WO2016113544A1

WO2016113544A1 - Treatment of diseases associated with mitochondrial dysfunction

Info

Publication number: WO2016113544A1
Application number: PCT/GB2016/050041
Authority: WO
Inventors: Tamara SIREY; Christopher Ponting
Original assignee: Isis Innovation Limited
Priority date: 2015-01-12
Filing date: 2016-01-08
Publication date: 2016-07-21
Also published as: GB201500427D0

Abstract

The invention relates to methods for increasing mitochondrial function in a cell or an individual. Kits for use in such methods are also provided.

Description

TREATMENT OF DISEASES ASSOCIATED WITH MITOCHONDRIAL DYSFUNCTION

Field of the Invention

Background of the Invention

Effective control of metabolic homeostasis is vital to maintain the physiological function and health of an organism. Metabolic homeostasis is coordinated by a combination of key transcription factors and regulatory noncoding ribonucleic acids (RNAs) that interact within intricate regulatory control circuits. MicroRNAs (miRNAs) are known to participate in the regulation of metabolic pathways including insulin signaling, glucose homeostasis, and lipid homeostasis (Esau et al, 2006; Trajkovski et al, 2011). Further complexity can be imposed on this post-transcriptional regulatory network by long noncoding RNAs (IncRNAs; >200 nucleotides). Presently, the few IncRNAs that have a demonstrated role in regulating metabolic processes have been implicated in the regulation of adipogenesis (Xu et al, 2010) and in the homeostasis of pancreatic tissues (Moran et al, 2012). Some IncRNAs act as competing endogenous RNAs (ceRNAs) that bridge between miRNA and IncRNA regulatory processes by acting as miRNA decoys in the cytoplasm. More specifically, they compete with mRNAs for binding to miRNAs, thereby freeing the protein coding mRNA from miRNA-mediated repression (Cesana et al, 2011; Karreth et al., 2011; Kumar et al., 2014; Sumazin et al., 2011; Tay et al, 2011). This miRNA :ceRNA: mRNA regulatory crosstalk can initiate rapid post- transcriptional responses that co-ordinately regulate the levels of transcripts whose proteins participate in the same complex or process (Tan et al., 2014).

Mitochondrial oxidative phosphorylation (OXPHOS) is one such crucial metabolic process coordinated by complex layers of regulation. In eukaryotes, OXPHOS is responsible for the majority of ATP production that fuels fundamental cellular reactions. OXPHOS is localised to the mitochondrial inner membrane and requires the activity of five key multi-protein complexes and two electron carriers. Together these complexes constitute the electron transport chain that transfers electrons from NADH (at complex I) and FADH₂ (at complex II) through a series of redox reactions to molecular oxygen as a final electron acceptor (at complex IV). In doing so, protons are pumped across the inner mitochondrial membrane to create a proton gradient that generates the motive force required for ATP-synthase to phosphorylate ADP to produce ATP. OXPHOS dysfunction is an important pathophysiological feature of many disparate diseases including type II diabetes (Lowell and Shulman, 2005; Petersen et al., 2003), heart disease (Heather et al., 2010), bipolar disorder (Andreazza et al., 2010; Konradi et al., 2004), schizophrenia (Karry et al, 2004; Rosenfeld et al., 2011) and neurodegenerative disorders such as Parkinson's (Janetzky et al, 1994; Schapira et al., 1990) and Alzheimer's diseases (Canevari et al, 1999).

The subunits comprising the different OXPHOS complexes are encoded by both mitochondrial and nuclear genomes. OXPHOS structural integrity and functional efficiency are maintained through tightly co-ordinated transcriptional regulation (Mootha et al, 2003; Van Waveren and Moraes, 2008) and specific sub-cytoplasmic colocalisation (Matsumoto et al, 2012; Michaud et al., 2014). The nuclear encoded subunits are imported into the mitochondria after translation in the cytoplasm and their complexes assembled with the mitochondrially encoded subunits in an intricate assembly process (Lazarou et al., 2009; Perales-Clemente et al., 2010; Vogel et al, 2007). This coordinated process of nuclear encoded subunit expression is post-transcriptionally regulated by miRNAs in various contexts including hypoxia (Chen et al, 2010; Semenza, 2011), during aging (Li et al, 2011), locally in axons (Aschrafi et al, 2012; Aschrafi et al., 2008), in the adult human brain (Boudreau et al, 2014) and in neural precursors derived from human umbilical cord stem cells (Chang et al, 2011). Post-transcriptional regulation of OXPHOS transcripts may be particularly important in tissues that have high metabolic energy demands, such as the brain which, in humans account for 10-fold more oxygen consumption than expected from its weight (Rolfe and Brown, 1997), and more specifically in cells with extensive and unmyelinated axonal arbours (Pissadaki and Bolam, 2013).

Summary of the Invention

The inventors have surprisingly shown that an unusually abundant and conserved long noncoding RNA (IncRNA), Ceroxl, modulates the levels of transcripts encoding 12 complex I subunits and substantially alters complex I enzymatic activity and oxidative stress. These enzymatic changes within the electron transport chain are similar in magnitude to those observed in disorders with mitochondrial dysfunction, such as Parkinson's and Alzheimer's diseases. Ceroxl sequesters microRNAs which otherwise bind complex I transcripts in both mouse and human cells, reducing their proteins' abundance. A single microRNA response element for microRNA-488 is required for Ceroxl to modulate these transcripts' levels. Ceroxl is the first IncRNA demonstrated to regulate mitochondrial energy metabolism.

Accordingly, the present invention provides a method of increasing mitochondrial function in a cell, the method comprising administering to the cell an inhibitor of miR-488-3p and thereby increasing mitochondrial function in the cell.

The invention also provides: a method of increasing mitochondrial function in a cell, the method comprising administering to the cell a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) and thereby increasing mitochondrial function in the cell;

a method of increasing mitochondrial function in an individual, the method comprising administering to the patient an inhibitor of miR-488-3p and thereby increasing mitochondrial function in the individual;

a method of increasing mitochondrial function in an individual, the method comprising administering to the patient a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) and thereby increasing mitochondrial function in the individual;

use of an inhibitor of miR-488-3p in the manufacture of a medicament for increasing mitochondrial function in an individual;

an inhibitor of miR-488-3p for use in a method of increasing mitochondrial function in an individual;

use of a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) in the manufacture of a medicament for increasing mitochondrial function in an individual;

a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) for use in a method of increasing mitochondrial function in an individual;

a kit for increasing mitochondrial function in a cell or a patient comprising (a) an inhibitor of miR-488-3p and (b) means for measuring mitochondrial function;

a kit for increasing mitochondrial function in a cell or an individual comprising (i) a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) and (ii) means for measuring mitochondrial function;

an in vitro method of determining whether or not a cell has decreased mitochondrial function or mitochondrial dysfunction, comprising measuring the amount of miR-488-3p and/or the amount of the long non-coding RNA (IncRNA) comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell, wherein an increased amount of miR-488-3p compared with a normal cell of the same type indicates that the cell has mitochondrial dysfunction and wherein a decreased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 compared with a normal cell of the same type indicates that the cell has decreased mitochondrial function or mitochondrial dysfunction;

a method of determining whether or not a patient has or is likely to develop a disease or disorder associated with mitochondrial dysfunction, comprising measuring the amount of miR- 488-3p and/or the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in a cell sample from the patient, wherein an increased amount of miR-488-3p compared with a normal cell sample of the same type indicates that the patient has or is likely to develop the disease or disorder associated with mitochondrial dysfunction and wherein a decreased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 compared with a normal cell sample of the same type indicates that the patient has or is likely to develop the disease or disorder associated with mitochondrial dysfunction;

- a method of treating or preventing a disease or disorder associated with mitochondrial dysfunction in a patient, comprising determining whether or not the patient has or is likely to develop a disease or disorder associated with mitochondrial dysfunction using a method of the invention and, if the patient has or is likely to develop the disease or disorder, administering to the patient an inhibitor of miR-488-3p or a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) and thereby treating or preventing the disease or disorder in the patient;

a method of identifying a compound which is capable of increasing mitochondrial function in a cell, comprising contacting the cell with the compound and measuring the amount of miR-488-3p and/or the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell, wherein a decreased amount of miR-488-3p in the presence of the compound compared with the absence of the compound indicates that the compound is capable of increasing mitochondrial function in the cell and wherein an increased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the presence of the compound compared with the absence of the compound indicates that the compound is capable of increasing mitochondrial function in the cell;

a method of identifying a compound as an inhibitor of miR-488-3p in a cell, comprising contacting the cell with the compound and measuring the amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell, wherein an increased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the presence of the compound compared with the absence of the compound indicates that the compound is capable of inhibiting miR-488-3p in the cell; and

a compound or an inhibitor identified using a method of the invention.

Description of the Figures

Figure 1. Ceroxl is conserved in mammals and highly expressed in the central nervous system, (a) The mouse Ceroxl locus (mm9 assembly). Sequence shaded highlights conservation within exon two among eutherian mammals, but not in non-mammalian vertebrates such as chicken and zebrafish. (b) Syntenic human locus (hgl9). This transcript was previously identified on the minus strand as LMF1 non-coding transcript 4, and is located within the intron of LMF1 non-coding transcript 2. The ENCODE H3K4me3 track (a chromatin mark primarily associated with promoter regions and transcriptional start sites), generated from seven cell lines, reveals peaks in HI -human embryonic stem cells, skeletal muscle, fibroblasts and umbilical vein endothelial cells, (c) Schematic representation of mouse Ceroxl transcript and the human orthologous sequence. Exon two contains blocks of 60-70% sequence identity; human CEROX1 has an additional 1235 bases of retrotransposed insertions at the 5' end. (d) Distributions of IncRNAs and protein-coding gene expression levels across individuals and tissues in mouse. The black arrow indicates the expression level of Ceroxl. TPM = tags per million, (e) Average expression levels of Ceroxl across mouse tissues. The lighter (third) bar highlights nervous tissue samples whose values for replicates among neurological tissues are shown in the inset: 1- Medulla oblongata, 2- Spinal cord, 3- Diencephalon, 4- Substantia nigra, 5- Microglia, 6- Raphe, 7- Dorsal spinal cord, 8- Corpora quadrigemina, 9- Cortex, 10- Corpus striatum, 11- Visual cortex, 12- Olfactory brain, 13- Cerebellum, 14- Neurospheres sympathetic neuron derived, 15- Neurospheres parasympathetic neuron derived, 16- Neurospheres enteric neuron derived, 17- Astrocytes (cerebellar), 18- Hippocampus, 19- Hippocampal, 20- Ventral spinal cord, 21- Astrocytes, 22- Pituitary gland, 23- Astrocytes (hippocampus), 24- Cortical neurons, 25- Striatal neurons, 26- Schwann cells, 27- Meningeal cells. Error bars indicate s.e.m. (f) Cytoplasmic localisation of mouse Ceroxl compared to a nuclear retained IncRNA, Malatl. Mouse Ceroxl is 15-fold enriched in the cytoplasm of N2A cells (n = 5; error bars s.e.m.).

Figure 2. Overexpression of mouse Ceroxl results in increased levels of transcripts encoding proteins involved in oxidative phosphorylation, (a) Gene ontology analysis indicates a significant enrichment of upregulated genes involved in mitochondrial electron transport, energy production and redox reactions, (b) Four membrane bound multi-subunit complexes (CI, CII, CIII, CIV) are embedded in the inner mitochondrial membrane and facilitate transfer of electrons; three of these subunits are also proton pumps which create the chemiosmotic gradient required for ATP synthase activity, with complex V being ATP synthase. The subunits vary in size and complexity with Complex I (NADH:ubiquinone oxidoreductase) consisting of 45 subunits, Complex II (succinate dehydrogenase) 4 subunits, Complex III (Ubiquinol: cytochrome c oxidoreductase) 11 subunits and Complex IV (Cytochrome c oxidase) 13 subunits. Of 15 oxidative phosphorylation genes whose transcripts were up-regulated following Ceroxl overexpression 53.3% were subunits of Complex I, 13.3% were subunits of Complex III and 33.3%) were subunits of Complex IV. * indicates core subunits that are essential for activity. Note: subunit NDUFA4 has recently been reassigned to mitochondrial complex IV (Balsa et al, 2012). (c) qPCR profiling of 35 complex I subunits and assembly factors (30 nuclear encoded complex I subunits and 5 assembly factors). Transcripts showing a 1.4 fold, or greater, change in expression after overexpression of Ceroxl are present within the boxed shaded area. The transcripts profiled can be characterised into six categories: Core-Q module, subunits responsible for the electron transfer to ubiquinone; Core-N module, subunits responsible for the oxidation of NADH; Supernumerary subunits- those that are additional to the core subunits required for the catalytic role of complex I, but do not play a catalytic role themselves. Many of these subunits may be performing a structural role, but the majority are of unknown function. The supernumerary subunits can be further subdivided into supernumerary - N module, those accessory subunits associated with the NADH oxidation module of CI; supernumerary ACP (acyl carrier protein) - in addition to being a non-catalytic subunit of CI, NDUFABl is also a carrier of the growing fatty acid chain in mitochondrial fatty acid biosynthesis; assembly factor, proteins that are required for the correct assembly and integration of CI. (d,e) Overexpression of Ceroxl results in large increases in the total protein levels of core subunits (d) NDUFSl (average densitometry 1.92, P = 0.0043) and (e) NDUFS3 (average densitometry 2.14, P = 0.013).

NDUFSl is one of three (NDUFSl, NDUFVl, NDUFV2) core components of the N-module of Complex I. NDUFS3 is one of four (NDUFS2, NDUFS3, NDUFS7, NDUFS8) core components of the Complex I Q-module. Error bars s.e.m. (n = 5 biological replicates for control and overexpression). 2-sided t-test; ** P < 0.01, * P < 0.05

Figure 3. Knockdown or overexpression of Ceroxl results in decrease or increase, respectively, in OXPHOS enzyme activity, (a) shRNA mediated knockdown of Ceroxl results in significant decreases of Complexes I and IV enzymatic activities 72 hours post transfection; no significant changes were observed for complex III or the citrate synthase control. Error bars s.e.m. {n = 8 biological replicates for control and overexpression). (b) Oxygen consumption, as measured using an extracellular oxygen sensitive probe, by Ceroxl knockdown N2A cells over a period of 360 mins. As a negative control, N2A cells were treated with the Complex III inhibitor antimycin A to completely impede the flow of electrons to complex IV. This indicates that the O2 consumption measurement specifically reported mitochondrial respiration. O2 consumption was significantly decreased in Ceroxl knockdown cells (P = 0.003, ANCOVA; Control and Overexpression, n = 12 biological replicates, Control + Antimycin A and Overexpression + Antimycin A, n = 3 biological replicates), (c) Enzyme activities in mouse N2A cells 72 hours post-transfection of Ceroxl overexpression construct. Mouse Ceroxl overexpression in N2A cells results in significant increases in the catalytic activities of complexes I (22% increase) and IV (50%) increase). Complexes II, III and citrate synthase show no significant change in activity. Error bars s.e.m. (n = 8 biological replicates), (d) Oxygen consumption, as measured using an extracellular oxygen sensitive probe, by N2A cells overexpressing Ceroxl over a period of 300 mins. O2 consumption was significantly increased in cells overexpressing Ceroxl (P < 0.0001, ANCOVA; Control and Overexpression, n = 12 biological replicates, Control + Antimycin A and Overexpression + Antimycin A, n = 3 biological replicates).

