerebrovascular ischemia. Preferably, the ischemia treated or prevented, preferably treated, is ischemic stroke. The used described herein preferably comprises administering an effective amount of a substantially pure population of the sEVs or exosomes of the invention, together with one or more pharmaceutically acceptable carriers. An “effective amount” is the amount sufficient to reduce, ameliorate, treat and / or prevent ischemia. The effective amount will vary depending upon several factors including the age and weight of the subject to be treated, how advanced the disease state is, the general health of the patient, the severity of the symptoms. As used herein, the phrase "subject in need thereof” includes subjects, such as mammalian subjects, preferably humans, that would benefit from administration of the population of sEVs or exosomes, or the compositions comprising them, disclosed herein. The subject may be a female, preferably human female. Preferably, the subject is a male, more preferably a human male. More preferably, the subject in need of a treatment for ischemia. The terms “individual”, “patient” or “subject” are used interchangeably in the present application and are not meant to be limiting in any way. The “individual”, “patient” or “subject” can be of any age, sex and physical condition. The administration route can be systemic or local. By “systemic administration route” is referred herein the administration of the composition into the circulatory system, directly or indirectly. Systemic administration routes comprise parenteral route such as intravascular, intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, or rectal. A preferred systemic route of administration is intravascular, which is herein understood as the administration within a vessel or vessels and typically includes intravenous or intraarterial administration. The use provided herein may be short or long term defined as a period of time exceeding three months. In a preferred embodiment, the use of the population of sEVs of the invention, preferably exosomes of the invention, is in combination with nicotine, wherein the combination is herein understood as the simultaneous or sequential administration, in any order, of a) the sEVs of the invention, and b) nicotine. Preferably, the use in combination comprises the simultaneous or sequential administration, in any order, of a) the exosomes of the invention, and b) nicotine. Preferably, the use in combination comprises the simultaneous or sequential administration, in any order, of a) the exosomes of the invention comprising a gene encoding for a protein as set forth in SEQ ID NO: 2, and b) nicotine. In a third aspect, the present invention provides a kit comprising the sEvs or the exosomes as defined in the first aspect or any of its embodiments, or a composition, preferably a pharmaceutical composition, comprising them. Preferably, the kit further comprises nicotine for the combined administration as defined above. In some embodiments, the kit comprises AMPKa2-DN as defined in the first aspect of the invention, but wherein said AMPKa2-DN is vehiculized in a different vector that is not sEVs, but for example, a viral vector. The following items are also comprised in the invention: 1. A population of small extracellular vesicles (sEVs) comprising at least one polynucleotide comprising a gene encoding a dominant negative AMP-activated protein kinase alpha 2 (AMPKa2-DN) mutant protein. 2. The population of small extracellular vesicles according to item 1, wherein the amino acid sequence of the AMPKa2-DN mutant protein comprises SEQ ID NO: 1, or a sequence with at least 85% sequence identity to SEQ ID NO: 1, with the proviso that the amino acid residue in position number 157 in SEQ ID NO: 1 is not an aspartic acid. 3. The population of small extracellular vesicles according to item 2, wherein the amino acid residue in position number 157 in SEQ ID NO: 1 is an alanine. 4. The population of small extracellular vesicles according to item 1, wherein the amino acid sequence of the AMPKa2-DN mutant protein comprises SEQ ID NO: 2, or a sequence with at least 85% sequence identity to SEQ ID NO: 2, with the proviso that the amino acid residue in position number 169 in SEQ ID NO: 2 is not an aspartic acid. 5. The population of small extracellular vesicles according to item 4, wherein the amino acid residue in position number 169 in SEQ ID NO: 2 is an alanine. 6. The population of small extracellular vesicles according to any one of items 1 to 5, wherein the gene encoding the AMPKa2-DN mutant protein is operably linked and under the control of a ubiquitous and constitutive promoter, preferably a cytomegalovirus promoter. 7. The population of small extracellular vesicles according to any one of items 1 to 6, the sEVs express in their outer membrane at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b. 8. The population of small extracellular vesicles according to item 7, wherein the fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprises SEQ ID NO: 5, or a sequence with at least 85% sequence identity to SEQ ID NO: 5. 9. The population according to any of items 1 to 8, wherein the small extracellular vesicles have a size distribution of between 30 and 150 nm. 10. The population according to any of items 1 to 9, wherein the small extracellular vesicles are exosomes. 11. A population of small extracellular vesicles as defined in any of items 1 to 10, for use in therapy. 12. A population of small extracellular vesicles as defined in any of items 1 to 10, for use in the treatment or prevention of ischemia or ischemia-reperfusion. 13. The population of small extracellular vesicles for use according to item 12, wherein the ischemia is a ischemic stroke or cerebral ischemia. 14. The population of small extracellular vesicles for use according to any one of items 11 to 13, wherein the administration of said small extracellular vesicles is via systemic route. 15. The population of small extracellular vesicles for use according to any one of items 11 to 14, wherein the use of the small extracellular vesicles, preferably exosomes, is in combination with nicotine, wherein the combination is herein understood as the simultaneous or sequential administration, in any order, of a) the population of small extracellular vesicles, and b) nicotine. 16. A dominant negative AMP-activated protein kinase alpha 2 (AMPKa2-DN) mutant protein, for use in the treatment or prevention of ischemia or ischemia-reperfusion. 17. The AMPKa2-DN for use according to item 16, with an amino acid sequence comprising SEQ ID NO: 1, or a sequence with at least 85% sequence identity to SEQ ID NO: 1, with the proviso that the amino acid residue in position number 157 in SEQ ID NO: 1 is not an aspartic acid. 18. The AMPKa2-DN for use according to item 17, wherein the amino acid residue in position number 157 in SEQ ID NO: 1 is substituted with an alanine. 