Patent Application
20170242034
The present invention relates to the field of in vitro testing methods for investigating impaired mitochondrial DNA (mtDNA) replication phenomena in biological samples collected from individuals, animals or humans, and in particular relates to methods that can be applied to the monitoring and/or diagnosing of the health status of a subject susceptible of suffering from physiological ageing (also referred to as chronological ageing or organismal ageing herein), or physiopathological ageing, in particular premature ageing or accelerated ageing or of a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof. The invention also relates to kits for performing the methods of the invention and their uses.
The present invention also relates to a method for treating or delaying the symptoms of a subject suffering from physiological ageing or pathophysiological ageing, in this last case in particular premature ageing or accelerated ageing, or of a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders, through administration of protease inhibitor(s), in particular serine protease inhibitor(s), having influence on the pathways associated with mtDNA replication, dysfunction and/or mismanagement of oxidative stress at the mitochondrial level, in particular defective pathways leading to abnormal levels of the functional POLG entity as defined hereafter, more particularly to the functional POLG1 protein, in particular abnormally low levels of POLG1 protein.
The present invention also relates to a nitroso-redox stress scavenger compound for use in a patient in need thereof to treat or delay Cockayne syndrome (CS) or symptoms thereof, and/or restore level(s) of protein(s) as disclosed herein, in particular to treat or delay Cockayne syndrome (CS) or symptoms thereof, said nitroso-redox stress scavenger compound being, according to a particular embodiment, MnTBAP.
The present invention also relates to a screening method for identifying particular protease inhibitor(s) and/or nitroso-redox stress scavenger compound(s) of interest within the context of the invention.
The present invention is of particular relevance in the context of diseases and symptoms directly associated with physiological ageing or physiopathological ageing, in particular premature ageing syndromes, especially progeroid syndromes—such as Cockayne syndrome (CS)—, which are a group of diseases all characterized by signs of premature ageing, and in the context of analysis of mitochondrial dysfunctions associated either with precocious ageing or observed during the onset and the establishment of neurodegenerative disorders.
The present invention relies on experiments emphasizing the relevance of the pathways associated with the management of oxidative stress in altered cells, in particular at the mitochondrial level.
Cockayne syndrome (CS), (OMIM entry 216400 http://www.omim.org/entry/216400, OMIM entry 133540 http://www.omim.org/entry/133540), also called Weber-Cockayne syndrome or Neill-Dingwall syndrome, is a rare genetic disorder characterized by neurological abnormalities and several growth and developmental defects, which include photosensitivity, hypersensitivity to oxidative damage, skeletal abnormalities, hearing loss, pigmentary retinopathy, progressive neurological disorders, mental retardation and premature ageing′.
CS is an autosomal recessive disorder with an incidence of 1 per 2.7 million births in western Europe2. The incidence is extremely high in immigrant populations, likely for the consanguineous marriages2.
CS is due to mutation in genes CSA (or ERCC8)3 and/or CSB (or ERCC6)4, which are required for nucleotide excision repair (NER), a DNA repair mechanism that removes bulky DNA adducts such as UV-induced DNA damage. About 70% of identified CS patients carry a mutation in CSB5. In particular, CSB is implicated in a sub type of NER, TC-NER (or transcription-coupled NER) also called TCR, that acts specifically on lesions located on the transcribed strand of expressed genes6. The other NER subtype GG-NER (or global genome NER) acts anywhere throughout the genome, and is normal in CS patients. The absence of TCR in cells treated by genotoxic agents leads to apoptosis. Mutations in the CSA or CSB genes are known to affect the proper performance repair of altered DNA structures resulting from UV damage, via transcription-coupled nucleotide excision repair (TC-NER) pathway. In a CS diagnosis situation however, the situation is complicated by the fact that some mutations in CSA exist, which affect the TC-NER pathway but do not affect the oxidative stress response, and are accordingly associated with a much milder syndrome (UV-sensitivity syndrome, UVSS) with no sign of precocious ageing11.