Enzyme activities are represented as a percentage of control enzyme activity. ** R < 0.01, * P < 0.05, ns not significant.

Figure 4. Ceroxl levels modulate cellular oxidative stress, (a) Knockdown of Ceroxl leads to a 20% increase in the production of reactive oxygen species, whilst Ceroxl

overexpression decreases reactive oxygen species production by 45% (error bars s.e.m, n = 12 biological replicates), (b) Protein oxidative damage was also decreased in the overexpression condition compared to the control, as measured by densitometry on western blots against carbonylation of amino acid side chains (error bars s.e.m., n = 6 biological replicates), (c)

Control and Ceroxl overexpressing and knockdown N2A cells were stressed by the addition of electron transport chain (ETC) inhibitors (rotenone, CI inhibitor; malonate, competitive inhibitor of CII; antimycin A, CIII inhibitor; sodium azide, CIV inhibitor; oligomycin, ATP synthase inhibitor), exposure to environmental stress (heat, ultraviolet radiation), or manipulation of extracellular osmolarity (NaCl) or extracellular calcium (CaCb) concentration, for 1 hour and then the viability of the cells measured using the fluorescent indicator Alamar Blue. Error bars s.e.m. {n = 6 biological replicates for control and overexpression). 2-sided t-test; *** P < 0.001, ** P < 0.01, * P < 0.05.

Figure 5. Ceroxl activity is mediated by miRNAs. (a) Overexpression of Ceroxl in mouse wildtype and Dicer^A/A embryonic stem (ES) cells. The overexpression of Ceroxl in wildtype mouse embryonic stem cells results in an increase in complex I subunit transcripts, with no observed change in expression of two control subunits (Ndufs2, Nduftl) that were also unaffected in N2A cells. Overexpression of Ceroxl in Dicer^A/A embryonic stem cells results in no increase in the expression of any complex I subunit. (b) Predicted MREs whose presence is conserved in both the mouse and human Ceroxl. Coloured MREs indicate those MREs whose presence is conserved between mouse and human and whose miRNAs are expressed in N2A cells. The grey predicted MREs represent those that are conserved, but whose miRNAs are not expressed in N2A cells (miR-125a-3p, miR-199/199-5p, miR-302ac/520f, miR-485/485-5p, miR-486/486-5p, miR-501/501-5p, miR-654-3p, miR-675/675-5p).

Figure 6. Mutation of MREs result in a loss of Ceroxl function, (a) Overexpression of the 5xMRE mutant failed to alter the expression of complex I subunits that previously had been shown to increase in expression following Ceroxl overexpression. The numbers of MREs predicted by TargetScan in these transcripts' 3'UTRs for the four conserved, N2A expressed miRNA families are indicated. Due to known widespread noncanonical miRNA binding(Helwak et al, 2013), predictions were extended across the gene body (bracketed MREs). (b)

Overexpression of the 5xMRE mutant failed to alter OXPHOS enzymatic activity compared to the control for any of the complexes measured. A one-way ANOVA was applied to test for differences in activities of the mitochondrial complexes between a control and overexpression of wildtype Ceroxl and the 5xMRE mutant. As expected, enzymatic activity was significantly different for complex I (F [2, 21] = 4.9, P = 0.017) and complex IV (F[2, 20] = 4.6, P = 0.033). A post-hoc Dunnett's test indicated that the overexpression of wildtype Ceroxl resulted in significantly increased complex I and IV activities, while the comparison of the 5xMRE mutant with the control was not significant. There was no significant difference in the activities of complex III ( [2,19]= 0.08, P = 0.5) or citrate synthase CF[2,20]=2.6, P = 0.42). Significance levels, one-way ANOVA, Dunnett's post hoc test * P < 0.05. (c) Schematic of the miR-488 miRNA recognition element in Ceroxl. (d) Luciferase destabilisation assay for both wildtype Ceroxl and the miR-488 MRE mutant Ceroxl. (e) Overexpression of a single miR-488 MRE mutant failed to alter expression of complex I subunits previously shown to increase in expression following Ceroxl overexpression. (f) Overexpression of the single miR-488 Ceroxl mutant failed to recapitulate the observed increase in complex I activity observed for the wildtype transcript. As expected, wildtype enzymatic activity was significantly different for complex I (F [2, 19] = 4=8.8, P = 0.019). A post-hoc Dunnett's test indicated that the overexpression of wildtype Ceroxl resulted in significantly increased complex I activity, while the comparison of miR-488 mRE mutant with the control was not significant. There was no significant difference in the activity of citrate synthase (i [2,21]=1.4, P = 0.28). Significance levels, one-way ANOVA, Dunnett's post hoc test * P < 0.05. (g) Overexpression of miR-488 knocks down all Cerox7-sensitive subunit transcripts, (h) Inhibition of miR-488 increases the expression of most target transcripts compared to the control. Figure 7. CEROXl is highly expressed and also regulates the enzymatic activity of mitochondrial complex I. (a) CEROXl is enriched in the cytoplasm, (b) Relative levels of IncRNA (dark) and protein-coding gene (light) expression across individuals and tissues in human. The black arrow indicates the expression level of CEROXl in the set of 5161 IncRNAs. RPKM: reads per kilobase per million reads, (c) Average expression levels of CEROXl in human tissues. The first, second and fifth bars highlight neurological tissues used to build the inset graph. The inset graphic represents the comparison of gene expression variation among individuals for neurological tissues: 1-Putamen, 2-Caudate nucleus, 3-Nucleus accumbens, 4- Cortex, 5-Substantia nigra, 6-Amygdala, 7-Hippocampus, 8-Spinal cord, 9-Anterior cingulate cortex, 10-Frontal cortex, 11-Hypothalamus, 12-Tibial nerve, 13-Cerebellum, 14-Pituitary gland, 15-Cerebellar hemisphere, (d) OXPHOS enzyme activities in human HEK293 cells after 72 hours of CEROXl overexpression. Overexpression of CEROXl results in significant increases in the activities of complexes I (31% increase) and III (18% increase), with no significant change in other enzyme activities. Error bars s.e.m. (n = 8 biological replicates), t-test: *** P < 0.001, ns=not significant, (e) Reciprocal overexpression of Ceroxl and CEROXl in human or mouse cells respectively results in elevated complex I activity. Error bars s.e.m. (n = 8 biological replicates), t-test: **** P O.0001, ** P < 0.01, * P< 0.05, ns=not significant.

Figure 8. Proposed model for Ceroxl as a post-transcriptional regulator of mitochondrial energy metabolism. We hypothesize that Ceroxl (a) post-transcriptionally maintains energy metabolism homeostasis through buffering the stable ETC transcripts against miRNA-mediated gene silencing. Overexpression of Ceroxl (b) leads to a depletion of the pool of miRNAs that bind ETC transcripts, and therefore a decrease in miRNA mediated gene silencing of the ETC protein-coding transcripts. This has two subsequent effects: 1) a further accumulation of ETC protein coding transcripts, and 2) an increase in the overall translation of ETC subunit proteins owing to decreased miRNA binding to ETC transcripts. More rapid replenishment by

undamaged subunits in mitochondrial complex I, leads to increased efficiency of complex I activity and hence an increase in overall oxygen consumption of the ETC.

Figure 9, Related to Figure 1. Transcript characterisation, (a) PhyloCSF tracks for mouse Ceroxl and Sox8 and human CEROXl AND SOX8 (b) tissue profiling of mouse Ceroxl or human CEROXl (c) transcript levels by quantitative PCR. (d) Expression levels inferred from FANTOM promoter atlas CAGE tag data for two separate Ceroxl transcriptional start sites. The minor 'long' isoform corresponds to NR 045176, whereas the major 'short' isoform corresponds to AK079380, which is referred to here as Ceroxl . Errors bars s.d. on samples with two or more biological samples, (e) Quantitative PCR confirmation of the transcription of two isoforms. Ceroxl is the dominant isoform, with NR 045176 being 10-fold more lowly expressed and is only detectable in brain tissue and N2A cells, (f) To measure the transcript turnover of Ceroxl we inhibited transcription in N2A cells by the addition of the transcriptional inhibitor actinomycin D, and measured the transcript half-life over a period of four hours. Following transcriptional inhibition the remaining transcript was measured by qPCR relative to the stable control gene a-tubulin over a period of 4 hours. Errors s.d. (n = 3 biological replicates), (g) qPCR analysis of sucrose gradient fractions to assess the distribution of Ceroxl and Gapdh transcripts within ribosomal fractions, n = 3, error bars s.e.m.

Figure 10, related to Figure 2. (a) Six shRNA constructs were tested for their ability to knock-down Ceroxl. shRNA sh92, on average, led to a -65% decrease in the level of Ceroxl expression compared to the control, (b) Ceroxl was cloned into a pCAGGS backbone and expression driven from the synthetic pCAG promoter resulting in a 6.8 fold increase in expression compared to the control in transiently transfected N2A cells, (c) Overexpression of Ceroxl has no significant effect on the expression of its close neighbouring protein coding gene, Sox8 or the downstream gene LMFl . (d) Overexpression of Ceroxl slows cell growth. Counting of cells previously seeded to the same density (0.1 x 10⁶ cells/well) 48 hours post transfection demonstrated that there were 45% fewer cells in wells overexpressing Ceroxl (P = 0.02,two- tailed Student's t-test, errors s.d, n = 6). (e) N2A cells overexpressing control and Ceroxl were stained with propidium iodide and subjected to fluorescent activated cell sorting to determine the proportion of cells in various phases of the cell cycle. In an asynchronously cycling cell population there was no significant difference (two sided t-test) between the proportion of cells in G0/G1, S and G2/M phases between control cells and those overexpressing Ceroxl. (f) qPCR validation of the expression increase in OXPHOS subunits indicated by microarray technology. Significant fold changes in gene expression from the microarray data were < 1.8 fold, therefore we set an arbitrary cut off of 1.4 fold change in expression. Errors bars s.e.m (n = 3 biological replicates), (g) Knock down of Ceroxl leads to a decrease in complex I subunit transcripts as measured by qPCR (errors s.e.m, n = 3 biological replicates).

Figure 11, related to Figure 3. Increases in mitochondrial complex I and complex IV activities are not due to an increase in mitochondrial copy number, (a) The mitochondrial to nuclear genome ratio was calculated by amplifying the nuclear genes β-actin and beclin-1 and the mitochondrial genes ND1 and ND2 and calculating the ratio. Primers were designed to regions in ND1 and ND2 that would not amplify any nuclear pseudogenes. No significant difference in mitochondrial genome number was found between control and Ceroxl

overexpression N2A cells (P = 0.99, two-tailed Student's t-test, errors s.d, n = 6). (b) Western blots were performed using an OXPHOS antibody cocktail on protein extracted from control and overexpression N2A cells. Densitometry analysis indicated no significant difference in the amount of OXPHOS complexes between control and experimental cell lines. CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV; CV, complex V; NDUFB8, NADH

dehydrogenase (ubiquinone) 1 beta subcomplex, 8; SDHB, succinate dehydrogenase complex, subunit B; UQCRC2, ubiquinol cytochrome c reductase core protein 2; MTCOl, mitochondrial cytochrome oxidase 1; ATP5A, ATP synthase alpha subunit

Figure 12, Related to Figure 5. (a) Four to six fold overexpression of each of four miRNAs with predicted MREs that are conserved in both mouse and human Ceroxl resulted in a decrease in Ceroxl transcript level. This was not observed when the miRNA miR-137, which has no predicted MREs in Ceroxl, was overexpressed. D(b) Overexpression of Ceroxl and subsequent measurement of mature miRNA levels in mouse N2A cells resulted in a significant decrease in the detectable amount of mature miR-488 (n = 3 biological replicates, error bars s.e.m.). t-test: *** P <0.001, ** P < 0.01. Description of the Sequence Listing

SEQ ID NO: 1 shows the sequence of human miR-488-3p.

SEQ ID NO: 2 shows the sequence of mouse miR-488-3p.

SEQ ID NO: 3 shows the reverse complement sequence of the human miR-488 seed sequence (nucleotides 2 to 8 of SEQ ID NO: 1) and mouse miR-488 seed sequence (nucleotides 2 to 8 of SEQ ID NO: 2). The sequence is CCUUUC A.

SEQ ID NO: 4 shows the cDNA sequence of human Cerox 1 (BC098409).

>gi|68533516|gb|BC098409.1 | Homo sapiens cDNA clone IMAGE:5285809.

SEQ ID NO: 5 shows the cDNA sequence of mouse Ceroxl (AK079380).

>gi|26098466|dbj | AK079380.11 Mus musculus 16 days neonate cerebellum cDNA, RIKEN full-length enriched library, clone:9630053J03 productunclassifiable, full insert sequence.

SEQ ID NO: 6 shows the cDNA sequence of an alternative transcript of mouse Ceroxl (NR_045176). >gi| 347582603 |ref|NR_045176.11 Mus musculus RIKEN cDNA 2810468N07 gene (2810468N07Rik), long non-coding RNA

SEQ ID NO: 7 shows the cDNA sequence of the 5x MRE mutant used in the Examples. It is the same sequence as SEQ ID NO: 5, except the MREs for five different miRNAs are inverted.

Detailed Description of the Invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an inhibitor" includes two or more such inhibitors, or reference to "an oligonucleotide" includes two or more such oligonucleotide and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Method increasing mitochondrial function in a cell

The invention concerns increasing mitochondrial function in a cell. An increased mitochondrial function is typically measured or determined in comparison with a cell of the same type which has not undergone the method of the invention. An increased mitochondrial function is typically measured or determined in comparison with a cell of the same type which has not been contacted with an inhibitor or a polynucleotide in accordance with the invention. An increased mitochondrial function is typically measured or determined in comparison with a normal cell of the same type. The comparison is typically made under the same conditions. Suitable conditions are discussed in more detail below. An increased mitochondrial function may be measured or determined by measuring or determining the mitochondrial function in the same cell in the presence and absence of the inhibitor or polynucleotide.

Mitochondrial function may be increased by any amount. For instance, the function may be increased by at least 10%, at least 20%, at least 30%> at least 40%, at least 50%, at least 60%>, at least 70%, at least 80%, at least 90%, by least 95%, by at least 100%, by at least 150%, by at least 200%), by at least 300%> or more. Mitchondrial function may be measured in any manner. Mitchondrial function is preferably measured as described in the Example.

The method of the invention preferably (a) increases the expression of mitochondrial complex I in the cell. This can be measured using routine methods. An increased expression of mitochrondrial complex I may be measured as an increase in the amount of mitochrondrial complex I messenger RNA and/or protein in the cell. The amount of mRNA can be measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR), such as real time qRT-PCR, northern blotting or microarrays. The amount of protein can be measured using immunohistochemistry, western blotting, mass spectrometry and FACS.

The method of the invention preferably (b) increases mitochondrial oxidative phosphorylation in the cell. This can be measured using an oxidative phosphorylation assay. Suitable assays are known in the art. Mitochondrial oxidative phosphorylation is preferably measured using the assay disclosed in the Example.

The method of the invention preferably (c) reduces or decreases oxidative stress in the cell. For instance, the method of the invention may reduce or decrease oxidative damage to proteins in the cell. Oxidative stress can be measured using standard assays, including the one in the Example.

The method of the invention preferably (d) reduces or decreases the amount of reactive oxygen species (ROS) in the cell. The production of hydrogen peroxide can be used to measure the amount of ROS in the cells. A suitable assay is disclosed in the Example.

The method of the invention preferably (e) increases the amount of glutathione in the cell.