19. The AMPKa2-DN for use according to item 16, with an amino acid sequence comprising SEQ ID NO: 1, or a sequence with at least 85% sequence identity to SEQ ID NO: 2, with the proviso that the amino acid residue in position number 169 in SEQ ID NO: 2 is not an aspartic acid. 20. The AMPKa2-DN for use according to item 19, wherein the amino acid residue in position number 169 in SEQ ID NO: 2 is substituted with an alanine. 21. The AMPKa2-DN for use according to any of items 16-20, comprising or consisting of SEQ ID NO: 2. 22. A vector encoding or comprising the AMPKa2-DN as defined in any one of items 16 to 21 or in the first aspect, for use in the treatment or prevention of ischemia or ischemiareperfusion. 23. The vector for use according to item 22, wherein the vector is a polynucleotide vector comprising in its sequence a gene encoding for the AMPKa2-DN as defined in any of items 16 to 21, and wherein the gene of AMPKa2-DN is operably linked and under the control of a ubiquitous and constitutive promoter, preferably a cytomegalovirus promoter. 24. The vector for use according to item 23, wherein the polynucleotide is comprised in sEVs, and wherein said sEVs preferably express in their outer membrane at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b. 25. The vector for use according to item 24, wherein the fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprises SEQ ID NO: 5, or a sequence with at least 85% sequence identity to SEQ ID NO: 5. 26. The vector for use according to any one of items 24 to 25, wherein the small extracellular vesicles have a size distribution of between 30 and 150 nm. 27. The vector for use according to any one of items 24 to 26, wherein the small extracellular vesicles are exosomes. 28. The AMPKa2-DN for use as defined in any of items 16 to 21, or the vector for use as defined in any of items 22 to 27, wherein the ischemia is ischemic stroke or cerebral ischemia. 29. The AMPKa2-DN or the vector for use according to item 28, wherein the administration of is via systemic route. 30. The AMPKa2-DN or the vector for use according to items 28 or 29, wherein the use is in combination with nicotine, wherein the combination is herein understood as the simultaneous or sequential administration, in any order, of a) the population of small extracellular vesicles, and b) nicotine. SEQUENCE LISTING SEQ ID NO: 1: wildtype APMKa2 MAEKQKHDGRVKIGHYVLGDTLGVGTFGKVKIGEHQLTGHKVAVKILNRQKIRSLDVVGKIKREIQNLKL FRHPHIIKLYQVISTPTDFFMVMEYVSGGELFDYICKHGRVEEVEARRLFQQILSAVDYCHRHMVVHRDL KPENVLLDAQMNAKIADFGLSNMMSDGEFLRTSCGSPNYAAPEVISGRLYAGPEVDIWSCGVILYALLCG TLPFDDEHVPTLFKKIRGGVFYIPDYLNRSVATLLMHMLQVDPLKRATIKDIREHEWFKQDLPSYLFPED PSYDANVIDDEAVKEVCEKFECTESEVMNSLYSGDPQDQLAVAYHLIIDNRRIMNQASEFYLASSPPSGS FMDDSAMHIPPGLKPHPERMPPLIADSPKARCPLDALNTTKPKSLAVKKAKWHLGIRSQSKACDIMAEV YRAMKQLGFEWKVVNAYHLRVRRKNPVTGNYVKMSLQLYLVDSRSYLLDFKSIDDEVVEQRSGSSTPQR SCSAAGLHRARSSFDSSTAENHSLSGSLTGSLTGSTLSSASPRLGSHTMDFFEMCASLITALAR SEQ ID NO: 2: AMPKa2-DN mutant protein amino acid sequence: Underlined : Myc tag bold: Linker bold and underlined: D->A substitution MEQKLISEEDLGGGEKQKHDGRVKIGHYVLGDTLGVGTFGKVKIGEHQLTGHKVAVKILNRQKIRSLDVV GKIKREIQNLKLFRHPHIIKLYQVISTPTDFFMVMEYVSGGELFDYICKHGRVEEVEARRLFQQILSAVDYCH RHMVVHRDLKPENVLLDAQMNAKIAAFGLSNMMSDGEFLRTSCGSPNYAPEVISGRLYAGPEVDIWSCG VILYALLCGTLPFDDEHVPTLFKKIRGGVFYIPEYLNRSIATLLMHMLQVDPLKRATIKDIREHEWFKQDLPSYL FPEDPSYDANVIDDEAVKEVCEKFECTESEVMNSLYSGDPQDQLAVAYHLIIDNRRIMNQASEFYLASSPPTGS FMDDSAMHIPPGLKPHPERMPPLIADSPKARCPLDALNTTKPKSLAVKKAKWHLGIRSQSKPYDIMAEVYRA MKQLDFEWKVVNAYHLRVRRKNPVTGNYVKMSLQLYLVDNRSYLLDFKSIDDEVVEQRSGSSTPQRSCSAAG LHRPRSSVDSSTAENHSLSGSLTGSLTGSTLSSASPRLGSHTMDFFEMCASLITALAR SEQ ID NO: 3: AMPKa2-DN mutant nucleotide sequence: GGCGAGAAGC AGAAGCACGA CGGGCGTGTG AAGATCGGAC ACTACGTGCT GGGGGACACC CTGGGCGTCG GCACCTTCGG CAAAGTGAAG ATTGGAGAAC ATCAATTGAC AGGCCATAAA GTGGCAGTTA AGATCTTAAA TAGACAGAAG ATTCGCAGTT TAGATGTTGT TGGAAAAATA AAACGAGAAA TTCAAAATCT TAAACTCTTT CGTCATCCTC ATATTATCAA ACTCTACCAA GTGATCAGCA CTCCAACAGA CTTTTTTATG GTAATGGAAT ATGTGTCTGG AGGTGAATTG TTCGACTACA TCTGTAAACA CGGGAGGGTT GAAGAGGTGG AAGCTCGCCG GCTCTTCCAG CAGATTCTGT CTGCCGTGGA CTACTGTCAC AGGCACATGG TTGTCCACAG GGACCTGAAG CCAGAGAACG TGTTGCTGGA CGCCCAGATG AATGCTAAGA TAGCTGCCTT CGGACTCTCT AATATGATGT CAGATGGTGA ATTTCTACGA ACTAGCTGTG GATCGCCAAA TTATGCAGCA CCGGAGGTCA TCTCAGGAAG GCTGTATGCG GGTCCTGAGG TTGATATCTG GAGCTGTGGT GTTATCCTGT ATGCCCTTCT CTGTGGCACC CTCCCGTTCG ACGATGAGCA CGTGCCTACG CTCTTTAAGA AGATCCGAGG GGGTGTGTTC TACATCCCGG AGTATCTCAA CCGTTCTATT GCCACTCTGC TGATGCACAT GCTGCAGGTG GACCCCTTGA AGCGAGCAAC TATCAAAGAC ATACGAGAGC ATGAATGGTT CAAGCAAGAT TTGCCCAGTT ACCTCTTTCC TGAAGACCCC TCCTATGATG CTAACGTCAT TGATGATGAG GCTGTGAAAG AAGTATGTGA AAAATTTGAG TGTACAGAAT CAGAAGTGAT GAACAGTTTA TACAGTGGTG ACCCTCAAGA CCAGCTCGCA GTGGCTTATC ATCTCATCAT TGACAATCGG AGAATAATGA ACCAAGCCAG TGAGTTCTAC CTCGCCTCCA GTCCTCCAAC GGGTTCCTTC ATGGACGATA GTGCCATGCA TATTCCCCCC GGCCTGAAAC CACATCCTGA AAGGATGCCA CCTCTCATAG CAGACAGCCC CAAAGCACGC TGTCCACTGG ATGCACTCAA CACAACTAAG CCCAAATCTT TAGCTGTGAA AAAAGCCAAG TGGCACCTTG GGATCCGAAG CCAGAGCAAA CCATACGACA TTATGGCGGA GGTGTACCGA GCTATGAAGC AGCTGGACTT TGAATGGAAG GTAGTGAATG CATACCATCT TCGAGTAAGA AGAAAAAACC CAGTGACTGG CAATTACGTG AAAATGAGCT TACAGCTTTA CCTGGTTGAC AATCGGAGCT ATCTTCTAGA CTTTAAAAGC ATCGATGATG AGGTGGTGGA GCAGAGGTCT GGTTCTTCAA CACCTCAGCG CTCCTGTTCT GCTGCCGGCC TCCACAGACC TCGGTCAAGT GTCGATTCCA GCACAGCCGA GAACCATTCA CTGTCTGGCT CTCTCACTGG TTCTTTGACT GGCAGCACTT TGTCCTCCGC TTCCCCGCGC CTGGGCAGTC ATACCATGGA TTTTTTTGAA ATGTGCGCCA GTCTTATCAC TGCTTTAGCC CGTTGATAAT AACTCAC SEQ ID NO: 4: CMV promoter nucleotide sequence: TACGGTAAATGGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTCGTT SEQ ID NO: 5: neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b). Note: (Not underlined: Lamp2b sequence) (Underlined: RVG sequence) MCLSPVKGAKLILIFLFLGAVQSNALIVNLTDSKGTCLYARYTIWMPENPRPGTPCDIFTNSR GKRASNGSGGAEWEMNFTITYETTNQTNKTITIAVPDKATHDGSSCGDDRNSAKIMIQFGFA VSWAVNFTKEASHYSIHDIVLSYNTSDSTVFPGAVAKGVHTVKNPENFKVPLDVIFKCNSVLT YNLTPVVQKYWGIHLQAFVQNGTVSKNEQVCEEDQTPTTVAPIIHTTAPSTTTTLTPTSTPTP TPTPTPTVGNYSIRNGNTTCLLATMGLQLNITEEKVPFIFNINPATTNFTGSCQPQSAQLRLN NSQIKYLDFIFAVKNEKRFYLKEVNVYMYLANGSAFNISNKNLSFWDAPLGSSYMCNKEQVL SVSRAFQINTFNLKVQPFNVTKGQYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYLIGRRKT YAGYQTL SEQ ID NO: 6: VQAd CMV AMPKa2DN 31487, see Fig. 10 1 TTAAGCTAGC ATCATCAATA ATATACCTTA TTTTGGATTG AAGCCAATAT 51 GATAATGAGG GGGTGGAGTT TGTGACGTGG CGCGGGGCGT GGGAACGGGG 101 CGGGTGACGT AGTAGTGTGG CGGAAGTGTG ATGTTGCAAG TGTGGCGGAA 151 CACATGTAAG CGACGGATGT GGCAAAAGTG ACGTTTTTGG TGTGCGCCGG 201 TGTACACAGG AAGTGACAAT TTTCGCGCGG TTTTAGGCGG ATGTTGTAGT 251 AAATTTGGGC GTAACCGAGT AAGATTTGGC CATTTTCGCG GGAAAACTGA 301 ATAAGAGGAA GTGAAATCTG AATAATTTTG TGTTACTCAT AGCGCGTAAT 351 ATTTGTCTAG GGAGATCAGC CTGCAGGTCG TTACATAACT TACGGTAAAT 401 GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT 451 GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT 501 GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT 551 CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC 601 CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT 651 ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG 701 TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT 751 CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG 801 ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT 851 AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTCGTT TAGTGAACCG 901 TCAGATGGTA CCGTTTAAAC TCGAGGTCGA CGGTATCGAT AAGCTTGATA 951 TCGAATTCGC CATGGAGCAG AAGCTTATCT CCGAGGAGGA CCTCGGTGGC 1001 GGCGAGAAGC AGAAGCACGA CGGGCGTGTG AAGATCGGAC ACTACGTGCT 1051 GGGGGACACC CTGGGCGTCG GCACCTTCGG CAAAGTGAAG ATTGGAGAAC 1101 ATCAATTGAC AGGCCATAAA GTGGCAGTTA AGATCTTAAA TAGACAGAAG 1151 ATTCGCAGTT TAGATGTTGT TGGAAAAATA AAACGAGAAA TTCAAAATCT 1201 TAAACTCTTT CGTCATCCTC ATATTATCAA ACTCTACCAA GTGATCAGCA 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 CTCCAACAGA TTCGACTACA GCTCTTCCAG TTGTCCACAG AATGCTAAGA ATTTCTACGA TCTCAGGAAG GTTATCCTGT CGTGCCTACG AGTATCTCAA GACCCCTTGA CAAGCAAGAT CTAACGTCAT TGTACAGAAT CCAGCTCGCA ACCAAGCCAG ATGGACGATA AAGGATGCCA ATGCACTCAA TGGCACCTTG GGTGTACCGA CATACCATCT AAAATGAGCT CTTTAAAAGC CACCTCAGCG GTCGATTCCA TTCTTTGACT ATACCATGGA CGTTGATAAT CCAGACATGA GCAGTGAAAA TTGTAACCAT CATTTTATGT GTAAAACCTC GCGTTTCGGT CGGTCACAGC GGCGCGTCAG TCTAGTCCCC CGCACCAGGT GCCTGTGATG TGGCCTGCAC GGTACTGAAA TGGGGGTCTT TGAGCACCAA CGCATGCCCC TGATGGTCGC CCGTGTCTGG GCTGCAGCCA GCTTGCAAGC CGGCTCTTTT TCTCAGCAGC CTCCCCTCCC GGATTTGGAT GCGCGGTAGG CTTTTTTATG TCTGTAAACA CAGATTCTGT GGACCTGAAG TAGCTGCCTT ACTAGCTGTG GCTGTATGCG ATGCCCTTCT CTCTTTAAGA CCGTTCTATT AGCGAGCAAC TTGCCCAGTT TGATGATGAG CAGAAGTGAT GTGGCTTATC TGAGTTCTAC GTGCCATGCA CCTCTCATAG CACAACTAAG GGATCCGAAG GCTATGAAGC TCGAGTAAGA TACAGCTTTA ATCGATGATG CTCCTGTTCT GCACAGCCGA GGCAGCACTT TTTTTTTGAA AACTCACCAC TAAGATACAT AAATGCTTTA TATAAGCTGC TTCAGGTTCA TACAAATGTG GATGACGGTG TTGTCTGTAA CGGGTGTTGG GCGGTGGCAG GCAGACCCTG CTGGATGTGA CCGCGCTGAG TGTGTGGGCG ATGTAGTTTT CTCGTTTGAT CATGGGCCGG CCCGTCCTGC AACGCCGTTG CCGCCCGCGG AGTGCAGCTT GGCACAATTG TGTTGGATCT AATGCGGTTT CAAGCAAGTG CCCGGGACCA GTAATGGAAT CGGGAGGGTT CTGCCGTGGA CCAGAGAACG CGGACTCTCT GATCGCCAAA GGTCCTGAGG CTGTGGCACC AGATCCGAGG GCCACTCTGC TATCAAAGAC ACCTCTTTCC GCTGTGAAAG GAACAGTTTA ATCTCATCAT CTCGCCTCCA TATTCCCCCC CAGACAGCCC CCCAAATCTT CCAGAGCAAA AGCTGGACTT AGAAAAAACC CCTGGTTGAC AGGTGGTGGA GCTGCCGGCC GAACCATTCA TGTCCTCCGC ATGTGCGCCA CGGCTAGAGC TGATGAGTTT TTTGTGAAAT AATAAACAAG GGGGGAGGTG GTATGGCTGA AAAACCTCTG GCGGATGCCG CGGGTGTCGG ATCTGGAAGG CGAGTGTGGC CCGAGGAGCT TTTGGCTCTA TGGCTTAAGG GTATCTGTTT GGAAGCATTG GGTGCGTCAG CCGCAAACTC GAGACTGCAG GATTGTGACT CCCGTTCATC GATTCTTTGA GCGCCAGCAG AAAACATAAA TCTTGCTGTC GCGGTCTCGG ATGTGTCTGG GAAGAGGTGG CTACTGTCAC TGTTGCTGGA AATATGATGT TTATGCAGCA TTGATATCTG CTCCCGTTCG GGGTGTGTTC TGATGCACAT ATACGAGAGC TGAAGACCCC AAGTATGTGA TACAGTGGTG TGACAATCGG GTCCTCCAAC GGCCTGAAAC CAAAGCACGC TAGCTGTGAA CCATACGACA TGAATGGAAG CAGTGACTGG AATCGGAGCT GCAGAGGTCT TCCACAGACC CTGTCTGGCT TTCCCCGCGC GTCTTATCAC GGCCGCCACA GGACAAACCA TTGTGATGCT TTAACAACAA TGGGAGGTTT TTATGATCCG ACACATGCAG GGAGCAGACA GGCGCAGCCA TGCTGAGGTA GGTAAACATA GAGGCCCGAT GCGATGAAGA GTGGGAAAGA TGCAGCAGCC TGAGCTCATA AATGTGATGG TACTACCTTG CCTCCGCCGC GACTTTGCTT CGCCCGCGAT CCCGGGAACT GTTTCTGCCC TAAAAAACCA TTTATTTAGG TCGTTGAGGG AGGTGAATTG AAGCTCGCCG AGGCACATGG CGCCCAGATG CAGATGGTGA CCGGAGGTCA GAGCTGTGGT ACGATGAGCA TACATCCCGG GCTGCAGGTG ATGAATGGTT TCCTATGATG AAAATTTGAG ACCCTCAAGA AGAATAATGA GGGTTCCTTC CACATCCTGA TGTCCACTGG AAAAGCCAAG TTATGGCGGA GTAGTGAATG CAATTACGTG ATCTTCTAGA GGTTCTTCAA TCGGTCAAGT CTCTCACTGG CTGGGCAGTC TGCTTTAGCC GCGGGGAGAT CAACTAGAAT ATTGCTTTAT CAATTGCATT TTTAAAGCAA GCTGCCTCGC CTCCCGGAGA AGCCCGTCAG TGAGGTCGAC CGATGAGACC TTAGGAACCA CACTTGGTGC TACAGATTGA ATATATAAGG GCCGCCGCCA TTTGACAACG GCTCCAGCAT ACCTACGAGA CGCTTCAGCC TCCTGAGCCC GACAAGTTGA TAATGTCGTT TGAAGGCTTC GACTCTGTTT GGTTTTGCGC TCCTGTGTAT 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 5901 5951 6001 6051 6101 6151 6201 6251 6301 6351 6401 6451 6501 6551 6601 tttttccagg TAAGCCCGTC GGGGTGGTGT CCTAAAAATG TGTAAGTGTT ATGAGATGCA ATCCCTCCGG TGCACTTGGG TTGGAGACGC GATGGCAATG CACTAACGTC ACAAAGCGCG CCCAGGGGCG CAGATGGGGG GGGGTAGGGG CTTACCGCAG GGTAGTTAAG TCGTTAAGCA AAGGCGCTCG TCAACGGTTT AGCAGTTCCA ATCCAGCATA CAGTAGTCGG GCAGGGTCCT GGCTGCGCGC GCGCTGCCGG TGTCATAGTC TTGGAGGAGG CTTGGGCGCG CCCCGCAGAC GGGTCAAAAA GGTTTCCATG CCCCGTATAC CTTCAACCCA CCGCACTTAT GCAGCGCTCT GATGATCGGC AAGCCTTCGT ATTATCGCCG CGCGACGCGA GCGGCATCGG GACGACCATC AAAGGCCGCG ATCACAAAAA TAAAGATACC TCCGACCCTG GCGTGGCGCT GTCGTTCGCT CCGCTGCGCC ACGACTTATC AGGTATGTAG CTACACTAGA CCTTCGGAAA GGTAGCGGTG ACGTGGTAAA TCTGGGGTGG TGTAGATGAT TCTTTCAGTA TACAAAGCGG TCTTGGACTG GGATTCATGT AAATTTGTCA CCTTGTGACC GGCCCACGGG ATAGTTGTGT GGCGGAGGGT TAGTTACCCT GATCATGTCT AGATCAGCTG CCGGTGGGCC AGAGCTGCAG TGTCCCTGAC CCGCCCAGCG GAGACCGTCC GGCGGTCCCA TCTCCTCGTT TGCTCGTCCA CGTCAGCGTA TGGCCAGGGT TCTTCGCCCT CAGCCCCTCC CGCCGCACGA AGAAATACCG GGTCTCGCAT CCAGGTTTCC AGCCGGTGTC AGACTTGAGA GTCAGCTCCT GACTGTCTTC GGGTCATTTT CTGTCGCTTG CACTGGTCCC GCATGGCGGC GGCTGGATGG GATGCCCGCG AGGGACAGCT TTGCTGGCGT TCGACGCTCA AGGCGTTTCC CCGCTTACCG TTCTCATAGC CCAAGCTGGG TTATCCGGTA GCCACTGGCA GCGGTGCTAC AGGACAGTAT AAGAGTTGGT GTTTTTTTGT GGTGACTCTG AGGTAGCACC CCAGTCGTAG GCAAGCTGAT TTAAGCTGGG TATTTTTAGG TGTGCAGAAC TGTAGCTTAG TCCAAGATTT CGGCGGCCTG TCCAGGATGA GCCAGACTGC CACAGATTTG ACCTGCGGGG GGAAGAAAGC CGTAAATCAC CTGCCGTCAT TCGCATGTTT ATAGCAGTTC GCCGTAGGCA CAGCTCGGTC TCGCGGGTTG GACGGGCCAG GTCTGGGTCA GCGCTTGAGG GCGCGTCGGC GCGGCGTGGC GGGGCAGTGC ATTCCGGGGA TCCACGAGCC CCCATGCTTT CACGCTCGGT GGCCTGTCCT TCCGGTGGGC TTTATCATGC CGGCGAGGAC CGGTATTCGG GCCACCAAAC CGACGCGCTG CCTTCCCCAT TTGCAGGCCA TCAAGGCCAG TTTTCCATAG AGTCAGAGGT CCCTGGAAGC GATACCTGTC TCACGCTGTA CTGTGTGCAC ACTATCGTCT GCAGCCACTG AGAGTTCTTG TTGGTATCTG AGCTCTTGAT TTGCAAGCAG GATGTTCAGA ACTGCAGAGC CAGGAGCGCT TGCCAGGGGC ATGGGTGCAT TTGGCTATGT CACCAGCACA AAGGAAATGC TCCATGCATT GGCGAAGATA GATCGTCATA GGTATAATGG CATTTCCCAC CGATGAAGAA AGGTTCCTGA ACCTATTACC CCCTGAGCAG TCCCTGACCA TTGCAAGGAA TGCTTTTGAG ACCTGCTCTA GGGCGGCTTT GGTCATGTCT CGGTGAAGGG CTGGTCCTGC CAGGTAGCAT CCTTGGCGCG AGACTTTTGA GTAGGCATCC AGGTGAGCTC TTGATGCGTT GACGAAAAGG CGACCGATGC GCGGGGCATG AACTCGTAGG CGCTTTCGCT AATCTTGCAC GTTTCGGCGA GGCTACGTCT TATGATTCTT TGCTGTCCAG CAAAAGGCCA GCTCCGCCCC GGCGAAACCC TCCCTCGTGC CGCCTTTCTC GGTATCTCAG GAACCCCCCG TGAGTCCAAC GTAACAGGAT AAGTGGTGGC CGCTCTGCTG CCGGCAAACA CAGATTACGC TACATGGGCA TTCATGCTGC GGGCGTGGTG AGGCCCTTGG ACGTGGGGAT TCCCAGCCAT GTGTATCCGG GTGGAAGAAC CGTCCATAAT TTTCTGGGAT GGCCATTTTT TTCCATCCGG GCTTTGAGTT AACGGTTTCC GCAGCTGCGA GGGTGCAACT GGGGGCCACT AATCCGCCAG GCAAAGTTTT CGTTTGACCA CGGCATCTCG CGCTGTACGG TTCCACGGGC GTGCGCTCCG TGGTGCTGAA TTGACCATGG CAGCTTGCCC GGGCGTAGAG GCGCCGCAGG TGGCCGTTCG TCTTACCTCT CTGTCCGTGT CCTTGAGAGC ACTATCGTCG ACAGGTGCCG GGAGCGCGAC GCCCTCGCTC GAAGCAGGCC