There are at least two forms of CS: type I, which is characterized by normal foetal growth and abnormalities appearing in the first two years or later and degeneration between 10-20 years, and type II, which is associated with little neurological development early after birth and death usually during the first decade.
It is important to note that currently neither large-scale diagnosis method nor a pre-natal test exists for CS, other than genotyping for CSA and CSB mutations. In spite of the devastating phenotype of CS, to date there is no indication of relevant, and easy to detect, cellular alterations in CS cells compared to UVSS and to normal cells.
According to another aspect, the Cockayne syndrome (CS) may be characterized by two major impairments, which are related to each other: appearance of premature ageing and neurological/developmental abnormalities.
In relation to these impairments, CS patients also display a variety of symptoms that include short stature, failure to gain weight and to thrive, microcephaly, hearing loss, eye abnormalities, severe tooth decay, bone abnormalities, and changes in the brain that can be detected on brain scans. These patients are also very sensitive to sun light (photosensitivity).
CS defects are strictly related to each other in a way that no indication enables to uncouple any of these defects from the other. Thus, treatments or conditions that alleviate or rescue the CS defects would be expected to act on multiple if not all the symptoms of CS. It happens that nervous cells and the nervous system are amongst the most sensitive targets of the CS impairment. Therefore treatments expected to act on CS can be advantageously considered both in the context of premature ageing and neurological abnormalities. Including the nervous system in the paradigm of CS results in an appropriate understanding and consequently enables the possibility of an appropriate care of this disease.
Therefore treatments that reverse or alleviate critical molecular impairment(s) in cells from individuals with CS, i.e., CS cells herein, extend to the cells of the nervous system, which are systematically included. It also has to be noted that to date, there is no perfect model to study neurodegenerative diseases in laboratory. In particular, analysis and treatment on, in particular, fibroblast cells, can be considered as representative of the impact of the same on neuron-type cells for CS-related alterations.
In addition, alterations observed in the premature ageing syndromes are considered to be representative, although at a much faster rate, of physiological deteriorations occurring during normal, i.e., physiological ageing, which includes neurodegeneration. Consequently, treatment(s) that reverse, at least partly, these impairments in disease should also be intended in the context of deteriorations due to normal ageing and neurodegeneration.
Turning to the molecular mechanism(s) involved in the CS, the current prevalent view is that TC-NER is impaired in CSA and CSB mutants, which results in the inability to repair the DNA lesions or clearing stalled RNA polymerase II in front of DNA damage, thereby blocking transcription after UV damage resulting in global transcription arrest. However, it seems that CSB-mutated cells, which have been extensively studied, have a transcription defect beyond the TC-NER impairment7. In this context, CSB appears as a transcription factor implicated in the activation of several genes and networks.
The severe phenotype of CS patients is however hard to reconcile with a sole defect in TC-NER and transcription7.
In fact, CSB cells, i.e. cells from patients known to have an impaired CSB gene, as disclosed in Nardo et al, 2009 PNAS 106 (15):620914, are also hypersensitive to oxidative damage. Indeed CSA and CSB appear to be involved in the repair of oxidative DNA lesions, produced by endogenous reactive oxygen species and normally repaired by the base excision repair (BER) pathway8. CSA/CSB modulate the BER pathway by direct interaction with BER proteins, and also by modulating the expression of BER genes. Both nuclear and mitochondrial BER are involved. Indeed CSA and CSB have been detected not only in the nucleus, but also in mitochondria9,10. The implication of CSA/CSB in the repair of oxidative stress could explain developmental defects and the neurological degeneration observed in CS. However, the impairment of BER due to CSA or CSB deficiency is mild, suggesting an additional role of the CS proteins in the response to oxidative stress and more in general in the etiology of CS8.
Interestingly, a mutation in CSA has been associated with the UV-sensitive syndrome (UVSS), an autosomal recessive disorder characterized by mild cutaneous symptoms and defective TC-NER, but not with other symptoms of the CS, in particular no hypersensitivity to oxidative damage and no precocious ageing11. Thus defects in TC-NER and oxidative damage repair have been uncoupled in the phenotype of CSA mutations, suggesting that much of CS symptoms are due to the management of oxidative stress rather than to transcription-related DNA repair.