Suitable assays for measuring glutathione are known in the art. For instance, it can be measured using the GSH-Glo™ Glutathione Assay from Promega®.

The method of the invention may comprise any combination of (a) to (e), including {a}, {b}, {c}, {d}, {e}, {a,b}, {a,c}, {a,d}, {a,e}, {b,c}, {b,d}, {b,e}, {c,d}, {c,e}, {d,e}, {a,b,c}, {a,b,d}, {a,b,e}, {a,c,d}, {a,c,e}, {a,d,e}, {b,c,d}, {b,c,e}, {b,d,e}, {c,d,e}, {a,b,c,d}, {a,b,c,e}, {a,b,d,e}, {a,c,d,e}, {b,c,d,e}, and {a,b,c,d,e}.

The effects in (a) to (e) are typically measured or determined in comparison with a cell of the same type which has not undergone the method of the invention. The effects in (a) to (e) are typically measured or determined in comparison with a cell of the same type which has not been contacted with an inhibitor or a polynucleotide in accordance with the invention. The effects in (a) to (e) are typically measured or determined in comparison with a normal cell of the same type. The comparison is typically made under the same conditions. Suitable conditions are discussed in more detail below. The effects in (a) to (e) may be measured or determined by measuring or determining the mitochondrial function in the same cell in the presence and absence of the inhibitor or polynucleotide.

The effects in (a), (b) and (e) may be may be increased by at least 10%, at least 20%, at least 30% at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, by least 95%, by at least 100%, by at least 150%, by at least 200%, by at least 300% or more.

The effects in (c) and (d) may be reduced or decreased by any amount. For instance, the effects may be reduced or decreased by at least 10%, at least 30% at least 40%, at least 50%, at least 60%), at least 70%, at least 80%, at least 90% or at least 95%. The method may abolish the effects in (c) and (d) (i.e. the effect is decreased by 100%).

The invention comprises contacting the cell with an inhibitor of miR-488-3p or a polynucleotide as defined above. The invention typically comprises introducing an inhibitor of miR-488-3p or a polynucleotide as defined above into the cell, i.e. into the cytoplasm and/or into the nucleus of the cell.

The method typically comprises contacting the cell with an effective amount of the inhibitor or polynucleotide. An effective amount is an amount which increases mitochondrial function in the cell as discussed above. An effective amount is an amount which typically has any of effects (a) to (e) or a combination thereof as discussed above.

The cell may be in vitro. If the method is carried out in vitro, the cell may be contacted with the inhibitor or polynucleotide by introducing the inhibitor or polynucleotide to the culture medium. Suitable cell culture media are known in the art, such as Dulbecco's Modified Eagle Medium (DMEM). Techniques for culturing cells are well known to a person skilled in the art. The cells are typically cultured under standard conditions of 37°C, 5% C0₂ in medium supplemented with serum. Suitable medium and conditions are disclosed in the Example.

An in vitro cell may be contacted with an oligonucleotide or polynucleotide using any of the methods discussed below. The oligonucleotide or polynucleotide may be introduced into or contacted with the cell directly, for instance by adding it to the culture medium. Suitable methods for ensureing the oligonucleotide or polynucleotide enters the cell are discussed below. The cell is preferably contacted with the oligonucleotide or polynucleotide by transfecting or transforming the cell such that it expresses or overexpresses the oligonucleotide or

polynucleotide. This is discussed in more detail below. Suitable transfection and transformation techniques are known in the art. The cell is typically transfected or transformed using a vector. Suitable vectors are known in the art (see, for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al, Eds., Greene Publishing and Wiley- lnterscience, New York (1995)) and are disclosed in the Example.

When in vitro, the method of the invention typically increases the capacity of the cell to survive and/or divide. The method inhibits senescence in the cell. The method of the invention typically increases the Hayflick limit of a cell population comprising the cell. The Hayflick limit is is the number of times a normal cell population (typically a normal human cell population) will divide until cell division stops and is discussed in Shay andWright, Nat Rev Mol Cell Biol. 2000 Oct; l(l):72-6.

The method of the invention may concern increasing mitochondrial function in two or more cells, such as 100 or more cells, 1000 or more cells, 5000 or more cells, 10000 or more cells, 5 x 10⁵ or more cells, 1 x 10⁶ or more cells, 2 x 10⁶ or more cells, 5 x 10⁶ or more cells, 1 x 10⁷ or more cells, 2 x 10⁷ or more cells, 5 x 10⁷ or more cells, 1 x 10⁸ or 2 x 10⁸ or more cells. In some instances, the method may increase the mitochondrial function is 1.0 x 10⁷ or more cells, 1.0 x 10⁸ or more cells, 1.0 x 10⁹ or more cells, 1.0 x 10¹⁰ or more cells, 1.0 x 10¹¹ or more cells or 1.0 x 10¹² or more cells or even more. The method comprises administering to the two or more cells an inhibitor or polynucleotide as defined above. The method increases the Hayflick limit of the two or more cells. The method has any of the any of effects (a) to (e) or a combination thereof as discussed above in the two or more cells.

Cell

The cell comprises at least one mitochondrion. The cell typically contains two or more mitochondria. The function of at least one of the mitochondria is increased in the cell. The function of all of the mitochondria in the cell is preferably increased.

The cell is typically derived from {i.e. obtained or extracted from) or present in any eukaryotic organism, such an an animal, a plant, a fungus and a protist.

The cell may be derived from or present in any tissue, such connective tissue, muscle tissue, nervous tissue, epithelial tissue or mineralised tissue. The cell may be derived from or present in any organ system, such as the cardiovascular system (typically including the lungs, heart, blood and blood vessels), digestive system (typically including the mouth, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum and anus), endocrine system (typically including the hypothalamus, pituitary gland, pineal body or pineal gland, thyroid, parathyroids and adrenal glands), excretory system (typically including the kidneys, ureters, bladder and urethra) and immune system (typically including the the lymph nodes, lymphatic system, immune cells, tonsils, adenoids, thymus and spleen), integumentary system (typically including the skin, hair and nails), muscular system, nervous system (typically including the brain, spinal cord and nerves), reproductive system (typically including the the ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, prostate and penis) the respiratory system (typically including the pharynx, larynx, trachea, bronchi, lungs and diaphragm) and the skeletal system (typically including bones, cartilage, ligaments and tendons).

The cell is typically human. However, the cell can be derived from or present in another animal or mammal, such as a commercially farmed animal, such as a horse, a cow, a sheep or a pig, a pet, such as a cat, a dog or a hamster, or a research animal, such as a rat or a mouse. The cell is preferably a human cell or a mouse cell.

Inhibitors of miR-488-3p

miR-488-3p is the 3' part of the miRNA miR-488. The sequence of human miR-488-3p is shown in SEQ ID NO: 1. The sequence of mouse miR-488-3p is shown in SEQ ID NO: 2. Both human and mouse miR-488-3p contain a seed sequence at nucleotides 2 to 8. The reverse complement of this sequence is shown in SEQ ID NO: 3.

The inhibitor is preferably inhbits miR-488-3p which comprises the sequence shown in

SEQ ID NO: 1 or SEQ ID NO: 2.

An inhibitor of miR-488-3p is any molecule that reduces the function of miR-488-3p.

The inhibitor may decrease the function of miR-488-3p by any amount. For instance, the function may be decreased by at least 10%, at least 20%, at least 30% at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. An inhibitor may abolish the function of miR-488-3p (i.e. the function is decreased by 100%). miR-488-3p function may be measured using known techniques. The extent to which an inhibitor affects miR-488-3p may be determined by measuring the function of miR-488-3p in a cell in the presence and absence of the inhibitor. The cell may be any of those discussed above.

The inhibitor may affect the function of miR-488-3p in any manner. For instance, the inhibitor may decrease the amount of miR-488-3p, for instance by decreasing the expression of or increasing the degradation of miR-488-3p. The inhibitor may decrease the activity of miR-

488-3p, for instance by binding to miR-488-3p or the molecule(s) which miR-488-3p interacts or by sequestering miR-488-3p.

The inhibitor may be a competitive inhibitor (which binds the active site of the molecule to which it binds) or an allosteric inhibitor (which does not bind the active site of the molecule to which it binds). The inhibitor may be reversible. The inhibitor may be irreversible.

The inhibitor may decrease the production or expression of miR-488-3p. The inhibitor may decrease the transcription of miR-488-3p. The inhibitor may disrupt the miR-488-3p DNA, for instance by site-specific mutagenesis using methods such as Zinc-finger nucleases or

CRISPR/Cas9 technology. The inhibitor may decrease the mRNA level of miR-488-3p or interfere with the processing of miR-488-3p mRNA, for instance by antisense RNA or RNA interference. This is discussed in more detail below.

The inhibitor may increase the degradation of miR-488-3p. The inhibitor may increase the level of natural inhibitors of miR-488-3p. The inhibitor may decrease the function of miR-

488-3p by binding to it and/or sequestering it or the like.

The inhibitor is preferably a small molecule inhibitor, a protein, an antibody, an oligonucleotide or a polynucleotide. The inhibitor may be an antisense RNA, small interfering

RNA (siRNA) or small hairpin RNA (shRNA). The inhibitor is preferably a long non-coding

RNA (IncRNA). The inhibitor may be identified using the screening methods of the invention discussed below. Antibodies

The inhibitor may be an antibody that specifically binds miR-488-3p or a fragment thereof. The fragment is typically at least 5 nucleotides in length, such as at least 6 or at least 7 nucleotides in length. The antibody preferably binds to the seed sequence of miR-488-3p, i.e. nucleotides 2 to 8 of SEQ ID NO: 1. An antibody "specifically binds" to miR-488-3p or a fragment thereof when it binds with preferential or high affinity to miR-488-3p or the fragment but does not substantially bind, does not bind or binds with only low affinity to other molecules, such as other miRNA. An antibody binds with preferential or high affinity if it binds with a Kd of 1 x 10-7 M or less, more preferably 5 x 10-8 M or less, more preferably 1 x 10-8 M or less or more preferably 5 x 10-9 M or less. An antibody binds with low affinity if it binds with a Kd of 1 x 10-6 M or more, more preferably 1 x 10-5 M or more, more preferably 1 x 10-4 M or more, more preferably 1 x 10-3 M or more, even more preferably 1 x 10-2 M or more. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of compounds, such as antibodies or antibody constructs and oligonucleotides are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993).

The antibody may be, for example, a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody, a CDR-grafted antibody or a humanized antibody. The antibody may be an intact immunoglobulin molecule or a fragment thereof such as a Fab, F(ab')2 or Fv fragment. The antibody may be a single chain antibody.

Antibodies for use in the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising an antibody in a host animal against miR-488-3p or a fragment thereof.

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with miR-488-3p or a fragment thereof and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with miR-488-3p or a fragment thereof, blood subsequently removed from the animal and the IgG fraction purified. A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat, mouse, guinea pig, chicken, sheep or horse. If desired, miR- 488-3p or a fragment thereof may be administered as a conjugate in which it is coupled to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

Protein and polynucleotide inhibitors

The inhibitor of miR-488-3p is preferably an oligonucleotide or a polynucleotide. An oligonucleotide is a short nucleotide polymer which typically has 50 or fewer nucleotides, such 40 or fewer, 30 or fewer, 22 or fewer, 21 or fewer, 20 or fewer, 10 or fewer or 5 or fewer nucleotides. The oligonucleotide used in the invention may be 20 to 25 nucleotides in length, more preferably 21 or 22 nucleotides in length. The nucleotides can be naturally occurring or artificial. Nucleotides and the ways in which they may be linked are defined below with reference to polynucleotides.

A polynucleotide, such as a nucleic acid, is a polymer comprising two or more nucleotides. The polynucleotide may be any length, i.e. may contain any number of nucleotides. The polynucleotide preferably comprises 200 or more nucleotides, such as 300 or more, 40 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more or 3000 or more nucleotides. Suitable lengths are discussed below with reference to the specific sequences used in the invention. The nucleotides can be naturally occurring or artificial.

A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'O-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a

ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (HDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5- methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP),

deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP),

deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate

(dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2' -deoxycytidine monophosphate, 5- methyl-2' -deoxycytidine diphosphate, 5 -methyl-2' -deoxycytidine triphosphate, 5- hydroxymethyl-2' -deoxycytidine monophosphate, 5 -hydroxymethyl-2' -deoxycytidine diphosphate and 5 -hydroxymethyl-2 '-deoxycytidine triphosphate. The nucleotides are preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.

The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'amino pyrimidines (such as 2'-amino cytidine and 2'-amino uridine), 2'-hyrdroxyl purines (such as , 2'-fluoro pyrimidines (such as 2'- fluorocytidine and 2'fluoro uridine), hydroxyl pyrimidines (such as 5'-a-P-borano uridine), 2'- O-methyl nucleotides (such as 2'-0-methyl adenosine, 2'-0-methyl guanosine, 2'-0-methyl cytidine and 2'-0-methyl uridine), 4'-thio pyrimidines (such as 4'-thio uridine and 4'-thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2'-deoxy uridine, 5-(3-aminopropyl)-uridine and l,6-diaminohexyl-N-5-carbamoylmethyl uridine).

One or more nucleotides in the oligonucleotide or polynucleotide can be oxidized or methylated. One or more nucleotides in the oligonucleotide or polynucleotide may be damaged. For instance, the oligonucleotide or polynucleotide may comprise a pyrimidine dimer. Such dimers are typically associated with damage by ultraviolet light.

The nucleotides in the oligonucleotide or polynucleotide may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.

The oligonucleotide or polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The oligonucleotide or polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), ηιο ΐιοΐίηο nucleic acid or other synthetic polymers with nucleotide side chains. The oligonucleotide or polynucleotide may be single stranded or double stranded. The oligonucleotide or polynucleotide may comprise a hairpin at one or both ends.

Oligonucleotide or polynucleotide sequences may be derived and replicated using standard methods in the art, for example using PCR involving specific primers. It is

straightforward to generate oligonucleotide or polynucleotide sequences using such standard techniques.

The amplified sequences may be incorporated into a recombinant replicable vector such as a cloning vector. The vector may be used to replicate the oligonucleotide or polynucleotide in a compatible host cell. Thus oligonucleotide or polynucleotide sequences may be made by introducing the oligonucleotide or polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells for cloning of oligonucleotides or polynucleotides are known in the art.

Alternatively, oligonucleotides or polynucleotides may be purchased. Suitable sources include, but are not limited to, Sigma- Aldrich®, Invitrogen® and Life Technologies®.

The oligonucleotide or polynucleotide used in the invention preferably specifically hybridises to miR-488-3p and/or a seed sequence contained therein. The oligonucleotide or polyucleotide used in the invention preferably specifically hybridises to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The oligonucleotide or polyucleotide used in the invention preferably specifically hybridises to the seed sequence shown in nucleotides 2 to 8 of SEQ ID NO: 1. miR-488-3p, the seed sequence contained therein, SEQ ID NO: 1 or 2 or the seed sequence shown in nucleotides 2 to 8 of SEQ ID NO: 1 are hereafter called the target sequence.

The oligonucleotide or polynucleotide may specifically hybridise to all of the target sequence. In such embodiments, the oligonucleotide or polynucleotide is typically the same length as or longer that the target sequence.

The oligonucleotide or polynucleotide may specifically hybridise to a part of the target sequence. The length of the part of the target sequence typically corresponds to the length of the oligonucleotide or polynucleotide. For instance, a 7 nucleotide oligonucleotide typically specifically hybridises to a 7 nucleotide target sequence within miR-488-3p. The part is typically at least 5 nucleotides in length, such as at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 nucleotides in length. Part of the oligonucleotide or polynucleotide may specifically hybridise to all of or a part of the target sequence.