TGCTGGCGTT CTCGCTTCCG GCAGGTAGAT GGAACCGTAA CCTGACGAGC GACAGGACTA GCTCTCCTGT CCTTCGGGAA TTCGGTGTAG TTCAGCCCGA CCGGTAAGAC TAGCAGAGCG CTAACTACGG AAGCCAGTTA AACCACCGCT GCAGAAAAAA 6651 AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT 6701 GGAACGAAAA CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG 6751 ATCTTCACCT AGATCCTTTT AAATTAAAAA TGAAGTTTTA AATCAATCTA 6801 AAGTATATAT GAGTAAACTT GGTCTGACAG TTACCAATGC TTAATCAGTG 6851 AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT AGTTGCCTGA 6901 CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC CATCTGGCCC 6951 CAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT 7001 CAGCAATAAA CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA 7051 ACTTTATCCG CCTCCATCCA GTCTATTAAT TGTTGCCGGG AAGCTAGAGT 7101 AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC ATTGCTGCAG 7151 GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT CAGCTCCGGT 7201 TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC 7251 GGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG 7301 TGTTATCACT CATGGTTATG GCAGCACTGC ATAATTCTCT TACTGTCATG 7351 CCATCCGTAA GATGCTTTTC TGTGACTGGT GAGTACTCAA CCAAGTCATT 7401 CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG GCGTCAACAC 7451 GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT CATCATTGGA 7501 AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC 7551 CAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA 7601 CTTTCACCAG CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA AAATGCCGCA 7651 AAAAAGGGAA TAAGGGCGAC ACGGAAATGT TGAATACTCA TACTCTTCCT 7701 TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC ATGAGCGGAT 7751 ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA 7801 TTTCCCCGAA AAGTGCCACC TGACGTCTAA GAAACCATTA TTATCATGAC 7851 ATTAACCTAT AAAAATAGGC GTATCACGAG GCCCTTTCGT CTTCAAGAAA 7901 ATTAA SEQ ID NO: 7: HIF nucleotide sequence ACGTGNNNNACGTG SEQ ID NO: 8: AMP-activated, alpha 2 catalytic subunit (Prkaa2), transcript variant 1, mRNA Mus musculus GGGTAGGCGGCGGCGGCGGCGGTGGCGGCGGTAGCGGCGGCGGCAGCGGTAGCGGCGGCGGCGCTCAGAG CCCGCGGCAGGGGGAGCCCGTGCGCCGAACATGGCTGAGAAGCAGAAGCACGACGGGCGGGTGAAGATCG GACACTACGTCCTGGGGGACACCCTGGGCGTCGGCACCTTCGGCAAAGTGAAGATTGGAGAACACCAATT GACAGGCCATAAAGTGGCAGTTAAGATCTTAAATAGACAGAAGATTCGCAGTTTAGATGTTGTTGGAAAA ATAAAACGAGAAATTCAAAATCTTAAACTCTTTCGTCATCCTCATATTATCAAACTCTACCAGGTGATCA GCACTCCGACAGACTTTTTTATGGTAATGGAATATGTGTCTGGAGGTGAATTGTTCGACTACATCTGCAA ACATGGGCGGGTTGAAGAGGTGGAAGCGCGCCGGCTCTTCCAGCAGATCCTGTCTGCCGTGGATTACTGT CACAGGCATATGGTTGTCCATAGGGACCTGAAGCCAGAGAATGTGCTGCTGGATGCCCAGATGAACGCTA AGATAGCTGACTTTGGACTATCTAATATGATGTCAGATGGTGAATTTCTACGAACTAGCTGTGGATCGCC AAATTATGCAGCACCTGAGGTCATCTCAGGAAGGCTGTATGCAGGTCCCGAGGTCGATATCTGGAGCTGT GGTGTCATCCTGTATGCCCTTCTCTGTGGCACCCTCCCTTTCGATGATGAGCACGTGCCTACGCTCTTCA AGAAGATCCGAGGGGGTGTGTTTTACATCCCAGACTATCTCAACCGTTCTGTCGCCACTCTGCTGATGCA CATGCTCCAGGTGGACCCCCTGAAGCGAGCGACTATCAAAGACATACGAGAACATGAATGGTTTAAACAG GATTTGCCCAGCTACCTATTTCCTGAAGACCCCTCCTACGATGCGAATGTCATTGACGATGAGGCTGTGA AGGAAGTCTGTGAGAAATTCGAGTGTACAGAGTCAGAAGTGATGAATAGTCTGTATAGTGGTGACCCTCA AGACCAGCTTGCAGTGGCTTATCATCTTATCATTGACAATCGGAGAATAATGAACCAAGCCAGTGAGTTC TACCTCGCCTCTAGTCCTCCATCAGGTTCTTTTATGGATGACAGCGCCATGCATATTCCTCCAGGCTTGA AACCACATCCAGAAAGGATGCCGCCTCTCATCGCAGACAGCCCCAAAGCACGCTGTCCACTGGATGCACT CAACACAACGAAGCCCAAGTCTCTAGCTGTGAAAAAAGCCAAGTGGCATCTTGGAATCCGAAGCCAGAGC AAAGCGTGTGACATTATGGCTGAAGTGTACCGAGCTATGAAGCAGCTGGGTTTTGAATGGAAGGTAGTGA ATGCATACCATCTTCGAGTAAGAAGAAAAAACCCAGTGACTGGCAACTATGTGAAAATGAGCTTACAGCT TTACCTGGTAGACAGTCGGAGCTATCTTCTGGACTTCAAAAGCATCGATGATGAGGTGGTGGAGCAGAGG TCTGGTTCTTCAACACCCCAGCGCTCCTGTTCTGCTGCGGGCCTCCACAGAGCACGGTCAAGTTTTGATT CCAGCACAGCTGAGAACCACTCCCTTTCTGGCTCTCTCACTGGCTCTTTGACTGGCAGCACTTTGTCCTC GGCATCCCCGCGCCTGGGCAGTCACACCATGGATTTTTTTGAAATGTGCGCCAGTCTTATCACTGCTTTA GCTCGTTGATTATCCACCCCTGGTCTCTGTCTTTCTGTCACCCCCCTGTGAAATCACATACACTCTTTAA ATTATTATCTCACTCGGTATTACGTGCTCTGCAATAGAAGTTGGTGTGAACATTCCCAGGTGACATGCAG TGTTATTGAAATCACCGAAAACACAGAAATCCAGCATTCTGTTTACTCGTAGAACTCTGTAACTCTCCTG TGCCTGTGACAAGTTTGCATGGGTAGTACTGGGTGAGTGTGGTGGCGCTTGCTAACTTACACCTGTGAAT TCGCTGTAGATGGCAAGCACACCTCACTGATGAACGAACCCGCTGAGGTAGGGGTGGTTCAATGGGCCTC CTTCCTTCATGTTTCCTGTACGTAAATCCGGTTTACCTCCTAAACTTCAGCAGCTGTTAGGCTGCCTGGG CACT CTT GCACAAGAAGATTAAGATATAGAATAACT GTTAGGGAAATATTATTT CAAT GTAGAAAT CT GA AAATCCTGTCCCCCTAAATATTAGAAACCAAAGTCTTTTAAATTATTTCTGCAAATACTGCCTAGTATTA GCCATTAAGACTGTGTTTCTGATAACAGGAGCCTAATCTCCCAGCTTCCTGACAGTTGTGCAAGGCGCCT AGT TAAAT CACAC CAC CAC CAAG CAAAACAAAAC CAT G CAAAAT TAGTACACAGTT CAAT GAGACAAG CA AAATACCAACAACAGTCTCTTAAAAGAAAATTCTACTCTATGATCTTGTTTGTGTTTATCAGACTGGGGT AGCTGGGAGGAGGGCCGGCAGTCATGGCCCTCTGGCATGTTTGCTAATTCCCTTAACATTTGTTATCGAT GCCACCTCCTCCCTCCGTCTTCCTCAGGAGCCTCTGTCTTGCTCAATGCTTTTCCCTTTCATTAATCCTT TAGCTAAAAACCTTGCTTTGGCCATCATGTCTTTGTATACTTCCAAAGCTTGATCTCTGTGACCTTCACT GTTGAACCTGATTGGACAGGGAAGCCTTAAATATTTAAAAGTATATTCTCCTGGAATACGTATATGTGTT GT T TACATATATATACATAT GTAT T GACACAT CAGTGCTTT TAAT CAAAAAAAAAC CAAACAT T T T T GT G CAAGTTTTTTTTTTAATTTAAGACAAAAAAGCACTATATTGCAAGATAGATTAAGTTAATTGATTATATG TTGTTTTGGTAAAAGTATTTTAAATTATGTGTAATTTATCTAATAATTATTTATTCATCTATTGTATTTA TGTTGTAATTTTAAAAATCATTGATCTGATTCACCCTAGAAAATATTAAGCTCTTGAAAAAAAAAAAAAG CAACATTGCCTTGACAGGTTTCTCTTCACCAAAAATTGTATATTTTCTATTTACCAAGAAGCAAATGAGA CTGTTAGACCAGGTTTCTCATGAGATTCCAGAAGACAAGTTTGTCTGTCTTTAAATAGCTTTTTGAATCA GGAATTTGCATCATATCACATCAGATAACTTTGTAGTTCTTTTAAAAAGATGGTCAATATTTATCACTCC AACTTCTCAATGGTAAAATAAAAGAATCTTTGCCCACTGATTGAGAAGGAGAGTGTATAATTTAAGGTAC CT TAAGT T TAAT CT T T GAAAGAT TGC CAT CT T GT GGCAATAGAT GGTAAT T T TAGTAGT TAT T T TAATAC CAATACTTAGTCCTGCCTGGGTTATCTAATGTGTTTTATGAATGTGGCATAGTTCTGACTTCTCCAAATG TGTCTGTATCCTTGTTTGTGGGGTGGGGGAGAGATCACCTTATGATGTTTGCCTAATCATGGCATGGAAG GCCTTGAAGTAAGGAACTGAATAGATTCTTTACCTCTAATAACAGCATCCACAACAGTGATGAAGGCAGG AAGGAGGCAGAATGCTTTTAGTCTAGTGAATTTAAACCAAAATTACTTTATATGAATGATTATGATGCTA TTCAAGAGCTAAAAGTTATTTACTATTGTCTTAATTCATTCCAGTATTATGAATACCCAGGTTGACAGTT ACCTGCTGAATTTCAGGAGAACTGAAGGTCATTTTGTTTCTTTTAACATTTTAAGACATAATTATTTAAT GAATCTAGATGATAGAGTAAGTTGGTTTTCAGAGAGAATTCAGTGACTCATCTCGAAAAGCAGAGGCAGG AATTCAGTCACAGCATAGGGATTTGTTGCTCTTGGAAACAGAGAAGGAAAGAATTCTTTGCAAATTTACA GTTCCTACATTTCAGTCAATCCTATGGGTATGGTAGGTACTGAGTTTAGAGCAGCCTATCCTCTTGGATC GTGCGGAGAATAACTATTAAGTTATAGATGTGGTATGTCTTGGAAACGGAGTCCATCTGTTTCCAAAATA GTATTTCCTAAAACAAAGAAATCCCACAAAATAATTTTGAAATACATTGTGTTCTGCTCTGCACCTTGCA GAGGACAGTGACTATTAACTCGAAAGACTTGCAGACTTTGAGAAAAAAACAAAACAAAAAGACCCCACAT AACTGCAAAAACCCTACTTTGCCCAAACTAGTTTACTCTTTTAAAACGTTTGCTGAGTCTGGTTGCTTAC CTGTAATACGACTGGGGAGTATGAGGCAGGAAGATACGTTGGGGCCTAAGAACTAGATAGATAGCAAGAC CTTGTTTCAAATTGAAACAATAAAGGAACTGTAGCATCCCATAAGAACATAGGAGAAACGATTGTCTCTG AGCGCCACGGTGTGGTCGTTAGTGACAGTGAGTAACACCTCTTCCCCAGAGCCTGATGCTAATTCAGATC TGCTCCATTGACCATGCACATAGGGTCATCCTGAGTCTGGGCTTGTACCACATGCTGGTTTTCCTAATCA TATGATGCTTTTTAGGTAGAACATCTATTCTTTTTTTCCCTTAGAGGGTGACTGAAAAGTCCTAGACAAG GTTACTTATGGAAAACCAGAAGAGTAAGAGAGCAGTGGCTTTCTGTTGTGGCTTCATGGATAGCAAGCCA GTTTTATTCATTTTTCAATGGCCACTGAACGTTTTCTAAGTCCTTTTATTTAGGAAACCAAAATTGAAGC