In fact, defects in NER are associated not only with CS, but also with xerodema pigmentosum (XP), characterized by increased sensitivity to tumors in sun-exposed areas of the skin, and trichothiodystropy (TTD), characterized by developmental and neurological abnormalities, and premature ageing12. CS and TTD are cancer-free disorders. XP and TTD are essentially associated with defective GG-NER12.
More generally, Cockayne syndrome is considered to pertain to progeroid syndromes, which are a group of diseases all characterized by signs of premature ageing. These syndromes include: Hutchinson-Guilford progeria syndrome (HGPS), Werner syndrome (WS), Bloom syndrome (BS), Rothmund-Thomson syndrome (RTS), Fanconi anemia (FA), Ataxia telangiectasia (A-T), Cockayne syndrome (CS), Xeroderma pigmentosum (XP) and trichothiodystropy (TTD)13.
Several of these syndromes are classified as segmental progeroid syndromes as multiple organs and tissues replicate phenotypes associated with normal ageing14.
HGPS and WS are two of the best characterized human progeroid diseases15. HGPS, which is one of the most severe forms of progeria, has an incidence of 1 in 4-8 million births and distinct clinical symptoms are developed during the first year of life, and patients die at a median age of 11-13 years. HGPS is also called “progeria of the childhood”. WS has an incidence of 1 per million births (but 1:100,000 in Japan), and Werner heterozygotes are 1/180 in the general population. Symptoms appear in the first-second decade and the life expectancy reaches 47-54 years. WS is also called “progeria of the adult”.
Most progeroid syndromes include defects in distinct repair systems such as NER, BER, and double strand break repair (DSB)13,15. The exact mechanism(s) by which these mutations lead to progeria is/are not known yet. Moreover, the extent of progeria is different among these syndromes. At present, there is no also no indication why mutation in some genes result in more severe progeria syndromes than mutations in other genes.
HGPS is mutated in LMNA that encodes for the four different types of lamins. Mutations activate a cryptic splice site that lead to deletion of 50 aminoacids (the deleted protein is called progerin) that cannot undergo further processing. Lamins constitute the major component of the nuclear lamina, which provide structure and shape to the nucleus and are also involved in chromatin organization and DNA replication, transcription, and repair. The prevalent view is that lamin mutations lead to deficient DNA damage response, probably by sequestering replication and repair factors, leading to stalled DNA replication forks that collapse into DSBs. Moreover, lamin defects would increase DNA damage signalling at the level of telomeres and reduce the telomere length, leading to early cell senescence.
The Werner syndrome is associated with mutation of WRN, an ATPase-helicase (of the family of RECQ helicases, which unwinds the DNA double strands by hydrolyzing ATP). WRN protein is involved in DNA replication, recombination and telomere maintenance and its impairment results in chromosomal aberrations.
The Bloom syndrome and the Rothmunds-Thomson syndrome are due to mutation in the RECQ helicases BLM and RECQ4, respectively. As with the WRN helicase, the RECQ4 and BLM helicases are necessary to maintain genome integrity, but they differ in their functions and in their interaction partners16. Bloom syndrome is characterized by a very high level of spontaneous sister chromatid exchanges (SCE).
At least 16 genes responsible for the Fanconi anemia have been identified, and they are all linked to a DNA damage signalling pathway (the FA pathway) that is activated in response to DNA damage, in particular DSBs. Eight FA proteins form a nuclear-localized complex with E3 ubiquitin ligase and thereby catalyze monoubiquitination in target proteins. This monoubiquitination does not lead to proteasomal degradation but it can alter cellular localization or the function of the target protein.
Ataxia-telangiectasia is due to mutation in the ATM gene, a serine threonine kinase that is important at the level of DNA damage (in particular DSB) signalling and activation of DNA repair mechanisms.
All progeroid diseases display clinical features mimicking physiological ageing at an early age. They might provide insights into the process of normal human ageing and/or dysfunctions linked to normal human ageing (also referred to as physiological or chronological or organismal ageing herein), which is itself characterized by dysfunction of several physiological processes, as well as insights into physiopathological ageing, in particular premature ageing, as disclosed herein15.