An oligonucleotide or polynucleotide "specifically hybridises" to a target sequence or a part thereof when it hybridises with preferential or high affinity to the target sequence or part thereof but does not substantially hybridise, does not hybridise or hybridises with only low affinity to other sequences.

An oligonucleotide or polynucleotide "specifically hybridises" if it hybridises to the target sequence or a part thereof with a melting temperature (T_m) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its T_m for other sequences. More preferably, the oligonucleotide or polynucleotide hybridises to the target sequence or a part thereof with a T_m that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its T_m for other sequences. Preferably, the oligonucleotide or polynucleotide hybridises to the target sequence or part thereof with a T_m that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its T_m for a sequence which differs from the target sequence or part thereof by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. The oligonucleotide or polynucleotide typically hybridises to the target sequence or part thereof with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C. T_m can be measured

experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available T_m calculators, such as those available over the internet.

Conditions that permit the hybridisation are well-known in the art (for example,

Sambrook et al., 2001, supra). Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCl and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na⁺) to 2X (0.33 M Na⁺) SSC (standard sodium citrate) at 50 °C. Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCl, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na⁺) to IX (0.1650 M Na⁺) SSC at 55 °C. Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37 °C, followed by a wash in 0.1X (0.0165 M Na⁺) SSC at 60 °C.

The oligonucleotide or polynucleotide may comprise a sequence which is substantially complementary to the target sequence or part thereof. Typically, the oligonucleotides or polynucleotides are 100% complementary to the target sequence or part thereof. However, lower levels of complementarity may also be acceptable, such as 95%, 90%, 85% and even 80%.

Complementarity below 100% is acceptable as long as the oligonucleotides or polynucleotides specifically hybridise to the target sequence or part thereof. An oligonucleotide may therefore have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches with the target sequence or part thereof.

The oligonucleotide or polynucleotide preferably comprises the sequence shown in SEQ

ID NO: 3 or a variant thereof which has 1, 2 or 3 nucleotide substitutions or deletions compared with SEQ ID NO: 3. SEQ ID NO: 3 shows the reverse complement to the same seed sequence in SEQ ID NOs: 1 and 2. SEQ ID NO: 3 specifically hybridises to the seed sequence in SEQ ID NOs: 1 and 2. The variant preferably has 1 or 2 nucleotide substitutions or deletions or only one nucleotide substitution or deletion compared with SEQ ID NO: 3. The variant preferably comprises nucleotides 1 to 6 of SEQ ID NO: 3 or nucleotides 2 to 7 of SEQ ID NO: 3. SEQ ID NO: 3 is RNA. The oligonucleotide or polynucleotide preferably comprises the sequence shown in SEQ ID NO: 3 in which uracil (U) is replaced by thymine (T) or a variant thereof which has 1,

2 or 3 nucleotide substitutions or deletions compared with SEQ ID NO: 3 in which uracil (U) is replaced by thymine (T). The variant preferably has 1 or 2 nucleotide substitutions or deletions or only one nucleotide substitution or deletion compared with SEQ ID NO: 3 in which uracil (U) is replaced by thymine (T). The variant preferably comprises nucleotides 1 to 6 of SEQ ID NO:

3 in which uracil (U) is replaced by thymine (T) or nucleotides 2 to 7 of SEQ ID NO: 3 in which uracil (U) is replaced by thymine (T).

The oligonucleotide or polynucleotide which specifically hybridises to the target sequence may be contacted with or introduced into the cell directly. Suitable methods for delivering oligonucleotides or polynucleotides into cells are known in the art. They include, but are not limited to, chemical-based methods, such as using cyclodextrin, cationic polymers (such as DEAE-dextran or polyethylenimine), liposomes, cationic lipsomes, nanoparticles, calcium phosphate or dendrimers, non-chemical methods, such as electroporation (gene electrotransfer), cell squeezing, sonoporation, optical transfection, impalefection and hydrodynamic delivery, and particle-based methods, such as using a gene gun, magnetofection (or magnet assisted transfection) and particle bombardment.

When introduced directly, the oligonucleotide or polynucleotide typically inhibits miR- 488-3p by specficially hybridising to miR-488-3p or a part thereof and preventing miR-488-3p from carrying out its function or action. The oligonucleotide or polynucleotide typically inhibits miR-488-3p by sequestering it.

The oligonucleotide or polynucleotide which specifically hybridises to the target sequence or part thereof may be expressed or overexpressed in the cell. For instance, the cell may be contacted, transfected or transformed with an oligonucleotide or polynucleotide which encodes the oligonucleotide or polynucleotide which specifically hybridises to the target sequence or part thereof. The encoding oligonucleotide or polynucleotide may be present in a vector as discussed above. An oligonucleotide or a polynucleotide is overexpressed if the cell normally expresses it but the level or amount of expression is increased in accordance with the invention. An oligonucleotide or a polynucleotide is overexpressed if the cell normally expresses it but the level or amount of its expression is increased following transfection or transformation of the cell with an oligonucletide or polyncueltoide which encodes it. The encoding oligonucleotide or polynucleotide or vector may be introduced into the cell using any of the techniques discussed above or below.

The polynucleotide preferably comprises the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence. If the polynucleotide or variant is RNA, the thymine (T) in the sequence shown in SEQ ID NO: 4, 5 or 6 is typically replaced by uracil (U).

SEQ ID NOs: 4, 5 and 6 show the cDNA sequences of the long non-coding RNAs (IncRNAs) human Cerox 1, mouse Ceroxl and an alternative transcript of mouse Ceroxl respectively. Human Cerox 1, mouse Ceroxl and the alternative transcript of mouse Ceroxl specifically hybridise to miR-488-3p.

The cell may be contacted directly with a polynucleotide which comprises the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence, i.e. without the need for its expression or overexpression. The polynucleotide or variant will then hybdrise to to miR-488-3p and inhibit it as discussed above.

A polynucleotide which comprises the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence is preferably expressed or overexpressed by the cell. The method preferably comprises transfecting or transforming the cell with a polynucleotide which encodes the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence. The method preferably comprises expressing or overexpressing in the cell a polynucleotide which encodes a IncRNA (human Cerox 1, mouse Ceroxl, the alternative transcript of mouse Ceroxl or a variant thereof) which comprises the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence. In the IncRNA, the thymine (T) in the sequence shown in SEQ ID NO: 4, 5 or 6 is typically replaced by uracil (U). The encoding sequence preferably comprises a sequence which is the complement of or is complementary to SEQ ID NO: 4, 5 or 6 or variant thereof as defined above. The encoding sequence preferably comprises a sequence which is the reverse complement of SEQ ID NO: 4, 5 or 6 or variant thereof as defined above. The encoding sequence is preferably DNA.

The variant of SEQ ID NO: 4, 5 or 6 specifically hybridises to miR-488-3p or a part thereof. Over the entire length of the nucleotide sequence of SEQ ID NO: 4, 5 or 6, the variant will preferably be at least 60% homologous to that sequence based on nucleotide identity. More preferably, the variant may be at least 70%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on nucleotide identity to the sequence of SEQ ID NO: 4, 5 or 6 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, nucleotide identity over a stretch of 500 or more, for example 800, 900, 100, 1500, 1800, 2000 or 2500 or more, contiguous nucleotides ("hard homology").

Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387- 395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information

(http://www.ncbi.nlm.nih.gov/).

The variant preferably comprises a sequence which specifically hybridises to miR-488-3p or a part thereof as discussed above. The variant preferably comprises a sequence which is complmentary to miR-488-3p or a part thereof as discussed above. The variant preferably comprises a sequence which specifically hybridises to or is complementary to the seed sequence of miR-488-3p, i.e. nucleotides 2 to 8 of SEQ ID NO: 1. The variant most preferably comprises the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 3 in which uracil (U) is replaced by thymine (T).

Antisense and RNAi

It is clear from above that the inhibition of miR-488-3p may involve antisense and RNA interference (RNAi) technology. This is well known in the art and standard methods can be employed to inhibit miR-488-3p. Both antisense and siRNA technology can be designed to interfere with miRNA such as miR-488-3p. Antisense oligonucleotides interfere with RNA by binding to (hybridising with) a section of the RNA. The antisense oligonucleotide is therefore designed to be complementary to the target RNA or a part of it (although the oligonucleotide does not have to be 100% complementary as discussed below). The antisense oligonucleotide may be a section of the cDNA which encodes miR-488-3p or any of the oligonucleotides discussed above which specifically hybridise to miR-488-3p. Again, the oligonucleotide sequence may not be 100% identical to the cDNA sequence.

RNAi involves the use of double-stranded RNA, such small interfering RNA (siRNA) or small hairpin RNA (shRNA), which can bind to the mRNA and inhibit protein expression.

The antisense or RNAi oligonucleotide may be contacted with or introduced into the cell. The antisense or RNAi oligonucleotide may be expressed or overexpressed by the cell as discussed above.

The antisense or RNAi oligonucleotide can be a nucleic acid, such as any of those discussed above. The oligonucleotide is preferably RNA.

The antisense or RNAi oligonucleotide may be single stranded. The oligonucleotide may be double stranded. The antisense or RNAi oligonucleotide may compirse a hairpin. SEQ ID NOs: 4, 5 and 6 and related sequences

The invention also concerns increasing mitochondrial function in a cell by contacting the cell with a polynucleotide which (a) comprises the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a). The sequence in (a) is preferably RNA. If the polynucleotide or variant is RNA, the thymine (T) in the sequence shown in SEQ ID NO: 4, 5 or 6 is typically replaced by uracil (U). The encoding sequence in (b) may be the complement of (or complementary to) or the reverse complement of a sequence of (a). The encoding sequence in (b) is preferably DNA. Polynucleotides, such as IncRNAs, comprising the sequences shown in SEQ ID NOs: 4, 5 and 6 are capable of binding sequences other than miR-488-3p and so may increase mitochondrial function by affecting those sequences. Any of the embodiments discussed above equally apply to this embodiment.

The invention also concerns increasing mitochondrial function in a cell by contacting the cell with a compound which increases the amount of the long non-coding RNA (IncRNA) comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell. In the IncRNA, the thymine (T) in the sequence shown in SEQ ID NO: 4, 5 or 6 is typically replaced by uracil (U). Suitable compounds and how they may be identified are discussed in more detail below.

Method of increasing mitochondrial function in an individual

The invention also concerns increasing mitochondrial function in an individual. Any of the methods discussed above may be used in vivo. The invention may concern increasing mitochondrial function in one or more cells or one or more tissues of the individual. The invention may concern increasing mitochondrial function in one or more of organs or one or more organ systems of the individual. Suitable tissues, organs and organ systems are discussed above. Any number and combination of the effects discussed above may occur in the one or more cells, tissues, organs or organ systems of the individual.

Individual

The invention may be carried out in any individual. The individual is typically human. However, the individual can be another animal or mammal, such as a commercially farmed animal, such as a horse, a cow, a sheep or a pig, a pet, such as a cat, a dog or a hamster, or a research animal, such as a rat or a mouse. The individual is preferably a human or a mouse.

The individual may have decreased mitochondrial function compared with a normal individual of the same species and gender. The decreased mitonchodrial function may be present in one or more cells, tissues, organs or organ systems. The individual may have a normal mitochondrial function.

Therapy

The invention preferably concerns treating or preventing a disease or disorder associated with mitochondrial dysfunction in a patient. The patient may be any of the individuals discussed above. Any disease or disorder associated with mitochondrial dysfunction may be treated or prevented. Any of the embodiments discussed above may be used to treat or prevent the disease or disorder.

The invention also provides use of an inhibitor of miR-488-3p in the manufacture of a medicament for treating or preventing a disease or disorder associated with mitochondrial dysfunction in a patient.

The invention also provides an inhibitor of miR-488-3p for use in a method of treating or preventing a disease or disorder associated with mitochondrial dysfunction in a patient.

The invention also provides use of a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) in the manufacture of a medicament for treating or preventing a disease or disorder associated with mitochondrial dysfunction in a patient.

The invention also provides a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) for use in a method of treating or preventing a disease or disorder associated with mitochondrial dysfunction in a patient.

The sequences in (a) and/or (b) may be any of those discussed above.

The disease or disorder is preferably neuropathic, retinopathic, hepatoneuropathic, neuropathic and myopathic, myopathic, cardiomyopathic, myopathic and immunopathic, neuropsychiatric, an autoimmune disease or disorder or nephropathic.

The neuropathic disease or disorder is preferably Alzheimer's disease (AD),

aminoglycoside-induced deafness (AID), amyotrophic lateral sclerosis (ALS), ataxia, myoclonus and deafness (AMDF), autosomal dominant cerebellar ataxia (ADCA), diabetes mellitus and deafness (DMDF), Fahr's syndrome (Fahr disease), Huntingdon's disease, late-onset

encephalomyopathies, Leber's hereditary optic neuropathy (LHON), Leigh syndrome (Leigh's disease), leukodystrophy, leukoencephalopathies, maternally inherited diabetes and deafness (MIDD), maternally inherited Leigh syndrome (MILS), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial familial infantile bilateral striatal necrosis (FSBN), myoclonic epilepsy and psychomotor regression (MEPR), Parkinson's disease (PD), progressive myoclonus epilepsy (PME), sensorineural hearing loss (S HL) or sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO).

The retinopathic disease or disorder is preferably deafness and retinitis pigmentosa, neuropathy, ataxia and retinitis pigmentosa (NARP) or progressive encephalopathy (PEM).

The hepatoneuropathic disease or disorder is preferably Alpers' disease.

The neuropathic and myopathic disease or disorder is preferably chronic intestinal pseudoobstruction with myopathy and ophthalmoplegia (CIPO), coenzyme Q deficiency, fatal infantile multisystem disorder or Leber's hereditary optic neuropathy and dystonia (LDYT), MERRF/MELAS overlap syndrome (MERME), mitochondrial cytopathy (MC), mitochondrial encephalomyopathy, mitochondrial encephalopathy, mitochondrial encephalopathy, lactate acidosis and stroke (MELAS), mitochondrial neurogastrointestinal encephalopathy (MNGIE), myoclonic epilepsy with ragged-red fibres (MERRF), Pearson syndrome, progressive dementia and chorea (DEMCHO) or progressive dystonia.

The myopathic disease or disorder is preferably chronic progressive external

ophthalmoplegia (CPEO), exercise intolerance (EXIT), fatal infantile myopathy, Kearns-Sayre syndrome (KSS) (oculocraniosomatic neuromuscular disorder with ragged red fibres), lethal infantile mitochondrial myopathy (LFMM), maternal inherited myopathy (MFM), mitochondrial myopathy (MM) or reversible infantile myopathy.

The cardiomyopathic disease or disorder is preferably dilated cardiomyopathy, fatal infantile cardiomyopathy plus (FICP), hypertrophic cardiomyopathy with myopathy, infantile histiocytoid cardiomyopathy, maternal inherited cardiomyopathy (MICM) or maternal inherited hypertrophic cardiomyopathy (MHMC).

The myopathic and immunopathic disease or disorder is preferably Barth syndrome (BTHS).

The neuropsychiatric disease or disorder is preferably bipolar disorder (BD), dysthymia (persistent depressive disorder), major depressive disorder (MDD), mood disorders, posttraumatic stress disorder (PTSD), recurrent depression or schizophrenia (SZ).