TATCTCAAAGTAAATCTACAGGGAGAGAGAAAACCAAACCATTCACAAGTTAGGGTTCTGTTTTGATCAA GAACTCTTGTAAAAGGCCCTTGCAACATCTGGTTATCTGCAGAGAGAAGCTAGGAGCCTAAGCTAAGCAG AAACATTAAGACCCTCTGTGTTAGGGAATTGGGTATATGTAGCACAGTAATTCTCTACTTTATGCAAGTA AGTATTCAAGAATATTTACTTAATTCTCAATCTACTCTTTTCTTAGCTAGTTTGTACTCAGGGAACACAA ATTTAATACAAATGAAATGAAACACAATTTGGAAAATAATCGTAAACAACCGAATCAGATTTTTATACTG CTTCATCTGTAGTCTCTGCTAACATGCAAGCATGCTTAGTTAATGTGGGCCCCACACTGATGTTCATAAG ACTTAACAGCCTGGAACATACCTAACACAGATACCACAGCTCTGCCTGGTCACTCCCAGGGTGAAGTCTC ATACCTAAATGTGAGACTGCCCGTAGACTTGTCTGCTTTGTGTTTGGTTCAGTCTGGCAACTTGTCCTTC AGTTCTCTGTGGCTTGTTAAGTAGGGCTTATGGCTTAGTAGTTCTTGGCAACTCTGTTGAAAACTCTGGA TAGTAAGTGTGATCTAGGACTTGGCCTCAATTTAAAACCCTAAAATGAAAGGGATGGACACAGAGCTATC GTCCCTCTGAGCTCTGAAGTCATGCCTTGTTTACTACTTCAGAGCGTCAGTGTGAGTGTTCACATTTTAT GTATTTGTTTGCTAGTATGAGTGTTTTTTCCTTAGTCCGAGGCATTTCAAAGGTACACTAACCAAAAGTA CTTGGACTACTGGAATGCAAAACTGAAAACATGGTGTGGATTTAGCATTCATTAGAAGATGACTGCTAGG TAGCAGTGGTGGGGCCCATGCATTTCCAGGTCTGCTAGTGCTCCCTCGCAGTACCCAGTCCAGTAAGCAG GCTCACTGCAGTTGATCATCTTCTGGGCTGAACAGAAAGAGTTTGTGAAACATCTGAGAGTCAAGTAATA AAAATAAACACT GGT GT GT T GT T T GTAAGT C CT GGAAT GACATATAGTAGT TATAAGT GAAT GTAGT TAG TCTAATTATAAAAGCTAGTTGATTGCACTAACTAGATGTAATTTTGTGAAATATTTATGATTATATTGAT TTTATTATAAGTATTTATAAGTTATTGAGGGTGCTTAAAATCCAGAAAAATCTCTAGGACATATTTTTTT TTCTGAAATTGTATTTATATGATTTGGGTGTAGGGAGTTTTATACTGGTGACTGAGCCCAGGCTTCCCGT GTAAGAAGCAAGGGCTTAACTTCAACCTCAATCCCCTGGCCCTCTACATGATTCTGAAAGTCCAGGCGCT CTTTGTCGAGGAAAATTAGCCCCAGCCCGATCTAAATGATCACAGTTTGTGACTGCTAGCTGAACTAGGC TTATCTAAATTTCATTTTGGCCATTTTTAATTTTGTGCATAAATGAGATGACACTGTTTTTATACAGTGT GTCTCTCTGAGCTACCTAATCACTATTGGCTCTGATAGAAGTTCACCAAGCCTTTTTAAACTTGGAAACG TAACTTAAGT TAAT GAGCT T TAAGT CAT GCATATAT GTAT GT GTATATATACATAT GGAT TAT GTAAACA AGTTAGCAGCGGCGTCTTTATCAGCGGCGCCTTTCCTTGAATATTTATGATGCGGTTGATGCTACTGAAC AGTAATACCGTGAGGAACAGGCCCCAGTCTAGACTCTAGACTGGATGGAGTTTTGATTAAGCTTCTTCAG CCATAACCTGTTCAGAATCAGGTCCAATCCAGACGTTATCAGACTGCAGGCCTGTATTTTGTGAGCCAAC ATTAATGTTTACTGCCAGCCTTGCATAGCAGTGCCGGTTGCGACTGCATGTGACTTTTAGTAACTGTAGT TTATGTCTAAAAGCAGTTCAAGTTTTATAGAGGTAATTCATGATTATTTGAACAAGCAGGGTGGGTAATT CAAATATATTTAAAGTGCAATTAAGGAAATTCTAGGGTATAATATTCTTTCCAGTTTTAAAAAAAATAGT TAAGGATTTTTAGAATGTCCTAGAATGAAAAAAATTTTTTTTGGACAGACTTTTTAAAAAACAAGGTTTA TACAGATTTTTTTTTTTAATTCAAGGTTTGCTACTTTGTGTTTATTTAAATTTGCAAAGAGAAACCGAAG CCTCCTAGGAAAGAGTTGCATTGTTATTTTTCTCCTGCTAGTCTGAATGTGCATTTTCACAGCTAAAAGT CACTTGTAAATAAGGTTTGTTACCACATTCGAAGCCCATGTTTTCTAAATGGAGAACATTTTCCCAGAAT CAAAGAGACATAGACCCACTGTCTACACAAAGAAAGAAAGCGTACCTGGTGCCTTCTCAAATAATTGTAG AGTCAAAGTGAACGTGTTCCAAGTCTGCCCCACCCCACCCCCTCTCCTGTTCCTCGGGTTCTCTGTGACC TACCTGTCCCTCCCACAGACGGTCACAGGATTTGCTTCTGCCCTTTCAGTTCTACTTGACCAAACCTCAC AGCATCACCAGTGACGTTCTTCACTGAATACATTTTCTCTTTCACTTATTTCACTTTTTAAAGCCACCTG GGAAAATATTTGGCTGCCTTCTTATGCTTTTAAAATGAATGCAATTATTAGCAGTGTTTATATTTGAAAG TTTATAAAGGAAAGAAATTCAGAGCTAAGGACTGACTCTTAATTGGATCTAATTCCCTGTGTGTTACGAT AGCAGCACAAGTTATGTAACCTTGTGTAGTTTTCTCTTGTACAAAATGTGGATGATAACCTACCTCACAG ATGTGTGTGAGAAGCCGTTTCAAAGCCCCTAAGTAGGGGTTTATAGTCCCATCCTCAGTGTCAATCCTCC CTTTTTTACTAGACACCTTTGAGAATGGTAGGATGAGTATCTTGTTTTTAGATGTCATATGTAATAAGGG CATAGTGCTGAGAATGTTTAAGACACAGTTTCACAAATCATAGTGTTCTTACTGTAAGACACTTAAAAAT TTCTTGAAAACCTCAAATGTTTAAATATCGAATCTGTCTTTTGTTTCAATAAATATTGTTATTCAAAGTG TTGTCTGAAGCTGCCTTAATGTACCAAAATACAACTGTCAAAACTGTTCACAACTATATTCATGGTTGTA CTGTTTTGAGTGTTCATTTTGAAGTTGCTATGAACTATGACCTTGAAGGATAGTATTTTGATATTATTTT GGTAGGAATAGTTGTGGTTTTTTTTTTCATCATGTTTAACAGTGACTTTTGTGTGTAAAAAATAAAACTG AAACTTGGCAG SEQ ID NO: 9: Hsp70 promoter CTAGAATCCCAAAACAAACTGGTTATTGTGGTAGGTCATTTGTTTGGCAGAAAGAAAACTCGAGAAA TTTCTCTGGCCGTTATTCGTTATTCTCTCTTTTCTTTTTGGGTCTCTCCCTCTCTGCACTAATGCTCTCTC ACTCTGTCACACAGTAAACGGCATACTGCTCTCGTTGGTTCGAGAGAGCGCGCCTCGAATGTTCGCG AAAAGAGCGCCGGAGTATAAATAGAGGCGCTTCGTCTACGGAGCGACAATTCAATTCAAACAAGCA AAGTGAACACGTCGCTAAGCGAAAGCTAAGCAAATAAACAAGCGCAGCTGAACAAGCTAAACAATC TG C AGTAAAGTG CAAGTTAAAGTG AATC AATTAAAAGTAACC AG CAACCAAGTAAATC AACTG CAAC TACTGAAATCTGCCAAGAAGTAATTATTGAATACAAGAAGAGAACTCTG It will be understood that particular embodiments described in the examples are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. EXAMPLES Abbrevations'. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR); acetil-CoA carboxilasa (ACC); Activating Transcription Factor 6 (ATF6a); AMP-activated protein kinase (AMPK), apparent diffusion coefficient (ADC); C / EBP homologous protein (CHOP); diffusion-weighted magnetic resonance imaging (DWI), echo time (ET); endoplasmic reticulum (ER); eukaryotic translation-initiation factor 2 (pelF2a); Fatty acid synthase (FAS); field of view (FOV); flip angle (FA); Magnetic resonance angiography (MRA); Magnetic resonance imaging (MRI); Mechanistic target of rapamycin (mTOR); midline deviation (MD); multi-echo multi-slice sequence (MSME); number of averages (NA); oxygen-glucose deprivation (OGD); p70S6 kinase (pS6K), phosphorylated Unc-51 Like Autophagy Activating Kinase 1 (pULK-1); protein kinase RNA-like ER kinase (PERK); Regulatory-associated protein of mTOR (RAPTOR); repetition time (RT); spectral bandwidth (SW); T2-weighted (T2W); transient occlusion of the middle cerebral artery (tMCAO); time-of-flight MR angiography (TOF-MRA). 1. MATERIALS AND METHODS 1.1 Animals Experimental protocols were approved by the local Animal Care Committee (Project ID 15010 / 14 / 006 and 15012 / 2020 / 010), according to the European Union (EU) rules (86 / 609 / CEE, 2003 / 65 / CE, and 2010 / 63 / EU). The study conformed to Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Male adult Sprague-Dawley (SD) rats and C57 / BL6 mice, provided by the Centro de Biomedicina Experimental (CEBEGA); Santiago de Compostela, with a weight of 200-250 g for rats and 25 g for mice were used. Rats were housed in a temperature-controlled (22-24°C) room with a 12-hour light, 12-hour dark cycle (8:00 AM-8:00 PM). Rats were fed with water and food ad libitum. Anesthesia was induced by the inhalation of 5% sevoflurane in a N2O / O2 mixture (70 / 30). Rectal temperature was monitored and maintained at 37 °C ± 0.5 °C by using a feedback-controlled heating system. At the end of the procedures, animals were sacrificed by cervical dislocation and subsequent decapitation according to the European Union (EU) rules (86 / 609 / CEE, 2003 / 65 / CE, and 2010 / 63 / EU). Brain was extracted, frozen in liquid nitrogen, and stored at 80°C until further processing. 