By “early age” within the expression “physiological ageing at an early age” recited above, it is meant an age that is earlier than the age of normal onset of the symptoms of physiological ageing or an age that is not consistent with increased frequency of a condition or a disease that is generally related to aging.
Clinical symptoms of physiological ageing or physiopathological ageing, in particular premature aging, include skin atrophy with loss of cutaneous elasticity, dysfunction of cutaneous appendices, degeneration of the central nervous system, neurodegenerative symptoms, diabetes mellitus, changes in the volume of the adipose tissue, pigmentary changes with hyper- and hypopigmentation of the skin (poikiloderma), regional skin fibrosis, premature hair graying or hair loss, osteoporosis, and in certain cases tumors typical of those seen in patients of older age13. These symptoms are also associated with physiological ageing, although they appear at a later age in normal individuals.
In this context, inventors' investigations focused on the mechanism(s) underlying the etiology of CS in particular, which are also applicable to all diseases displaying symptoms of physiological ageing or physiopathological ageing, in particular premature ageing. These investigations have put in light dysfunctional mitochondrial pathways, in particular associated with ageing in general, and a completely new mechanism that may in particular explain defects in CS cells.
Back to the considerations introduced above regarding the management of oxidative stress, mitochondria are largely responsible for the production of oxidative stress, and CS patients are highly sensitive to oxidative damage. Oxidative damage is known to affect replication and transcription of mitochondrial DNA resulting in a decline of the mitochondrial function18. A large set of data suggests that oxidative damage is also associated with physiological ageing.18 Therefore, alterations in syndromes of precocious ageing like CS are considered informative also for understanding physiological ageing, since they recapitulate the dysfunction(s) observed in physiological ageing (Dreesen and Stewart, 2011 Aging, 3:889-895; Scaffidi and Misteli, 2006 Science 312: 1059-1063).
The present invention is based on the findings of new elements paving the way to a better diagnosis and treatment of symptoms of physiopathological ageing, in particular premature ageing or accelerated ageing or diagnosis and treatment of progeroid syndrome(s), such as Cockayne syndrome (CS), or neurodegenerative disorders or associated symptoms.
The invention therefore relates to an in vitro method for investigating mtDNA replication dysfunction (defective mtDNA replication or, differently said, mtDNA replication impairment or dysfunction of the mtDNA replication apparatus and/or machinery) in a biological sample removed from a subject susceptible of suffering from physiological ageing or physiopathological conditions related to physiological ageing, or physiopathological ageing or associated symptoms or conditions, in particular premature ageing or accelerated ageing, or of a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof, said method comprising the steps of:
Within the context of the invention, it has been shown by the inventors that dysfunctional mtDNA replication can be correlated to the observed levels of the expressed POLG entity, as defined herein, in particular the expressed POLG1 or POLG1 and POLG2, although not due to a mutation affecting the POLG entity in way rendering this entity, in particular the POLG1 protein or POLG1 and POLG2, dysfunctional in themselves. It is documented that mutations affecting POLG are associated to mtDNA depletion syndromes. However, within the context of the invention, impaired mtDNA replication due to reduced level of the key replicating enzyme (which otherwise works well), which is POLG1, can be evidenced.
Mitochondrial DNA replication is catalyzed by a mitochondria-specific mitochondrial complex comprising the so-called mitochondrial DNA polymerase gamma (γ) holoenzyme, which is an heterotrimer consisting of a single 140 kDa catalytic unit (encoded by the POLG1 gene at the nuclear chromosomal locus 15q25) and a 55 kDa accessory subunit that forms a tight dimer (encoded by the POLG2 gene at nuclear chromosomal locus 17q).