The autoimmune disease or disorder is lupus, multiple sclerosis, rheumatoid arthritis or Sjogren's syndrome.

The nephropathic disease or disorder is focal segmental glomerulosclerosis (FSGS) or tubulointerstitial nephritis.

The disease or disorder is preferably cancer, cardiovascular disease (CVD), carnitine palmitoyltransferase I/II deficiencies, coronary heart disease (CHD), diabetes mellitus (DM), GRACILE syndrome, hypoxia, metabolic syndrome, myocardial infarction (MI), myoglobinuria, obesity, stroke or sudden infant death syndrome (SIDS).

Administration

In the method of the invention, the inhibitor or polynucleotide is administered to the individual or patient. An inhibitor or polynucleotide may be administered to the individual or patient in any appropriate way. The inhibitor or polynucleotide may be administered in a variety of dosage forms. Thus, it can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. It may also be administered byenteral or parenteral routes such as via buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical or other appropriate administration routes. The inhibitor or polynucleotide may be administered directly into the cell, tissue, organ or organ system to be treated. A physician will be able to determine the required route of administration for each particular individual or patient.

The formulation of an inhibitor or polynucleotide will depend upon factors such as the nature of the exact inhibitor or polynucleotide, etc. An inhibitor or polynucleotide may be formulated for simultaneous, separate or sequential use with other inhibitors or polynucleotides defined herein or with other treatments as discussed in more detail below.

An inhibitor or polynucleotide is typically formulated for administration with a pharmaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active substance, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, lauryl sulphates; and, in general, non-toxic and

pharmacologically inactive substances used in pharmaceutical formulations. Such

pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.

Oral formulations include such normally employed excipients as, for example,

pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.

Capsules, tablets and pills for oral administration to an individual may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.

Polynucleotides or oligonucleotides may be naked nucleotide sequences or be in combination with cationic lipids, polymers or any of the other targeting systems discussed above. They may be delivered by any available technique. For example, the polynucleotide or oligonucleotide may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the polynucleotide or oligonucleotide may be delivered directly across the skin using a delivery device such as particle-mediated gene delivery. The polynucleotide or oligonucleotide may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, or intrarectal administration.

Uptake of polynucleotide or oligonucleotide constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents.

Examples of these agents include cationic agents, for example, calcium phosphate and DEAE- Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the polynucleotide or oligonucleotide to be administered can be altered.

An effective amount of the inhibitor or polynucleotide is typically administered to the individual or patient. An effective amount is an amount effective to increase the mitochondrial function in the releavant cell, tissue, organ or organ system of the individual or patient.

A therapeutically effective amount of the inhibitor or polynucleotide is typically administered to the individual or patient. A therapeutically effective amount is an amount effective to ameliorate one or more symptoms of the disease or disorder. A therapeutically effective amount is preferably an amount effective to abolish one or more of, or preferably all of, the symptoms of the disease or disorder.

A prophylactically effective amount of the the inhibitor or polynucleotide may be administered to the individual or patient. A prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the disease or disorder.

The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the individual or patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular individual or patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific inhibitor or polynucleotide, the age, weight and conditions of the subject to be treated and the frequency and route of administration. The dose may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered hourly. Preferably, dosage levels of inhibitors are from 5 mg to 2 g.

Typically polynucleotide or oligonucleotide inhibitors are administered in the range of 1 pg to 1 mg, preferably to 1 pg to 10 μg nucleic acid for particle mediated delivery and 10 μg to 1 mg for other routes.

Senescence and ageing Decreased mitochondrial function is associated with senescence and ageing (Brunk and Terman, European Journal of Biochemistry, Volume 269, Issue 8, pages 1996-2002, April 2002). Senescence is the gradual deterioration of function of cells and the organism comprising those cells. The invention may therefore concern inhibiting senescence in one or more cells, tissues, organs or organ systems of the individual. The invention may therefore concern inhibiting senescence in the individual. The one or more cells, tissues, organs or organ systems may be any of those discussed above. The invention also concerns inhibiting ageing.

Combination therapy

The inhibitor or polynucleotide may be used in combination with one or more other therapies intended to treat the same patient or disease or disorder. By a combination is meant that the therapies may be administered simultaneously, in a combined or separate form, to the patient. The therapies may be administered separately or sequentially to the patient as part of the same therapeutic regimen. For example, an inhibitor or polynucleotide may be used in combination with another therapy intended to treat the disease or disorder. The other therapy may be a general therapy aimed at treating or improving the condition of the individual or patient. For example, treatment with methotrexate, glucocorticoids, salicylates, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, other DMARDs, aminosalicylates,

corticosteroids, and/or immunomodulatory agents (e.g., 6-mercaptopurine and azathioprine) may be combined with the inhibitor or polynucleotide. The other therapy may be a specific treatment directed at the disease or disorder suffered by the individual or patient, or directed at a particular symptom of the disease or disorder.

If the disease is cancer, the inhibitor may be used in combination with chemotherapy, radiation therapy and surgery. The inhibitor may also be used in combination with other cancer drugs.

Diagnostic methods

The invention also concerns determining whether or not a cell has decreased

mitochondrial function or mitochondrial dysfunction. The amount of miR-488-3p and/or the amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell is measured. Since the IncRNA is RNA, the thymine (T) in the sequence shown in SEQ ID NO: 4, 5 or 6 is typically replaced by uracil (U).

An increased amount of miR-488-3p compared with a normal cell of the same type indicates that the cell has decreased mitochondrial function or mitochondrial dysfunction. A decreased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 compared with a normal cell of the same type indicates that the cell has decreased mitochondrial function or mitochondrial dysfunction. The cell may have both an increased amount of miR- 488-3p and a decreased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 compared with a normal cell of the same type. miR-488-3p preferably comprises SEQ ID NO: 1 or SEQ ID NO: 2. If the cell is human, the invention preferably concerns measuring the amount of SEQ ID NO: 1 or the IncRNA comprising the sequence shown in SEQ ID NO: 4. If the cell is a mouse cell, the invention preferably concerns measuring the amount of SEQ ID NO: 2 or the IncRNA comprising the sequence shown in SEQ ID NO: 5 or 6. The cell may be any of those discussed above and may present in or derived from any of the tissues, organs or organs systems discussed above. The method may be carried out in vitro.

The amount of the relevant RNA in the cell can be measured using known techniques, such as quantitative reverse transcription polymerase chain reaction (qRT-PCR), such as real time qRT-PCR, northern blotting or microarrays.

If the cell is determined as having a decreased mitochondrial function or mitochondrial dysfunction, the mitochondrial function in the cell can be increased as discussed above.

The invention also concerns determining whether or not a patient has or is likely to develop a disease or disorder associated with mitochondrial dysfunction. The invention therefore concerns diagnosing or prognosing diseases or disorders associated with mitochondrial dysfunction. The amount of miR-488-3p and/or the amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 is measured in a cell sample from the patient. The sample typically comprises one or more cells in which mitochondrial function is expected to be decreased by the disease or disorder. An increased amount of miR-488-3p compared with a normal cell sample of the same type, i.e. a cell sample of the same type from a patient without the disease or disorder, indicates that the patient has or is likely to develop the disease or disorder. A decreased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 compared with a normal cell sample of the same type indicates that the patient has or is likely to develop the disease or disorder. The cell sample may have both an increased amount of miR-488-3p and a decreased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 compared with a normal cell sample of the same type. The disease or disorder may be any of those discussed above.

miR-488-3p preferably comprises SEQ ID NO: 1 or SEQ ID NO: 2. If the patient is human, the invention preferably concerns measuring the amount of SEQ ID NO: 1 or the IncRNA comprising the sequence shown in SEQ ID NO: 4. If the patient is a mouse, the invention preferably concerns measuring the amount of SEQ ID NO: 2 or the IncRNA comprising the sequence shown in SEQ ID NO: 5 or 6. Any of the methods of measuring RNA discussed above may be used.

The cell sample may comprise any of the cells discussed above and may derived from any of the tissues, organs or organs systems discussed above.

Diagnosis and treatment

In a preferred embodiment, the invention concerns treating or preventing a disease or disorder associated with mitochondrial dysfunction in a patient by determining whether or not the patient has or is likely to develop a disease or disorder associated with mitochondrial dysfunction using the method of the invention and, if the patient has or is likely to develop the disease or disorder, administering to the patient an inhibitor of miR-488-3p or a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a). Any of the diagnostic, prognostic, therapeutic and prophylactic embodiments discussed above equally apply to this embodiment. The sequences in (a) and/or (b) may be any of those discussed above.

Kits

The present invention also provides kits for increasing mitochondrial function in a cell or a patient. The kit may comprise (a) an inhibitor of miR-488-3p and (b) means for measuring mitochondrial function. The kit may comprise (i) a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) and (ii) means for measuring mitochondrial function. The sequences in (a) and/or (b) may be any of those discussed above. The inhibitor or polynucleotide may be any of those discussed above.

The kit comprises a means (or reagent) for measuring mitochondrial function. This means (or reagent) may be any suitable means or reagent for the use in determining

mitochondrial function as described above. The kit may comprise an antibody which

specifically binds mitochondrial complex I, an oligonucleotide or polynucleotide probe for mitochrondrial complex I, one or more components of a mitochondrial oxidative

phosphorylation assay, one or more components of an oxidative stress or reactive oxygen species (ROS) assay or one or more components of a glutathione assay. Suitable components are disclosed in the Example. The kit may additionally comprise one or more other reagents or instruments which enables the method mentioned above to be carried out. Such reagents include means for taking a sample from the patient, suitable buffers, means to extract/isolate RNA or DNA from a sample or a support comprising wells on which quantitative reactions can be done. The kit may, optionally, comprise instructions to enable the kit to be used in the method of invention or details regarding patients on which the method may be carried out. The kit may comprise primers and reagents for PCR, qPCR (quantitative PCR), RT-PCR (reverse-transcription PCR), qRT-PCR (quantitative reverse-transcription PCR) reaction or RNA sequencing. Screening methods

The invention also provides screening methods. One method of the invention concerns identifying a compound which is capable of increasing mitochondrial function in a cell. The compound may be any of those discussed above with reference to inhibitors of miR-488-3p. The cell may be any of those discussed above. The method comprises contacting the cell with the compound and measuring the amount of miR-488-3p and/or the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell. In the IncRNA, the thymine (T) in the sequence shown in SEQ ID NO: 4, 5 or 6 is typically replaced by uracil (U).

Suitable measurement methods are discussed above. A decreased amount of miR-488-3p in the presence of the compound compared with the absence of the compound indicates that the compound is capable of increasing mitochondrial function in the cell. An increased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the presence of the compound compared with the absence of the compound indicates that the compound is capable of increasing mitochondrial function in the cell.

Another method concerns identifying a compound as an inhibitor of miR-488-3p in a cell. The compound may be any of those discussed above with reference to inhibitors of miR- 488-3p. The cell may be any of those discussed above. The method comprises contacting the cell with the compound and measuring the amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell. In the IncRNA, the thymine (T) in the sequence shown in SEQ ID NO: 4, 5 or 6 is typically replaced by uracil (U).

Suitable measurement methods are discussed above. An increased amount of the

IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the presence of the compound compared with the absence of the compound indicates that the compound is capable of inhibiting miR-488-3p in the cell.

The screening methods of the invention can be carried out using standard screening methods, such as those used to screen stimulators of utrophin expression. The invention also provides a compound or an inhibitor identified using a method of the invention. The compounds or inhibitors of the invention can be used in any of the methods disclosed above.

Example

Here we describe a novel mammalian conserved IncRNA, termed Ceroxl (competitive endogenous regulator of oxidative phosphorylation 1), which co-ordinately regulates the abundance of at least 12 transcripts encoding complex I subunits of the mitochondrial electron transport chain. In both mouse and human cells Ceroxl regulates complex I catalytic activity by modulating complex I transcript levels via a miRNA-mediated decoying mechanism. Ceroxl knockdown decreases enzymatic activities of complexes I and IV. Conversely, elevation of Ceroxl levels increases enzymatic activities of these complexes, halves cellular oxidative stress, and protects cells against the cytotoxic effects of the complex I inhibitor rotenone. Ceroxl is the first, to our knowledge, reported IncRNA modulator of normal mitochondrial energy metabolism homeostasis and cellular redox state. Its miRNA-dependent role both illustrates how RNA- interaction networks regulate energy homeostasis and that IncRNAs represent novel targets for modulating OXPHOS activity.

EXPERIMENTAL PROCEDURES

Human and mouse gene expression profiling

5' and 3' ends of the mouse and human IncRNAs were confirmed by 5' and 3' RACE using the GeneRacer™ Kit (Invitrogen) according to manufacturer's instructions. RNA from cell lines was extracted using RNeasy mini kit (Qiagen) and cDNA synthesis for all samples was performed using a QuantiTect Reverse Transcription kit (Qiagen). Differentially expressed genes (Benjamini-Hochberg adjusted -value <0.05) were identified on mouse gene 1.0 ST arrays (Affymetrix) between mouse IncRNA overexpression and control cells using Limma from the bioconductor package. All subsequent gene expression levels were determined by real-time quantitative PCR, using SYBR® Green Master Mix (Applied Biosystems). Average expression across 46 human tissues and individuals according to the Pilot 1 data from the GTEx Consortium (http://www.gtexportal.org/home/, GTEx Consortium 2013) was computed for both protein- coding genes and intergenic long non-coding RNAs from the Ensembl release 75 annotation (Flicek et al., 2014). For mouse, normalized numbers of CAGE tags across 399 cell types and tissues from the FANTOM5 Consortium (Kawaji et al, 2014) were used to approximate expression levels for protein-coding genes and intergenic IncRNAs from the Ensembl release 75 annotation. For genes with multiple promoters, their maximum average tag number was used. Tissue culture

Mouse Neuro-2a neuroblastoma cells (N2A) and human embryonic kidney (HEK) cells were grown at 37°C in a humidified incubator supplemented with 5% CO2. Both cell lines were grown in Dulbecco's modified eagle medium containing penicillin/streptomycin (lOOU/ml, lOOug/ml respectively) and 10% fetal calf serum. Mouse embryonic stem cells and dicer knockout embryonic stem cells were maintained as described previously (Nesterova et al, 2008).

Transcript localisation and RNA turnover

Cells were fractionated into nuclear and cytoplasmic fractions by differential

centrifugation. Half-life of the IncRNA was determined after treatment of the cells with the transcriptional inhibitor actinomycin D.

Constructs

Inserts were cloned into the pCAGGs vector for overexpression, and shRNAs were expressed using the BLOCK-iT™ U6 vector (Invitrogen). miRNAs were expressed using the BLOCK-iT™ Pol II miR RNAi expression vector (Invitrogen).

Western blots

Proteins were detected with the following antibodies: anti- DUFSl (abl69540), anti- DUFS3 (abl 10246), anti-alpha tubulin loading control (ab7291). Blots were imaged using ECL prime (GE Healthcare) and an ImageQuant LAS 4000, and signals were normalised to the loading control. Prediction ofMREs and production ofMRE mutants

MREs were predicted using TargetScan in the 3'UTR (longest annotated UTR,

ENSEMBL build 70) of protein coding genes, and across the entire transcript for IncRNAs. The 5x MRE mutant was custom produced by Biomatik. Mutagenesis of single MRE sites was performed using the PCR.