1.2 Middle Cerebral Artery Occlusion Transient focal ischemia was induced in rats by using the transient middle cerebral artery occlusion (tMCAO) model following the surgical procedures previously described [1-3], In brief, under an operating microscope, tMCAO was performed by exposing the left common, external and internal carotid arteries through a midline neck incision. The left external carotid artery and pterygopalatine artery of the internal carotid artery were separated and ligated by 6-0 silk sutures. A silicon rubber-coated filament (403512PK5Re; Doccol Corporation; Sharon, Massachusetts, USA) was inserted through the external carotid into the left common carotid artery and advanced into the internal carotid artery to 20 mm from the bifurcation to occlude the origin of the MCA. A laser-Doppler flow probe (tip diameter 1 mm) attached to a flowmeter (PeriFlux 5000; Perimed AB; Stockholm, Sweden) was located over the thinned skull in the MCA territory (4 mm lateral to the bregma) to obtain a continuous measure of relative cerebral flow during the arterial occlusion. Once the artery occlusion had been achieved, as indicated by Doppler signal reduction, each animal was carefully moved from the surgical bench to the Magnetic Resonance Imaging (MRI) system for ischemic lesion assessment using apparent diffusion coefficient (ADC) maps (defined as TO ischemic basal lesion). MR angiography (MRA) was also performed to ensure that the artery remained occluded throughout the MR procedure. After MR analysis, the animals were returned to the surgical bench and the Doppler probe was repositioned. The filament was removed after 75 minutes of occlusion. After suture removal, the left pterygopalatine artery was reperfused while left external carotid (used to introduce the suture) remained tied to avoid bleeding. As we have described in elsewhere [2], this surgical ischemic protocol, based on the combination of laser Doppler monitoring in combination with basal ADC and MRA assessment represents a reliable inclusion protocol during ischemic surgery to reduce variability intergroup and guarantees the reproducible of the infarct volumes. According to our reported protocol [2], the following exclusion criteria were used: i) less than 70% reduction in relative cerebral blood flow; ii) vascular abnormalities, as determined by MRA; iii) baseline lesion volume of less than 25% or greater than 45% with respect to the ipsilateral hemisphere, as measured using ADC maps, and iv) absence of reperfusion or prolonged reperfusion (more than 10 minutes until achievement of at least 50% of the baseline cerebral blood flow) after filament removal. Sham control groups were animals subjected to the same surgery procedure, but without occlusion of the middle cerebral artery (MCA). Experimental procedures were performed following criteria derived from the Stroke Therapy Academic Industry Roundtable group guidelines for preclinical evaluation of stroke therapeutics [4,5] which are: i) cerebral blood flow was measured to confirm the vascular occlusion as an index of the reliability of the ischemic model; ii) animals were randomly assigned to the experimental groups using the GraphPad software randomization tool (www.qraphpad.com); iii) researchers were blinded to treatment administration; iv) researchers were blinded to treatments during outcome assessment; and v) temperature was controlled during the ischemic period. 1.3 Experimental protocol The study was divided into 3 steps: a) a comparative study between ischemic rats fed with standard diet (STD) vs. SHAM; b) neuroprotective study of nicotine treatment in both ischemic STD group; and c) we conducted a study with AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide; TRC Inc., Canada) treatment, a direct activator of AMPK, in only STD group in order to elucidate that AMPK mediates protective effects of nicotine. Nicotine (nicotine-hydrogen-tartrate salt; Sigma-Aldrich; St. Louis, MO, USA) or vehicle (saline) were given subcutaneous (2 mg / kg per 12 h) in a total volume of 100 pL after one hour of reperfusion until the end of the follow-up period. During the whole period of treatment (vehicle, nicotine, or withdrawal), rats were maintained either on STD. AICAR was given subcutaneous (500 mg / Kg per24h) after one hour of reperfusion until the end of the follow-up period (7 days). The sample size of all groups (n) was always equal to or greater than 8 animals per group. 1.4 Magnetic resonance imaging and image analysis MRI studies were conducted on a 9.4 T horizontal bore magnet MRI system, Biospec 94 / 20USR (Bruker BioSpin; Ettlingen, Germany), with a 20 cm wide actively shielded gradient coils (440 mT / m). Radiofrequency transmission was achieved with a birdcage volume resonator; signal was detected using a four-element surface coil, positioned over the head of the animal, which was fixed with a tooth bar, earplugs, and adhesive tape. Transmission and reception coils were actively decoupled from each other. Gradient-echo pilot scans were performed at the beginning of each imaging session for accurate positioning of the animal inside the magnet bore. ADC maps were acquired during tMCAO from DWI using a spin echo echo-planar imaging sequence with the following acquisition parameters: echo time (ET) = 24.89 ms, repetition time (RT) = 4.5 s, spectral bandwidth (SW) = 200 kHz, seven b values of 0, 300, 600, 900, 1200, 1600, and 2000 s mm 2, flip angle (FA) = 90°, number of averages (NA) = 3, 14 consecutive slices of 1 mm, 24 x 16 mm2 field of view (FOV) (with saturation bands to suppress signal outside this FOV), a matrix size of 96 x 64 (isotropic in-plane resolution of 250 pm pixel-1) and implemented with fat suppression option. MCA status was evaluated in a non-invasive manner with the time-of-flight MR angiography (TOF-MRA). The TOF-MRA scan was performed with a 3D-Flash sequence with an ET = 2.8 ms, RT = 15 ms, FA = 30°, NA = 2, SW = 98 kHz, 1 slice of 14 mm, 30.72 x 30.72 x 14 mm3 FOV (with saturation bands to suppress signal outside this FOV), a matrix size of 256 x 256 x 58 (resolution of 120 pm pixel-1 x 120 pm pixel-1 x 241 pm pixel-1) and implemented without fat suppression option. The progression of ischemic lesions and infarct volumes were determined from T2 - maps calculated from T2-weighted (T2W) images acquired 24 and 72 hours, and 7, 14 and 28 days after the onset of ischemia using a multi-echo multi-slice sequence (MSME) sequence with an ET = 9 ms, RT = 3 s, 16 echoes with 9 ms echo spacing, FA = 180°, NA = 2, SW = 75 kHz, 14 slices of 1 mm, 19.2 x 19.2 mm2 FOV (with saturation bands to suppress signal outside this FOV), a matrix size of 192 x 192 (isotropic in-plane resolution of 100 pm pixel-1) and implemented without fat suppression option. All images were processed, and maps were constructed with Imaged (https: / / imagej.nih.gov / ij / ). Infarct volumes were determined from ADC maps and T2 relaxation maps by manually selecting areas of reduced ADC values or hyperintense T2 signal by a researcher blinded to the animal protocols [6,7], Infarct size was indicated as the percentage of ischemic damage with respect to the ipsilateral hemispheric volume, corrected for brain edema. For each brain slice, the total areas of both hemispheres and the areas of infarction were calculated. An edema index was measured by quantifying the midline deviation (MD), calculated as the ratio between the volume of the ipsilateral hemisphere and the volume of the contralateral hemisphere. The infarct size was adjusted for edema by dividing the area of infarction by the edema index [mm3 / MD], Finally, the presented infarct volume was calculated as following: (infarct volume [mm3 / MD] / ipsilateral hemispheric area [mm3]) x 100. 