The POLG1 protein (SEQ ID NO:1) (NCBI Reference POLG1: Gene ID 5428 http://www.ncbi.nlm.nih.gov/gene/5428, Primary source HGNC:9179, POLG1 protein NCBI Reference Sequence: NP_001119603.1 http://www.ncbi.nlm.nih.gov/protein/NP_001119603.1) is therefore the catalytic subunit of the so-called mitochondrial DNA polymerase gamma (γ), needed for mtDNA replication. The POLG1 protein is also referred to in the literature under the common name POLG, or POLGA (see the synonymy annotation at http://www.ncbi.nlm.nih.gov/gene/?term=NP_001119603.1). Human POLG1 (SEQ ID NO:2) is discussed in Stumpf and Copeland, 2011, Cell. Mol. Life Science 68:219-233 or Ropp & Copeland, 1996 Genomics 36:449-458. As shown on page http://www.ensembl.org/Homo_sapiens/Gene/Family?family=ENSFM00610 000964734; g=EN5G00000140521; r=15:89859534-89878092, there are several protein transcripts (splicing variants) corresponding to human POLG1. Accordingly, by POLG1 protein as referred to herein, it is meant the native form of the protein having a sequence as disclosed in databases and/or literature and/or herein, but also isoforms or variants thereof having a polypeptidic sequence showing 60% or 70% or 80% or 90% or 95% and up to 99% identity with the polypeptidic sequence of the native POLG1 protein.
POLG2 protein (SEQ ID NO:3) (NCBI Reference POLG2: Gene ID 11232 http://www.ncbi.nlm.nih.gov/gene/11232, Primary source HGNC:9180, POLG2 protein NCBI Reference Sequence: NP_009146.2 http://www.ncbi.nlm.nih.gov/protein/NP_009146.2), also called mitochondrial DNA polymerase subunit gamma-2, is a protein that in humans is encoded by the POLG2 gene, and is an accessory protein that increases the processivity of the catalytic subunit of the POLG protein. The POLG2 protein is also referred to in the literature under the common name POLGB (see the synonymy annotation at http://www.ncbi.nlm.nih.gov/gene/?term=NP_009146.2). Human POLG2 (SEQ ID NO:4) is discussed in Young et al, 2011 Human Molecular Genetics 20 (15):3052-3066. As shown on page http://www.ensembl.org/Homo_sapiens/Gene/Family/Genes?cdb=compara; db=core; family=ENSFM00250000007196; g=ENSG00000256525; r=17:624 73902-62493154, there are several protein transcripts (splicing variants) corresponding to human POLG2. Accordingly, by POLG2 protein as referred to herein, it is meant the native form of the protein having a sequence as disclosed in databases and/or literature and/or herein, but also isoforms or variants thereof having a polypeptidic sequence showing 60%, 70% or 80% or 90% or 95% and up to 99% identity with the polypeptidic sequence of the native POLG2 protein.
When reference is made herein to the POLG entity, reference is made to the POLG1 protein, or the POLG2 protein, or both.
HTRA3 protein (SEQ ID NO:5) (NCBI Reference HTRA3: Gene ID 94031 http://www.ncbi.nlm.nih.gov/gene/94031, Primary source HGNC:9180, HTRA3 protein NCBI Reference Sequence: NP_444272.1 http://www.ncbi.nlm.nih.gov/protein/NP_444272.1) is a serine peptidase (or serine protease) that is a member of the mammalian HTRA family. Human HTRA3 (SEQ ID NO:6) and human HTRA3 are discussed in Nie et al, 2003 Biochemical Journal 371:39-48 or Narkiewicz et al, 2009 Oncology reports 21: 1529-1237. As shown on page http://www.ensembl.org/Homo_sapiens/Transcript/Summary?db=core; g=E NSG00000170801; r=4:8271492-8308838; t=EN5T00000307358, there are several protein transcripts (splicing variants) corresponding to human HTRA3. More particularly, there are two (2) transcripts for HTRA3 (different splicing), giving a long (L) and a short (S) form, which are both discussed herein. Accordingly, by HTRA3 protein as referred to herein, it is meant the native form of the protein having a sequence as disclosed in databases and/or literature and/or herein, but also isoforms or variants thereof having a polypeptidic sequence showing 60%, 70% or 80% or 90% or 95% and up to 99% identity with the polypeptidic sequence of the native HTRA3 protein.