Oxidative phosphorylation enzyme assays

All assays were performed using a Shimadzu UV-1800 spectrophotometer. Activities of complexes were determined using the following: Complex I, oxidation of NADH to NAD⁺ at 340nm at 30°C; Complex II, reduction of DCPIP at 600 nm at 30°C; Complex III, decylubiquinol mediated reduction of cytochrome c at 550nm; Complex IV, the oxidation of cytochrome c at 550 nm, 30°C for 3 min, and Citrate synthase, activity assayed at 412 nm at 30°C.

Oxidative stress measurements

Hydrogen peroxide production was assessed as a marker of reactive oxygen species generation using the fluorescent indicator Amplex Red (10 μΜ, Invitrogen) in combination with horseradish peroxidise (0.1 units ml^"1). Total amount of H2O2 produced was normalised to mg of protein added. Total cellular glutathione content was determined using a glutathione assay kit (Cayman Chemical), and normalized to total cell number. Protein carbonylation was detected using the OxyBlot protein oxidative detection kit (Millipore).

Supplemental Experimental Procedures

Gene expression profiling

5' and 3' ends of the mouse and human IncRNA transcripts were confirmed by 5' and 3' RACE using the GeneRacer™ Kit (Invitrogen) according to manufacturer' s instructions. Total RNA from twenty normal human tissues (adipose, bladder, brain, cervix, colon, oesophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, trachea) were obtained from FirstChoice® Human Total RNA Survey Panel (Invitrogen). Total RNA from twelve mouse tissues (bladder, brain, colon, heart, kidney, liver, pancreas, skeletal muscle, small intestine, stomach and testis) were obtained from Mouse Tissue Total RNA Panel (Amsbio). RNA from cell lines was extracted using RNeasy mini kit (Qiagen) according to manufacturer's instructions, using the optional on column DNase digest. cDNA synthesis for all samples was performed on ^g of total RNA using a QuantiTect Reverse Transcription kit (Qiagen) according to manufacturer's instructions. RNA was extracted from samples used for the detection of miRNAs using the miRNeasy mini kit (Qiagen) according to manufacturer's instructions (with on column DNAse digest). All RNA samples were quantified using the 260/280 absorbance ratio, and RNA quality assessed using a Tapestation (Agilent). RNA samples with an RNA integrity number (RIN) >8.5 were reverse transcribed, ^g of total RNA from the miRNA samples were reverse transcribed using the NCode VILO miRNA cDNA synthesis kit. Expression levels were determined by real-time quantitative PCR, using SYBR® Green Master Mix (Applied Biosystems) and standard cycling parameters (95°C 10 min; 40 cycles 95°C 15s, 60°C 1 min) followed by a melt curve using a StepOne™ thermal cycler (Applied Biosystems). All amplification reactions were performed in triplicate using gene specific primers. Multiple reference genes were assessed for lack of variability using geNorm (Vandesompele et al., 2002). Human expression data were normalised to a-tubulin and RNA polymerase II, whilst mouse expression data was normalised to TATA box binding protein and RNA polymerase II.

Tissue culture and flow cytometry

Mouse Neuro-2a neuroblastoma cells (N2A) and human embryonic kidney (HEK) cells were grown at 37°C in a humidified incubator supplemented with 5% C0₂. Both cell lines were grown in Dulbecco's modified eagle medium containing penicillin/streptomycin (lOOU/ml, lOOug/ml respectively) and 10% fetal calf serum. Cells were seeded at the following densities: 6 well dish, 0.3 x 10⁶; 48 well dish, 0.2 x 10⁴; T75 flask 2.1 x 10⁶. Mouse embryonic stem cells and dicer knock-out embryonic stem cells were maintained as described previously (Nesterova et al, 2008). Cells were counted using standard haemocytometry. For flow cytometry the cells were harvested by trypsinization, washed twice with PBS and fixed in 70% ethanol (filtered, - 20°C). The cell suspension was incubated at 4°C for 10 min and the cells pelleted, treated with 40 μg/ml RNase A and propidium iodide (40 μg/ml) for 30 min at room temperature. Cells were analysed using a FACSCalibur (BD-Biosciences) flow cytometer.

Transcript localisation and RNA turnover

In order to determine the predominant cellular localization of the lncRNA transcripts cells were fractionated into nuclear and cytoplasmic fractions. Briefly, -2.8 x 10⁶ cells were collected by trypsinization, washed three times in PBS and pelleted at 1000 g for 5 min at 4°C. The cell pellet was resuspended in 5 volumes of lysis buffer (10 mM Tris-HCl, pH 7.5, 3 mM MgCl₂, 10 mM NaCl, 5 mM EGTA, 0.05% NP40, and protease inhibitors [Roche, complete mini] ) and incubated on ice for 15 min. Lysed cells were then centrifuged at 2000 g for 10 min at 4°C, and the supernatant collected as the cytoplasmic fraction. Nuclei were washed three times in nuclei wash buffer (10 mM HEPES, pH 6.8, 300 mM sucrose, 3 mM MgC , 25 mM NaCl, 1 mM EGTA), and pellet by centrifugation at 400g, 1 min at 4°C. Nuclei were extracted by resuspension of the nuclei pellet in 200 μΐ of nuclei wash buffer containing 0.5% Triton X- 100 and 700 units/ml of DNase I and incubated on ice for 30 mins. Nucleoplasm fractions were collected by centrifugation at 17 OOOg for 20 min at 4°C.

To determine the stability of the lncRNA transcripts, cells were cultured to -50 % confluency and then transcription was inhibited by the addition of 10 μg/ml actinomycin D (Sigma) in DMSO. Control cells were treated with equivalent volumes of DMSO.

Transcriptional inhibition of the N2A cells was conducted for 16 hours with samples harvested at 0 hrs, 30 mins, 1 hr, 2 hrs, 4 hrs, 8 hrs and 16 hrs. RNA samples for fractionation and turnover experiments were collected in Trizol (Invitrogen) and RNA purified and DNAse treated using the RNeasy mini kit (Qiagen). Reverse transcription for cellular localisation and turnover experiments was performed as previously described.

Constructs

PCR primers modified to contain BgUI and Xhol sites were used to amplify the full length mouse Ceroxl (SEQ ID NO: 5), whilst human CEROX1 (SEQ ID NO: 4) and the mouse 5x MRE mutant (SEQ ID NO: 7) were synthesised by Biomatik, and also contained BgUI and Xhol sites at the 5' and 3' ends respectively. All other MRE mutants were produced using overlapping PCR site directed mutagenesis to mutate 3 bases of the miRNA seed region. All purified products were ligated into the prepared backbone and then transformed by heat shock into chemically competent DH5a, and plated on selective media. All constructs were confirmed by sequencing. All full length IncRNAs were cloned into the pCAGGS overexpression vector under the actin/ -globin promoter. As an overexpression/transfection control EGFP was cloned into the pCAGGS backbone. Short hairpin RNAs specific to the transcripts were designed using a combination of the RNAi design tool (Invitrogen) and the siRNA selection program from the Whitehead Institute (Yuan et al., 2004). shRNA oligos to the target genes and β-galactosidase control oligos were annealed to create double-stranded oligos and cloned into the BLOCK-iT™ U6 vector (Invitrogen), according to manufacturer's instructions. miRNA expression constructs were generated and cloned into the BLOCK-iT™ Pol II miR RNAi expression vector (Invitrogen) according to manufacturer's instructions.

One day prior to transfection cells were either seeded in 6 well dishes (0.3 x 10⁶ cells/well), or in T75 flasks (2.1 x 10⁶ cells/flask). Twenty-four hours later cells in 6 well dishes were transfected with ^g of shRNA, miRNA or overexpression construct and their respective control constructs using FuGENE® 6 (Promega) according to manufacturers guidelines. Cells in T75 flasks were transfected with 8μg of experimental or control constructs. Transfected cells were grown for 48 hours under standard conditions, and then harvested for either gene expression studies or biochemical characterisation. Transcripts for the luciferase destabilisation assays were cloned into the pmirGLO miRNA target expression vector (Promega) and assayed using the dual-luciferase® reporter assay system (Promega). The 5x MRE mutant was custom produced by Biomatik. Mutagenesis of single MRE sites was performed using the PCR, and the resulting modified IncRNA cloned into the appropriate backbone (pCAGG or pmirGLO). miRNA inhibitors were purchased from Ambion, and utilised according to manufacturers instructions.

Efficacy of the overexpression and silencing constructs was determined by real-time quantitative PCR.

Computational techniques

Ceroxl and CEROX1 share binding sites for 12 miRNAs (miR-125a-3p, miR-138, miR- 199/199-5p, miR-28/28-5p/708, miR-302ac/520f, miR-370, miR-485/485-5p, miR-486/486-5p, miR-488, miR-501/501-5p, miR-654-3p, miR-675/675-5p). Of the twelve, miR-138, miR28/28- 5p/708, miR-370 and miR-488 are expressed in N2A (Landgraf et al, 2007). We used

TargetScan (Friedman et al, 2009) to predict the binding sites of these 4 miRNAs in the IncRNAs and the 3'UTR (longest annotated UTR, ENSEMBL build 70) of protein-coding OXPHOS genes.

The average expression across 46 human tissues and individuals according to the Pilot 1 data from the GTEx Consortium (Lonsdale et al, 2013) was computed for both protein-coding genes and intergenic long non-coding RNAs from the Ensembl release 75 annotation (Flicek et al, 2014). We used the normalized number of CAGE tags across 399 mouse cells and tissues from the FANTOM5 Consortium (http://fantom.gsc. riken.jp)(Kawaji et al, 2014) as an approximation of expression levels for protein-coding genes and intergenic IncRNAs from the Ensembl release 75 annotation. If multiple promoters were associated with a gene, we selected the promoter with the highest average tag number.

Conserved sequence blocks in IncRNA sequence were identified using LALIGN (Goujon et al, 2010). Microarray analysis

Microarray analysis was performed on 16 samples (four overexpression/four

overexpression controls; four knock-down/ four knock-down controls), and hybridizations were performed by the OXION array facility (University of Oxford). Data were analysed using the web-based bioconductor interface, CARMAweb (Rainer et al., 2006). Differentially expressed genes (Benjamini-Hochberg -value <0.05) were identified between mouse IncRNA

overexpression and control cells using Limma from the bioconductor package between the experimental samples and the respective controls.

Oxidative phosphorylation enzyme assays

Cell lysates were prepared 48 hours post-transfection, by harvesting cells by trypsinisation, washing three times in ice cold phosphate buffered saline followed by centrifugation to pellet the cells (2 mins, 1000 g). Cell pellets were resuspended to homogeneity in KME buffer (100 mM KCL, 50 mM MOPS, 0.5 mM EGTA, pH 7.4) and protein

concentrations were determined using a BCA protein assay detection kit (Pierce). Cell lysates were flash frozen in liquid nitrogen, and freeze-thawed three times prior to assay. 300-500 μg of cell lysate was added per assay, and assays were normalised to the total amount of protein added.

All assays were performed using a Shimadzu UV-1800 spectrophotometer, and all samples were measured in duplicate. Activity of Complex I (CI, NADH:ubiquinone

oxidoreductase) was determined by measuring the oxidation of NADH to NAD⁺ at 340nm at 30°C in an assay mixture containing 25 mM potassium phosphate buffer (pH 7.2), 5 mM MgCl₂, 2.5 mg/ml fatty acid free albumin, 0.13 mM NADH, 65 μΜ coenzyme Q and 2 μg/ml antimycin A. The decrease in absorbance was measured for 3 mins, after which 10 μΜ of rotenone was added and the absorbance measured for a further 2 mins. The specific complex I rate was calculated as the rotenone- sensitive rate minus the rotenone-insensitive rate. Complex II (CII, succinate dehydrogenase) activity was determined by measuring the oxidation of DCPIP at 600 nm at 30°C. Lysates were added to an assay mixture containing 25 mM potassium phosphate buffer (pH 7.2) and 2 mM sodium succinate and incubated at 30°C for 10 mins, after which the following components were added, 2 μg/ml antimycin A, 2 μg/ml rotenone, 50 μΜ DCPIP and the decrease in absorbance was measured for 2 mins. Complex III (CIII, Ubiquinol: cytochrome c oxidoreductase) activity was determined by measuring the oxidation of decylubiquinol, with cytochrome c as the electron acceptor at 550nm. The assay cuvettes contained 25 mM potassium phosphate buffer (pH 7.2), 3 mM sodium azide, 10 mM rotenone and 50 μΜ oxidized

cytochrome c. Decylubiquinol was synthesized by acidifying decylubiquinone (lOmM) with HC1 (6M) and reducing the quinine with sodium borohydride. After the addition of 35 μΜ decylubiquinol, the increase in absorbance was measured for 2 mins. Activity of Complex IV (CIV, cytochrome c oxidase) was measured by monitoring the oxidation of cytochrome c at 550 nm, 30°C for 3 min. A 0.83 mM solution of reduced cytochrome c was prepared by dissolving 100 mg of cytochrome c in 10ml of potassium phosphate buffer, and adding sodium ascorbate to a final concentration of 5 mM. The resulting solution was added into SnakeSkin dialysis tubing (7000 molecular weight cutoff, Thermo Scientific) and dialyzed against potassium phosphate buffer, with three changes at 4°C for 24 hrs. The redox state of the cytochrome c was assessed by assessing the absorbance spectra from 500-600 nm. The assay buffer contained 25 mM potassium phosphate buffer (pH 7.0) and 50 μΜ reduced cytochrome c. The decrease in absorbance at 550 nm was recorded for 3 mins. As a control the enzymatic activity of the tricarboxylic acid cycle enzyme, citrate synthase was assayed at 412 nm at 30°C in a buffer containing 100 mM Tris-HCl (pH 8.0), 100 μΜ DT B (5,5-dithiobis[2-nitrobenzoic acid]), 50 μΜ acetyl coenzyme A, 0.1% (w/v) Triton X-100 and 250 μΜ oxaloacetate. The increase in absorbance was monitored for 2 mins.

The following extinction coefficients were applied: CI, ε = 6.81 mM^"1 cm^"1, CII, ε =

21.0 mM^"1 cm^"1; CIII, ε = 19.1 mM^"1 cm^"1; CIV, ε = 21.84 mM^"1 cm^"1 (the difference between reduced and oxidised cytochrome c at 550 nm); CS, ε = 13.6 mM^"1 cm^"1

Western blots

Total protein was quantified using a BCA protein assay kit (Pierce). lOug of protein was loaded per well, and samples were separated on 12% SDS-PAGE gels in Tris-glycine running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). Proteins were then electroblotted onto PVDF membrane (40V, 3 hrs) in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol), the membrane blocked in TBS-T (50 mM Tris-Hcl, 150 mM NaCl, 0.1% Tween 20) with 5% non -fat milk powder for 1 hour. The membrane was incubated with primary antibodies overnight at 4°C with the following dilutions: anti- DUFSl (rabbit monoclonal, ab 169540, 1 :30,000), anti- DUFS3 (mouse monoclonal, 0.15 mg/ml, abl 10246), anti-alpha tubulin loading control (mouse monoclonal, ab7291, 1 :30,000). Following incubation with the primary antibodies, blots were washed 3x 5 min, and 2 x 15 mins in TBS-T and incubated with the appropriate secondary antibody for 1 hour at room temperature: goat anti-rabbit HRP

(Invitrogen) 1 :30,0000; goat anti-mouse HRP (Dako) 1 :3,000. After secondary antibody incubations, blots were washed and proteins of interested detected using ECL prime

chemiluminescent detection reagent (GE Healthcare) and the blots imaged using an ImageQuant LAS 4000 (GE Healthcare). Signals were normalised to the loading control.