1.5 Behavioral tests Sensorimotor deficits were assessed using the Bederson test, which is a validated scale to evaluate neurological deficits after cerebral ischemia. This test is based on the presence or absence of a reflex or spontaneous activity of the animal [8], Bederson test was performed by a blind researcher to the treatment 1 day before MCAO, and at 24 and 72 h, as well as at days 7, 14 and 28 after ischemia induction during the darkness cycle. The Open Field test was carried out to determine different locomotor parameters of the mice, such as speed, distance traveled and immobile times, among others. The test was recorded and analyzed using Anymaze software. 1.6 Western blot analysis Protein expression was detected in rat brains by western blot. All rats were sacrificed at day 28 after MCAO, except for the rats included in the AICAR treatment protocol that were sacrificed 7 days after tMCAO for being the temporal point with the highest peak of brain lesion volume, and their brains were then processed. All mice were sacrificed at day 7 after MCAO, and their brains were then processed. First, the portion of the brain of interest for the WB analysis was separated, and the rest of the sample was refrozen at -80 °C. Specifically, using a brain matrix, a 2 mm thick axial section was extracted from the ipsilateral hemisphere corresponding to the middle area of the ischemic lesion. After that, this ipsilateral hemisphere brain section was initially pulverized in liquid nitrogen using a mortar and pestle followed by homogenization in lysis buffer (InvitrogenTM, Life Technologies, USA) supplemented with the protease’s inhibitor cocktail Complete Mini (Roche Applied Science; Switzerland). Following homogenization, the samples were then centrifuged at 12,000 rpm at 4°C for 30 min and supernatant protein concentration was quantified using the Bradford assay (Bio-Rad Protein Assay Kit, Bio-Rad, USA). The protein concentration was determined by the Bradford Method (Protein assay dye concentrate, Bio-Rad Laboratories; Hercules, CA, USA), and the total protein content of the tissues was calculated. The protein lysates were subjected to SDS-PAGE, electrotransferred to polyvinylidene difluoride membranes (PVDF; Millipore; Billerica, MA, USA) with a semidry blotter and probed with primary antibodies against acetyl-CoA carboxylase (ACC) and AMP-activated protein kinase alpha 2 (AMPKa2) (Millipore; Billerica, MA, USA); pACC, pAMPK, phospho-mammalian target of rapamycin (pmTOR), phospho-ribosomal protein S6 kinase (pS6K), phospho-regulatory associated protein of mTOR (pRAPTOR), phopho-Unc-51 like autophagy activating kinase 1 (pUIkl), Caspase-3 and cleaved Caspase-3 (Cell Signaling; Danvers; MA, USA); activating transcription factor 6 (ATF6a), phospho-PKR-like endoplasmic reticulum kinase (pPERK), phospho-eukaryotic translation initiation factor 2 alpha (pelF2a), and C / EBP homologous protein (CHOP) (Santa Cruz; Santa Cruz, CA,USA); and phosphoiron-responsive element-binding protein (pIRE) (Abeam; Cambridge, UK), or p-actin (Sigma-Aldrich; St. Louis, MO, USA) as described [9-12], Each membrane was then incubated with the corresponding secondary antibody: anti-mouse, anti-rabbit or anti-goat (all of them from DAKO; Glostrup, Denmark), as shown [14-17], The membranes were exposed to an X-ray film (Fujifilm; Tokyo, Japan) and developed using developer (Developer G150; AGFA Healthcare: Mortsel, Belgium) and Fixator (Manual Fixing G354; AGFA Healthcare: Mortsel, Belgium). Autoradiographic films were scanned and the bands signal was quantified by densitometry using lmageJ-1.33 software (NIH; Bethesda, MD, USA), as shown [9-12], Values were expressed in relation to p-actin (brain). Representative images for all proteins are shown; in the case of the loading controls a representative gel is displayed, although each protein was corrected by its own internal control (P-actin), as explained above. In all the Figures showing images of gels, all the bands for each picture come always from the same gel, although they may be spliced for clarity. 1.7 Statistical analysis All data were presented as the mean ± standard error of the mean (mean±SEM). Oneway or two-way analysis of variance (ANOVA) followed by post-hoc Bonferroni evaluation was used for multiple groups to determine significant differences. Student's t-test was used to test the differences between two groups. Statistical significance was set at *p<0.05, **p<0.01, and ***p<0.001. The statistical analysis was conducted using GraphPad Prism v.8 for Macintosh / Windows (GraphPad, La Jolla, CA, USA). A minimal n=8 animals per group were required to detect the desired effect with a power (1-b) of 0.8 and an a of 0.05. N was calculated using EPIDAT software (http: / / www.sergas.es). 2. RESULTS 2.1 Nicotine treatment reduces brain ischemic injury and improves neurological recovery after cerebral ischemia Next, we examined the potential neuroprotective effect of nicotine treatment in control (STD) ischemic rats. Notably, when ischemic brain injury was examined, our data showed that nicotine treatment induced a significant reduction of lesion volume from 24h after tMCAO until the end of the follow-up period of 28 days in the STD rats (Fig. 2A, B). These neuroprotective effects on the lesion volume were also reflected in a reduction in the edema volume at 3 days after tMCAO (Fig. 2A, C), as well as in a significant reduction of neurological deficit evaluated by the Bederson at day 7 after tMCAO (Fig. 2D). All these results together show that nicotine treatment exerts a neuroprotection. 2.2 Nicotine treatment inhibits the activation of AMPK signaling after cerebral ischemia Similar to previous studies in the hypothalamus [11,12], STD ischemic rats treated with nicotine showed a reduction in the levels of pAMPK and its surrogate marker pACC, despite of increased expression of AMPKa2, likely due to compensation, in the extracts of brain injured area (Fig. 3A-H). Changes in pAMPK were associated to decreased ER stress markers levels (such as peiF2a and pIRE) in STD ischemic rats treated with nicotine. Finally, in keeping with the neurological improvement and reduction of lesion volume, nicotine treatment induced a reduction in the caspase-3 levels in both control and obese ischemic rats treated with nicotine (Fig. 3G-H). Altogether, these results indicated that nicotine treatment, besides beneficial therapeutic effects, also inhibits AMPK signaling, ER stress and apoptosis in the brain-injured areas induced by the ischemia. 2.3 The neuroprotective effects of nicotine treatment after cerebral ischemia depend on the inhibition of AMPK The obtained evidence indicates that nicotine-induced neuroprotection after cerebral ischemia was associated with an inhibitory effect on AMPK, as well as downstream targets (ER stress), and apoptosis. Therefore, to address whether nicotine protective actions were mechanistically linked to AMPK inhibition, we treated STD ischemic rats with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) treatment, a direct activator of AMPK