In a particular embodiment, the conclusion of step d) of the method of investigating the existence of a mtDNA replication dysfunction of the invention is made if the level of each species selected to carry out the above disclosed steps is as follows:
By “mtDNA replication dysfunction”, it is meant that mtDNA replication is altered or is totally impaired in cells of the assayed biological sample. This expression is used herein as a synonym of defective mtDNA replication or mtDNA replication impairment. Indeed, the inventors showed that in cells of a sample wherein mtDNA replication is dysfunctional, only the amount of the enzyme responsible for synthesizing of mtDNA that is POLG1, is lower or much lower than in healthy control cells. Moreover the mtDNA content and the levels of TFAM, which is a factor involved in mtDNA transcription and maintenance is generally altered in one sense or the other, indicating that, within the context of the invention, it is the process of mtDNA replication that is affected, and not POLG1 that is defective in itself. Finally, the inventors have also evidenced that ATP production by mitochondria is reduced in all tested CS samples that have POLG defects, showing that the mitochondrial function itself is affected in the tested cases. These methods may be used for demonstrating that mtDNA replication is impaired in a given assayed sample.
As a synonym for “mtDNA replication dysfunction” can also be used the expression “dysfunction of the mtDNA replication apparatus and/or machinery”. Indeed, POLG1 is the key replication enzyme for mtDNA replication. If POLG1 declines, it is a fact that the replication apparatus is dysfunctional, as illustrated above. In addition, it is stated by the inventors that POLG1 decrease results in altered mtDNA content (essentially decrease but also increase). Therefore, there is a direct relationship between the levels of the species monitored within the present invention and the fact that mtDNA replication is impaired, which is also correlated with a dysfunctional mtDNA replication apparatus and/or machinery. Impaired mtDNA replication is indeed evidenced by a dysfunctional mtDNA replication apparatus and/or machinery, as illustrated above.
By “biological sample” it is meant a sample originating from the sampling of biological tissue(s) or fluid(s), especially body tissue(s) or fluid(s), which is therefore substantially constituted of cells, for example bodily fluid such as a cerebrospinal fluid, saliva, mucus, urine or blood sample, or include a cell lysate of the same origin, and/or include a conditioned culture medium, and is optionally derived from a tissue (e.g., a tissue homogenate), a biopsy.
In a particular embodiment, the assayed biological sample comprises or contains fibroblasts or culture(s) thereof, or consists of isolated cells, in particular fibroblasts, or culture(s) thereof. However, other cells should also be considered as much as they grow in culture or they are used as isolated cells from a biological sample (for example from a body fluid), as described above.
According to a particular embodiment, the in vitro method of the invention is used within a prenatal testing procedure, wherein the tested subject is an embryo or a foetus. In addition to samples generally used for this type of testing (such as amniotic tissue), it is also possible to test parent(s) material (such as fibroblasts, cells from biopsies). Parents may be heterozygotes for the CSA or CSB mutation and their POLG/HTRA3 values might be different from controls (and from CS). This seems indeed the case for the parent that the inventors have tested (CS358), as described herein, and who has no CS phenotype.
By “physiopathological ageing or associated symptoms or conditions, in particular premature ageing or accelerated ageing, or of a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof”, it is meant all conditions disclosed above and herein, which are not clinically consistent with the age of the subject, in particular a condition resulting in skin atrophy with loss of cutaneous elasticity, dysfunction of cutaneous appendices, degeneration of the central nervous system, neurodegenerative symptoms, diabetes mellitus, changes in the volume of the adipose tissue, pigmentary changes with hyper- and hypopigmentation of the skin (poikiloderma), regional skin fibrosis, premature hair graying or hair loss, osteoporosis, muscle atrophy, weight loss, alopecia, kyphosis, anaemia, reduced fertility, and in certain cases tumors typical of those seen in patients of older age, as well as symptoms typical of neurodegenerative disorders like ophtalmoplegia, ataxic neuropathy, inflammation, cerebellar ataxia, cachexia, neuron loss, deterioration of cognitive and kinetic body response.