Oxidative stress measurements

Hydrogen peroxide production was assessed as a marker of reactive oxygen species generation using the fluorescent indicator Amplex Red (10 μΜ, Invitrogen) in combination with horseradish peroxidise (0.1 units ml^"1). Total amount of H2O2 produced was normalised to mg of protein added. Total cellular glutathione content was determined using a glutathione assay kit (Cayman Chemical) according to manufacturer's instructions, and normalized to total cell number. Protein carbonylation was assessed using the OxyBlot protein oxidation detection kit (Merck Millipore), and differential carbonylation was assessed by densitometry. The cell stress assay was performed on cells seeded in 48 well plates, and assayed 12 hours later by the addition of (final concentration): rotenone (5 μΜ), malonate (40 μΜ), Antimycin A (500 μΜ),

Oligomycin (500 μΜ), Sodium Azide (3 mM), NaCl (300 mM), CaCl₂ (5.4 mM). Cells were heat shocked at 42°C and UV irradiated using a Stratlinker UV Crosslinker for 10 minutes (2.4 J cm^"2). Cell viability was assessed by the addition of Alamar Blue (Invitrogen) according to manufacturer's instructions.

RESULTS

Ceroxl is a conserved, ubiquitously expressed long noncoding RNA

Ceroxl was selected for further investigation from among a set of central nervous system-derived long non-coding RNAs identified by cDNA sequencing (GenBank Accession AK079380, 2810468N07Rik)(Carninci et al, 2000; Ponjavic et al, 2007). Mouse Ceroxl is a 1.2 kb, two exon, intergenic transcript which shares a bidirectional promoter with the SRY (sex determining region Y)-box 8 (Sox8) gene (Fig. la). Ceroxl exons are conserved among eutherian mammals but not with non-eutherian vertebrates (Fig. la). A human orthologous transcript (CEROXI, GenBank Accession BC098409) was identified by sequence similarity and conserved synteny (60-70% nucleotide identity within alignable regions, Fig. lb,c). Both mouse and human transcripts had low coding potential (Methods, Fig. 9a) and no evidence for translation from available proteomic datasets

By surveying expression data from 399 tissues and cell lines (Kawaji et al, 2014) we determined that mouse Ceroxl expression levels are exceptionally high, within the top 13% of a set of 879 IncRNAs with associated Cap analysis of gene expression (CAGE) clusters and are, indeed, higher than for 46% of protein-coding genes (Fig. Id), with brain expression higher than 64.3%) of all protein coding genes. The high expression of Ceroxl in brain regions (Fig. le) and in other tissues was confirmed using quantitative real-time PCR (qPCR) for both mouse and human orthologous transcripts (Fig. 9b, c). CAGE tags indicated the presence of two Ceroxl transcriptional start sites. The shorter variant was the dominant isoform by CAGE tag count and qPCR (Fig. 9d, e), and thus was chosen for further investigation. In mouse N2A cells this transcript was strongly enriched in the cytoplasmic fraction (Fig. If) with a short half-life of of 35.5 ± 16 mins (Fig. 9f) and, in contrast to the protein coding transcript Gapdh, was mostly associated with the pre-ribosome fraction (Fig. 9g).

The high level of expression and predominant cytoplasmic localisation of Ceroxl are consistent with it acting not in the nucleus as a regulator of transcription, but rather in the cytoplasm in a post-transcriptional manner. Ceroxl expression modulates levels of oxidative phosphorylation transcripts The intracellular role of Ceroxl was initially investigated in vitro using microarray based transcriptional profiling. Knockdown of Ceroxl levels by approximately 65% (shRNA sh92; Fig. 10a) using short hairpin RNAs in mouse neuroblastoma (N2A) cells yielded no genes that survived a stringent genome-wide significance test for gene expression change. In contrast, overexpression of Ceroxl, by approximately 7-fold (Fig. 10b), resulted in the differential expression of 286 genes (q < 0.05, Benjamini-Hochberg multiple testing correction), of which an unexpectedly large and significant majority (237; 83%) were upregulated (P < 10^"6; binomial test). Gene Ontology (GO) analysis indicated that these upregulated genes were greatly enriched for respiratory chain genes (16 of 64 genes; 25%; Fig. 2a). Although IncRNA loci can regulate their neighbouring protein coding genes in cis (Bond et al, 2009; Maamar et al, 2013; Wang et al, 2011), altered Ceroxl expression had no effect on the expression of flanking protein coding genes (Fig. 10c) suggesting that, alternatively, it may act in trans. Interestingly, cells overexpressing Ceroxl exhibited a substantially reduced rate of cell division, without deviating from normal cell cycle proportions (Fig. 10d,e), suggesting that Ceroxl may play an important cellular role.

The mitochondrial electron transport chain (ETC) consists of five multi-subunit complexes encoded by approximately 100 genes of which only 13 are located in the

mitochondrial genome. Of the 15 subunits whose transcripts show statistically significant differential expression after Ceroxl overexpression, all are nuclear encoded, and principally encode either mitochondrial complex I or IV subunits (Fig. 2b). Eleven of 15 observed gene expression increases were validated by qPCR, of which 6 had a fold increase exceeding 1.4 (Fig. lOf), with the greatest changes observed for complex I subunit transcripts. Twelve of 35 nuclear encoded complex I subunits or assembly factors (36%) transcripts increased by at least 1.4 fold following Ceroxl overexpression, including 3 of 7 nuclear encoded core subunits that are essential for catalytic function (Hirst, 2013) (Fig. 2c). These 12 transcripts represent gene expression biomarkers for Ceroxl activity in this system. We further observed that 8 of the 12 mitochondrial complex I transcripts were reduced in cells expressing Ceroxl shRNA (Fig. lOg). These results indicate that Ceroxl is a positive regulator of the levels of multiple mitochondrial complex I transcripts.

We next investigated whether the observed elevation in OXPHOS subunit transcripts results in increased protein levels for those subunits. To do this we analysed the key complex I catalytic core proteins NDUFS1 and DUFS3, whose transcripts' levels had increased 1.4 fold. Surprisingly, compared to their transcript level changes we observed a disproportionately large, near two-fold, increase in NDUFSl and NDUFS3 protein levels (Fig. 2d,e). Ceroxl transcript abundance is therefore coupled to OXPHOS transcript levels and to their availability for translation, resulting in an amplification of the amount of protein produced from its transcripts. Given the cytoplasmic localisation of the Ceroxl transcript (Fig. If), these results indicate that Ceroxl post-transcriptionally regulates the transcript levels of OXPHOS subunits, leading to an increase in the amount of specific proteins produced.

Ceroxl can regulate mitochondrial OXPHOS enzyme activity

We next tested whether the observed increases in OXPHOS subunit transcript and protein levels alter OXPHOS enzymatic activity. It is known that the increased translation of a subset of complex I transcripts leads to an increase in respiration (Shyh-Chang et al, 2013), or more specifically an increase in the enzymatic activity of complex I (Alvarez-Fischer et al, 2011). We measured the catalytic activities of four specific OXPHOS enzymes (NADH:ubiquinone oxidoreductase, complex I; succinate dehydrogenase, complex II; ubiquinol-cytochrome c reductase, complex III; cytochrome c oxidase, complex IV) in response to altered Ceroxl levels in N2A cells.

After Ceroxl knockdown, complex I and complex IV enzymatic activities decreased significantly (by 11%, P = 0.03 and 19%, P = 0.02, respectively) (Fig. 3a), with knockdown cells consuming less oxygen (Fig. 3b). Conversely, after Ceroxl overexpression complex I and complex IV activities increased significantly (by 22%, P = 0.01; by 30%, P = 0.003,

respectively; 2-tailed Student's t-test), (Fig. 3c). Significantly, oxygen consumption increased, mirroring the elevations in complex I and complex IV activities (Fig. 3d). To further understand the basis of the observed increase in enzyme activity we also measured a number of other mitochondrial parameters. The enzymatic activity of complexes II, III and citrate synthase (Fig. 3c), the mitochondrial number, and the mitochondrial -to-nuclear genome ratio all remained unaltered by changes in Ceroxl levels (Fig. 11a, b). This suggests that the observed increases in enzymatic activity are not due to an overall change in the number of mitochondria, and that

Ceroxl specifically regulates the catalytic activities of complex I and complex IV in mouse N2A cells.

Ceroxl expression can protect cells from oxidative stress

To further assess the effect of Ceroxl induced ETC activity we measured the relative levels of reactive oxygen species (ROS) production and oxidative stress in N2A cells. In Ceroxl knockdown cells ROS levels increased by almost 20% (P = 4.2 x 10^"6, 2-tailed Student's t-test; Fig. 4a). Conversely, in cells overexpressing Ceroxl, ROS production was nearly halved (P = 3.5 x 10^"7, 2-tailed Student's t-test, Fig. 4a). Protein carbonylation, a measure of ROS-induced damage, was reduced by 35% in CeroxZ-overexpressing cells (P = 0.001, 2-tailed Student's t- test, Fig. 4b). The observed CeroxZ-dependent decrease in ROS levels is of particular interest because mitochondrial complex I is a major producer of ROS which triggers cellular oxidative stress and damage, and an increase in ROS production is a common feature of mitochondrial dysfunction in disease (Murphy, 2009).

We next tested whether CeroxZ-induced increases in complex I and complex IV activities might protect cells against the effects of specific mitochondrial complex inhibitors. Strikingly, CeroxZ-overexpressing cells showed reduced cytotoxicity when challenged with rotenone and sodium azide (complex I and complex IV inhibitors respectively); conversely, Ceroxl- knockdown cells were significantly more sensitive to rotenone (P < 0.001, Fig. 4c). From these results we conclude that increased Ceroxl expression leads to decreased ROS production, decreased levels of oxidative damage to proteins and can confer protective effects against complex I and complex IV inhibitors.

Increased OXPHOS enzymatic activity is dependent upon miRNA binding to Ceroxl

From its cytoplasmic localisation we hypothesised that Ceroxl acts by competing with complex I subunit transcripts for the binding of particular miRNAs. To investigate this hypothesis, we firstly compared the effect of Ceroxl overexpression in mouse wildtype and Dicer-deficient (Dicer⁰¹⁰) embryonic stem cells (Nesterova et al, 2008) (which are deficient in miRNA biogenesis) on the transcript levels of six complex I subunits, four of which had previously shown significant changes in N2A cells after Ceroxl overexpression (Fig. 2c). In wildtype mouse ES cells Ceroxl overexpression recapitulated the results previously seen in N2A cells. In contrast, in Dicer^a/a cells overexpression did not lead to an increase in the level of these transcripts (Fig. 5a). These results strongly suggest that Ceroxf s ability to alter mitochondrial metabolism involves a miRNA-dependent process.

To understand which miRNAs could be directly interacting with Ceroxl we performed a

TargetScan(Friedman et al, 2009) prediction of miRNA binding sites. Identifying target sites for miRNAs can be challenging due to the low sensitivity of current miRNA recognition element prediction (MRE) algorithms (Maziere & Enright, 2007). From the TargetScan prediction results we identified five MREs for further investigation whose presence was conserved between Ceroxl and human CEROX1 and that were expressed in N2A cells. The five MREs identified are from four specific miRNA groups: miR-138, miR-28/28-5p/708, miR-370, and miR-488 (Fig. 5b). Overexpression of each of the four miRNAs in N2A cells caused a significant reduction in Ceroxl levels. Nevertheless, overexpression of miR-488 showed the greatest effect, with a striking 13-fold reduction in Ceroxl (Fig. 12a). Conversely, Ceroxl overexpression decreases the level of miR-488, but not of miR-138, miR-28/28-5p/708 or miR-370 (Fig. 12b). We then further confirmed the requirement of miRNA-binding for Ceroxl activity by overexpressing a 5xMRE mutant Ceroxl transcript in which each MRE had been mutated by inversion of its seed region, and observed changes in neither transcript levels nor enzyme activities (Fig. 6a, b). Next, we individually investigated the miRNA of greatest effect, miR- 488, chosen from its ability to strongly deplete Ceroxl levels (Fig. 12), by creating a Ceroxl transcript containing three mutated nucleotides in its single miR-488 MRE. The interaction of miR-488 with Ceroxl is predicted to involve a 7mer-8m seed site, where the heptamer sequence of the seed is complementary to nucleotides 2-8 of the miRNA (Fig. 6c). Luciferase

destabilisation assays indicated that miR-488 destabilises wildtype Ceroxl, but not the Ceroxl transcript with a mutated miR-488 MRE seed region (Fig. 6d). Remarkably, overexpression of this miR-488 MRE mutant transcript in N2A cells resulted in no change in CeroxZ-sensitive transcript levels (Fig. 6e), and no change in complex I enzymatic activity (Fig. 6f). Furthermore, overexpression of miR-488 downregulated all 12 CeroxZ-sensitive complex I transcripts (Fig. 6g); conversely inhibition of miR-488 resulted in an increase in CeroxZ-sensitive transcripts (Fig. 6h). These findings indicate that (i) Ceroxl post-transcriptionally regulates OXPHOS enzymatic activity as a miRNA decoy, and {if) miR-488 plays a direct role in regulating the transcript levels of at least one-third of all complex I subunit genes in N2A cells.

Ceroxl is an evolutionarily conserved regulator of mitochondrial complex I activity

To investigate the evolutionary conservation of Ceroxl activity we considered whether the syntenic human transcript performs the functionally equivalent role of regulating

mitochondrial complex I activity in humans. Similar to mouse Ceroxl, human CEROXl is highly expressed in brain tissue, is otherwise ubiquitously expressed (Fig. 9b), and is also enriched in the cytoplasm (Fig. 7a). Using pilot data from the GTEx consortium (GTEx consortium 2013), human CEROXl was found to be expressed at very high levels: it occurs among the top 0.3% of all expressed IncRNAs (5161 IncRNAs in total; Fig. 7b) and was more highly expressed, averaged across individuals, than 87.5% of all protein coding genes. Its expression was highest within brain tissues, more specifically within both the basal ganglia (putamen and caudate nucleus as part of the dorsal striatum, and nucleus accumbens as part of the ventral striatum and substantia nigra) and the cortex (Fig. 7c inset).

Importantly, CEROXl overexpression in human embryonic kidney (HEK293) cells led to significant and substantial increases in the activities of mitochondrial complexes I and III (Fig. 7d; 31% increase, P = 6.4 x 10^"5; 18% increase, P = 2.7 x 10^"7, respectively). CEROXl overexpression had a greater effect on complex I activity than the mouse orthologous sequence and also increased the activity of complex III, rather than complex IV activity, in these cells. This last observation poses many interesting questions regarding the evolution and conservation of noncoding RNAs. One possibility is that it could reflect the mouse and human cell lines containing different miRNA pools and/or, due to rapid IncRNA sequence evolution (Marques and Ponting, 2009), the presence of different MREs in the IncRNA and human OXPHOS transcripts. A further possibility is that of a human complex I-III interaction that is absent in mouse cells. Importantly, reciprocal expression of Ceroxl and CEROX1 in human and mouse cell lines respectively, recapitulates the previously observed increase in complex I activity (Fig. 7e). The exceptionally high in vivo expression of both Ceroxl and CEROX1 and their ability to modulate the activity of mitochondrial complex I in mouse and human cells indicate that their function has been conserved over 90 million years since the common ancestor of mouse and human.