[13] , Our data showed that the reduction of lesion volume induced by nicotine treatment was reversed by AICAR (Fig. 4A-C). Likewise, neurological deficits were also worsened after AICAR administration in nicotine-treated ischemic rats (Fig. 4D). On the other hand, western blot analyses of protein extracts on the ipsilateral hemisphere corresponded to the middle area of the ischemic lesion show that, as expected, AICAR reversed the inhibitory effects of nicotine on pAMPK(Fig. 5A-B) and downstream targets, such as pACC (Fig. 5C-D). Importantly, nicotine-induced reduction of some ER stress markers (Fig. 5E-F) and caspase 3 (Fig. 5G-H) were also blunted by AICAR. Interestingly, AICAR activator also modulates nicotine action on pmTOR pathway (Fig. 6A-B) Overall, these results indicate the activation of AMPK reverses the beneficious action of nicotine on cerebral ischemia, and consequently that AMPK is mediating the neuroprotective effect of nicotine on ischemic brain injured areas. 2.4 CMV-AMPKa2-DN extracellular vesicles (EVs) downregulate downstream AMPK targets in the brain Small extracellular vesicles (sEVs) comprising a gene encoding for AMPKa2-DN as defined in SEQ ID NO: 2 were generated. The expression of said AM PKa2-DN mutant protein was driven the CMV promoter of SEQ ID NO: 4. The sEVs also expressed the fusion protein Lamp2b-RVG of SEQ ID NO: 5 to provide the sEVs with tropism to cerebral tissues.Two doses of sEVs were administered through the jugular at 4 hours and 48 hours after reperfusion of the animals. After 7 days of follow-up, the animals were sacrificed and the brains were extracted for analysis by Western Blot. A significant reduction in the infarct volumes of the mice was observed, a trend that was maintained from the first 24 h of occlusion until 7 days of follow-up (Fig. 7 A-C and Fig. 11). 2.5 CMV-AMPKa2-DN EVs treatment improves cognitive impairment promoted by cerebral ischemia Interestingly, as in the study of the ischemia area, a significant cognitive improvement is observed 24 hours after the first treatment. Specifically, a reduction in cognitive impairment, analyzed by the Bederson test, is observed in animals treated with CMV-AMPKa2-DN EVs compared to animals treated with empty EVs (Fig. 8 A and 12). Furthermore, when analyzing different locomotor parameters by the Open Field test, a deterioration was observed in all groups subjected to tMCAO compared to the Sham group. However, when analyzing the tMCAO groups, a significantly lower deterioration was observed in the CMV-AMPKa2-DN EVs animals compared to the empty EVs, both in distance traveled (Fig. 8 B and 13), average speed (Fig. 8 C and 14), maximum speed (Fig. 8 D and 15), such as rotations about the same body axis (Fig. 8 E and 16) 2.6 CMV-AMPKa2-DN EVs treatment improves cognitive impairment promoted by cerebral ischemia When analyzing the brain extracts after 7 days, a reduction in ACC phosphorylation, a target of the AMPK pathway, was observed, indicating that systemic treatment with CMV-AMPKa2-DN EVs inhibits the activation of the AMPK pathway in the brain (Fig. 9 A and 17). Furthermore, a reduction of active Caspase-3 is observed in the CMV-AMPKa2-DN EVs group, reaching levels similar to those of the Sham group, unlike the group treated with Empty EVs where the levels are significantly higher (Fig. 9B and 18). Our data indicate that systemic treatment with CMV-AMPKa2-DN EVs inhibits the activation of AMPK in areas of ischemic brain tissue, reducing the affected area, improving the underlying cognitive impairment after ischemic, and reducing apoptotic processes derived from the injury. 3. REFERENCES [1] Campos F, Sobrino T, Ramos-Cabrer P, Argibay B, Aguila J, Perez-Mato M, et al. Neuroprotection by glutamate oxaloacetate transaminase in ischemic stroke: An experimental study. Journal of Cerebral Blood Flow and Metabolism 2011;31:1378-86. https: / / doi.Org / 10.1038 / JCBFM.2011.3. [2] Fernandez-Susavila H, Iglesias-Rey R, Dopico-Lopez A, Perez-Mato M, Sobrino T, Castillo J, et al. Inclusion criteria update for the rat intraluminal ischaemic model for preclinical studies 2017;10:1433-8. https: / / doi.org / 10.1242 / DMM.029868. [3] Perez-Mato M, Ramos-Cabrer P, Sobrino, Blanco M, Ruban A, Mirelman D, et al. 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Claims

1. A population of small extracellular vesicles (sEVs) for use in the treatment or prevention of ischemia or ischemia-reperfusion, wherein the population of sEVs comprises at least one polynucleotide comprising a gene encoding a dominant negative AMP-activated protein kinase alpha 2 (AMPKa2-DN) mutant protein comprising SEQ ID NO: 1, wherein the amino acid residue in position number 157 in SEQ ID NO: 1 is substituted by an alanine.

2. The population for use according to claim 1, wherein the AMPKo2-DN consists of SEQ ID NO: 2.

3. The population for use according to any one of claims 1 or 2, wherein the gene encoding the AMPKa2-DN mutant protein is operably linked and under the control of a ubiquitous and constitutive promoter, preferably a cytomegalovirus promoter.

4. The population for use according to any one of claims 1 to 3, wherein the sEVs express in their outer membrane at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b.

5. The population for use according to claim 4, wherein the fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprises SEQ ID NO: 5, or a sequence with at least 85% sequence identity to SEQ ID NO: 5.

6. The population for use according to any of claims 1 to 5, wherein the small extracellular vesicles have a size distribution of between 30 and 150 nm.

7. The population for use to any of claims 1 to 6, wherein the small extracellular vesicles are exosomes.

8. The population for use according to any of claims 1 to 7, wherein the ischemia is ischemic stroke or cerebral ischemia.

9. The population for use according to any one of claims 1 to 8, wherein the administration of said small extracellular vesicles is via systemic route.

10. The population for use according to any one of claims 1 to 9, wherein the use of the small extracellular vesicles, preferably exosomes, is in combination with nicotine, wherein the combination is herein understood as the simultaneous or sequential administration, in any order, of a) the population of small extracellular vesicles, and b) 5            nicotine.