DISCUSSION

Ceroxl is the first IncRNA, to our knowledge, demonstrated to be involved in the regulation of mitochondrial energy metabolism. It is predominantly located in the cytoplasm where, our data indicate, it post-transcriptionally regulates the levels of mitochondrial OXPHOS subunit transcripts and proteins by acting as a miRNA-decoy (Fig. 8). Changes in Ceroxl abundance are mirrored by altered levels of mitochondrial OXPHOS subunit transcripts and, more importantly, larger changes in their protein subunits levels, leading to corresponding changes in mitochondrial complex I catalytic activity. Significantly, the observed changes in catalytic activity are in line with the degree of change seen in diseases exhibiting mitochondrial dysfunction. Overexpression of Ceroxl in N2A cells leads to an increase in oxidative

metabolism, a decrease in cellular oxidative stress and enhanced protection against the complex I inhibitor rotenone. In mouse cells the effect of Ceroxl on complex I subunit transcript levels can be explained by their competition for binding to MREs within Ceroxl, most notably miR-488, which buffers the OXPHOS transcripts against miRNA-mediated repression.

The dynamics and mechanism of IncRNA competition for miRNA binding have been both modelled mathematically (Ala et al, 2013; Figliuzzi et al, 2013) and addressed

experimentally (Bosson et al, 2014; Denzler et al, 2014) with differing conclusions. It has been proposed that for a single endogenous transcript to relieve miR-122 mediated target repression, expression of this transcript would need to be at physiologically unfeasibly high levels (Denzler et al, 2014). However, hepatitis C virus RNA sequestration of miR-122 has been shown to de- repress miR-122 targets after an only two fold reduction in miR-122 suggesting that functional decoying occurs at much lower levels of endogenous expression (Luna et al, 2015).

Furthermore, it has been demonstrated that the addition of high affinity target sites effectively decoys miRNAs with low miRNA-to-target ratios (Bosson et al, 2014). Other under-appreciated factors that may influence the dynamics of ceRNA crosstalk are miRNA target site accessibility, degree of sequence pairing, RNA secondary structures, miRNA turnover and the association of the RNA with RNA binding proteins (Guo et al, 2015). Our experimental findings indicate that Ceroxl provides an example wherein target competition substantially perturbs a post- transcriptional gene regulatory network. This model is consistent with the relatively low expression of miR-488 (Kozomara & Griffiths- Jones, 2014) and the high in vivo expression of both Ceroxl and OXPHOS transcripts (Schwanhausser et al, 2011; Vogel et al, 2010).

Furthermore, high levels of Ceroxl in central nervous system tissues support the notion that Ceroxl supports OXPHOS homeostasis in cells with sustained high metabolic activity and high energy requirements (Sokoloff, 1977).

We have demonstrated that the post-transcriptional regulation of a subset of complex I subunits by Ceroxl leads to an increase in oxygen consumption. How an increase in translation of only a subset of OXPHOS transcripts leads to an increase in a specific complex's enzymatic activity remains unclear, yet has been observed previously in mouse dopaminergic neurons (Alvarez-Fischer et al, 2011) and tissues (Shyh-Chang et al, 2013). Observed increases in OXPHOS activity may reflect some subunits of the complex I holo-enzyme (including NDUFS3 and NDUFA2) being present as a monomer pool and therefore being available for direct exchange without being integrated into assembly intermediates (Dieteren et al, 2012). This monomer pool facilitates the rapid swapping out of oxidatively damaged complex I subunits (Dieteren et al, 2012), and a Ceroxl mediated expansion of the pool may thereby improve the efficiency of complex I catalysis.

An increase in the enzymatic activity of specific ETC complexes has particular biomedical relevance. Mitochondrial dysfunction is a feature of many disorders and often manifests as decreases in the catalytic activities of particular mitochondrial complexes. A decrease in catalytic activity can result in an increase in ROS production, leading to oxidative damage of lipids, DNA, and proteins, with OXPHOS complexes themselves being particularly susceptible to such damage (Musatov & Robinson, 2012). Parkinson's and Alzheimer's diseases both feature pathophysiology associated with oxidative damage resulting from increased ROS production and decreased complex I and IV activities (a reduction of 30% and 40%,

respectively)(Canevari et al, 1999; Keeney et al, 2006; Schapira et al, 1990). Currently no effective treatments exist that help to restore mitochondrial function. Importantly, a 20% increase in complex I activity is sufficient to protect mouse midbrain dopaminergic neurons against MPP+, a complex I inhibitor and a chemical model of Parkinson's disease (Alvarez- Fischer et al, 2011). We note that highest expression of CEROX1 occurs in the basal ganglia, which contains the substantia nigra in which the dopaminergic neurons that are particularly sensitive to degeneration in Parkinson's disease are located. It has been hypothesized that susceptibility of these neurons to this disease is due to the energy stress they experience arising from their unmyelinated axonal arbour (Pissadaki & Bolam, 2013). The high levels of observed CEROXl expression may reflect these cells' greater requirement for post-transcriptional regulation of energy metabolism and may contribute in vivo to energy metabolism homeostasis specifically in central nervous system tissues with high energy demands. The ability of CEROXl to increase mitochondrial complex I activity makes it an exciting new target for investigating new treatments for mitochondrial dysfunction. The identification of regulatory RNAs such as CEROXl offers new opportunities to develop novel pharmacological approaches to restore mitochondrial function and highlights a growing interest in the potential therapeutic value of IncRNA gene expression manipulation (Wahlestedt, 2013).

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134.

Claims

1. A method of increasing mitochondrial function in a cell, the method comprising administering to the cell an inhibitor of miR-488-3p and thereby increasing mitochondrial function in the cell.

2. A method according to claim 1, wherein the miR-488-3p comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

3. A method according to claim 1 or 2, wherein the inhibitor is a small molecule inhibitor, a protein, an antibody, an oligonucleotide or a polynucleotide.

4. A method according to any one of the preceding claims, wherein the oligonucleotide or polynucleotide specifically hybridises to miR-488-3p and/or a seed sequence contained therein.

5. A method according to claim 4, wherein the oligonucleotide or polynucleotide specifically hybridises to the seed sequence shown in nucleotides 2 to 8 of SEQ ID NO: 1.

6. A method according to claim 5, wherein the oligonucleotide or polynucleotide comprises the sequence shown in SEQ ID NO: 3 or a variant thereof which has 1, 2 or 3 nucleotide substitutions or deletions compared with SEQ ID NO: 3.

7. A method according to claim 6, wherein the oligonucleotide or polynucleotide comprises nucleotides 1 to 6 of SEQ ID NO: 3 or nucleotides 2 to 7 of SEQ ID NO: 3.

8. A method according to claim 6 or 7, wherein the polynucleotide comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a).

9. A method of increasing mitochondrial function in a cell, the method comprising administering to the cell a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) and thereby increasing mitochondrial function in the cell.

10. A method according to any one of claims 3 to 9, wherein the oligonucleotide or polynucleotide is expressed or overexpressed by the cell.

11. A method according to any one of claims 3 to 10, wherein the oligonucleotide or polynucleotide is a ribonucleic acid (RNA).

12. A method according to any one of the preceding claims, wherein the method (a) increases the expression of mitochondrial complex I in the cell, (b) increases mitochondrial oxidative phosphorylation in the cell; (c) reduces oxidative stress in the cell, (d) reduces the amount of reactive oxygen species (ROS) in the cell, (e) increases the amount of glutathione in the cell or (f) any combination of (a) to (e).

13. A method according to any one of the preceding claims, wherein the cell is a human cell or a mouse cell.

14. A method according to any one of the preceding claims, wherein the method is carried out in vitro.

15. A method of increasing mitochondrial function in an individual, the method comprising administering to the patient an inhibitor of miR-488-3p and thereby increasing mitochondrial function in the individual.

16. A method of increasing mitochondrial function in an individual, the method comprising administering to the patient a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) and thereby increasing mitochondrial function in the individual.

17. A method according to claim 15 or 16, wherein the method is for treating or preventing a disease or disorder associated with mitochondrial dysfunction in a patient.

18. A method according to claim 17, wherein the disease or disorder is neuropathic, retinopathic, hepatoneuropathic, neuropathic and myopathic, myopathic, cardiomyopathic, myopathic and immunopathic, neuropsychiatric, an autoimmune disease or disorder or nephropathic.

19. A method according to claim 18, wherein:

(a) the neuropathic disease or disorder is Alzheimer's disease (AD), aminoglycoside- induced deafness (AID), amyotrophic lateral sclerosis (ALS), ataxia, myoclonus and deafness (AMDF), autosomal dominant cerebellar ataxia (ADCA), diabetes mellitus and deafness (DMDF), Fahr's syndrome (Fahr disease), Huntingdon's disease, late-onset

encephalomyopathies, Leber's hereditary optic neuropathy (LHON), Leigh syndrome (Leigh's disease), leukodystrophy, leukoencephalopathies, maternally inherited diabetes and deafness (MIDD), maternally inherited Leigh syndrome (MILS), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial familial infantile bilateral striatal necrosis (FSBN), myoclonic epilepsy and psychomotor regression (MEPR), Parkinson's disease (PD), progressive myoclonus epilepsy (PME), sensorineural hearing loss (SNHL) or sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO);

(b) the retinopathic disease or disorder is deafness and retinitis pigmentosa, neuropathy, ataxia and retinitis pigmentosa (NARP) or progressive encephalopathy (PEM);

(c) the hepatoneuropathic disease or disorder is Alpers' disease;

(d) the neuropathic and myopathic disease or disorder is chronic intestinal

pseudoobstruction with myopathy and ophthalmoplegia (CIPO), coenzyme Q deficiency, fatal infantile multisystem disorder or Leber's hereditary optic neuropathy and dystonia (LDYT), MERRE/MELAS overlap syndrome (MERME), mitochondrial cytopathy (MC), mitochondrial encephalomyopathy, mitochondrial encephalopathy, mitochondrial

encephalopathy, lactate acidosis and stroke (MELAS), mitochondrial neurogastrointestinal encephalopathy (MNGIE), myoclonic epilepsy with ragged-red fibres (MERRF), Pearson syndrome, progressive dementia and chorea (DEMCHO) or progressive dystonia;

(e) the myopathic disease or disorder is chronic progressive external ophthalmoplegia (CPEO), exercise intolerance (EXIT), fatal infantile myopathy, Kearns-Sayre syndrome (KSS) (oculocraniosomatic neuromuscular disorder with ragged red fibres), lethal infantile mitochondrial myopathy (LIMM), maternal inherited myopathy (MFM), mitochondrial myopathy (MM) or reversible infantile myopathy;

(f) the cardiomyopathic disease or disorder is dilated cardiomyopathy, fatal infantile cardiomyopathy plus (FICP), hypertrophic cardiomyopathy with myopathy, infantile histiocytoid cardiomyopathy, maternal inherited cardiomyopathy (MICM) or maternal inherited hypertrophic cardiomyopathy (MHMC);

(g) the myopathic and immunopathic disease or disorder is Barth syndrome (BTHS); the neuropsychiatric disease or disorder is bipolar disorder (BD), dysthymia (persistent depressive disorder), major depressive disorder (MDD), mood disorders, post-traumatic stress disorder (PTSD), recurrent depression or schizophrenia (SZ);

(h) the autoimmune disease or disorder is lupus, multiple sclerosis, rheumatoid arthritis or Sjogren's syndrome; or

(i) the nephropathic disease or disorder is focal segmental glomerulosclerosis (FSGS) or tubulointerstitial nephritis.

20. A method according to claim 17, wherein the disease or disorder is cancer, cardiovascular disease (CVD), carnitine palmitoyltransferase I/II deficiencies, coronary heart disease (CHD), diabetes mellitus (DM), GRACILE syndrome, hypoxia, metabolic syndrome, myocardial infarction (MI), myoglobinuria, obesity, stroke or sudden infant death syndrome (SIDS).

21. A method according to claim 15 or 16, wherein the method is for inhibiting senescence (a) of one or more cells, tissues, organs or organ systems in the individual or (b) in the individual.

22. A method according to claim 15 or 16, wherein the method is for inhibiting aging in the individual.

23. A method according to any one of claims 15 to 22, wherein the inhibitor is as defined in any one of claims 3 to 8 and 11.

24. A method according to any one of claims 15 to 23, wherein the method results in any of effects (a) to (f) as defined in claim 12 in one or more cells, tissues, organs or organ systems of the individual or patient.

25. A method according to any one of claims 15 to 24, wherein the individual or patient is human.

26. Use of an inhibitor of miR-488-3p in the manufacture of a medicament for increasing mitochondrial function in an individual.

27. An inhibitor of miR-488-3p for use in a method of increasing mitochondrial function in an individual.

28. Use of a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) in the manufacture of a medicament for increasing mitochondrial function in an individual.

29. A polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) for use in a method of increasing mitochondrial function in an individual.

30. A kit for increasing mitochondrial function in a cell or a patient comprising (a) an inhibitor of miR-488-3p and (b) means for measuring mitochondrial function.

31. A kit for increasing mitochondrial function in a cell or an individual comprising (i) a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) and (ii) means for measuring mitochondrial function.

32. An in vitro method of determining whether or not a cell has decreased mitochondrial function or mitochondrial dysfunction, comprising measuring the amount of miR-488-3p and/or the amount of the long non-coding RNA (IncRNA) comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell, wherein an increased amount of miR-488-3p compared with a normal cell of the same type indicates that the cell has mitochondrial dysfunction and wherein a decreased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 compared with a normal cell of the same type indicates that the cell has decreased mitochondrial function or mitochondrial dysfunction.

33. A method of determining whether or not a patient has or is likely to develop a disease or disorder associated with mitochondrial dysfunction, comprising measuring the amount of miR-488-3p and/or the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in a cell sample from the patient, wherein an increased amount of miR-488-3p compared with a normal cell sample of the same type indicates that the patient has or is likely to develop the disease or disorder associated with mitochondrial dysfunction and wherein a decreased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 compared with a normal cell sample of the same type indicates that the patient has or is likely to develop the disease or disorder associated with mitochondrial dysfunction.

34. A method of treating or preventing a disease or disorder associated with

mitochondrial dysfunction in a patient, comprising determining whether or not the patient has or is likely to develop a disease or disorder associated with mitochondrial dysfunction using a method according to claim 33 and, if the patient has or is likely to develop the disease or disorder, administering to the patient an inhibitor of miR-488-3p or a polynucleotide which comprises (a) the sequence shown in SEQ ID NO: 4, 5 or 6 or a variant thereof which has at least 50% homology to SEQ ID NO: 4, 5 or 6 based on nucleotide identity over its entire sequence or (b) a sequence which encodes a sequence of (a) and thereby treating or preventing the disease or disorder in the patient.

35. A method of identifying a compound which is capable of increasing mitochondrial function in a cell, comprising contacting the cell with the compound and measuring the amount of miR-488-3p and/or the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell, wherein a decreased amount of miR-488-3p in the presence of the compound compared with the absence of the compound indicates that the compound is capable of increasing mitochondrial function in the cell and wherein an increased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the presence of the compound compared with the absence of the compound indicates that the compound is capable of increasing mitochondrial function in the cell.

36. A method of identifying a compound as an inhibitor of miR-488-3p in a cell, comprising contacting the cell with the compound and measuring the amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the cell, wherein an increased amount of the IncRNA comprising the sequence shown in SEQ ID NO: 4, 5 or 6 in the presence of the compound compared with the absence of the compound indicates that the compound is capable of inhibiting miR-488-3p in the cell.

7. A compound or an inhibitor identified using a method according to claim 35 or 36.