According to a particular embodiment, the methods and means described herein are applied to subjects undergoing physiological ageing or suffering therefrom, with related detrimental consequences on their health or their capabilities or appearance. In this context, “physiological ageing” or “physiopathological condition related to physiological ageing” means the deterioration of the physiological processes, which appears with the aging of a subject, i.e., in an extent compatible with the observations commonly made on persons subject to normal ageing, when their age is taken in account. In a more particular embodiment, the state of “physiological ageing” in a subject can be determined by symptoms of hair and/or skin damage, including loss of cutaneous elasticity and/or observation of hair and/or cutaneous alterations that are nevertheless in correlation with the age of the subject.
According to another particular embodiment, the methods and means described herein are applied to subjects susceptible of suffering from physiopathological ageing. In this context, “physiopathological ageing” means the deterioration of the physiological processes appearing in a subject, which can be qualified as going beyond the observations that can be made, in average, on normal subjects having around the same age, and not diagnosed with a health disorder or condition, than the observed subject. This category includes subjects having diseases as invalidating as diseases entering in the category of premature ageing or accelerated ageing, or of a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof.
By “marker specific for at least one species selected in the group of: POLG1 protein, POLG1 RNA, POLG2 protein, protease(s) which have POLG as a target, in particular serine protease(s) such as HTRA3 protein, HTRA2 protein and, HTRA3 RNA, HTRA2 RNA”, it is meant a marker suitable for directly or indirectly specifically revealing the qualitative and/or quantitative presence of, respectively, one of the following species: POLG1 protein, POLG1 RNA, POLG2 protein, protease(s) which have POLG as a target, in particular serine protease(s) such as HTRA3 protein, HTRA2 protein and, HTRA3 RNA, HTRA2 RNA when further performing detection methods, in particular methods such as immunofluorescence, Western Blotting, ELISA or a PCR-based amplification method, such as RT-qPCR.
The specificity of the marker is assessed with respect to its ability to react with its target but not to react in a detectable or in a functionally effective manner with other compounds of the sample.
For example, nucleotide probes specifically binding to DNAs or RNAs and carrying a ligand at one end, which is recognized by a specific antibody, have been developed. Molecular beacons (antisense nucleotide probes targeting DNA or RNA with a fluorophore and a quencher at opposite ends), which emit a signal when hybridizing their targets (and are thus detected by fluorescence) have also been developed. These embodiments are encompassed in the definition provided above with respect to the markers that can appropriately be used within the present invention.
In the case of, but not exclusively, proteins, in particular POLG1, POLG2, protease(s) as identified above, HTRA3, HTRA2, such marker(s) may be an antibody specific for said protein(s), in particular POLG2 and/or HTRA3 protein(s) or a combination of several antibodies altogether specific for said protein(s), and, optionally, one or several of secondary antibody(ies) or reagent(s) (such as dye(s)) to reveal a complex between specific antibody(ies) recited above and its(their) target.
In case of RNA determination, in particular POLG1 RNA or HTRA3 RNA, such marker(s) may be at least one pair of specific oligonucleotide primers specific for hybridization (by base pairing) with the cDNA corresponding to the RNA target (e.g. POLG1 or HTRA3 RNA) or at least one pair of specific oligonucleotide primers specific for directly hybridizing with the corresponding target RNA, and, optionally, at least one label or marker for detection of nucleic acids, in particular a dye detectable in a real-time PCR equipment, for revealing the HTRA3 RNA or cDNA marker. According to a particular embodiment, the pair of specific oligonucleotide primers that is used is capable to hybridize both with the target RNA and the cDNA synthesized using the target RNA as a template.
According to a particular embodiment, protein(s) as defined above are detected together with RNA(s) as defined above, and use is made of a suitable combination of markers for this purpose: the markers used in combination can therefore, according to this embodiment, be all those mentioned above in all combinations thereof. They may in particular consist of at least one antibody specific for a protein selected amongst: POLG1, POLG2, HTRA3, HTRA2 or a combination of several antibodies specific respectively for POLG1, POLG2, HTRA3, HTRA2 and, at least one pair of specific oligonucleotide primers specific for hybridization with HTRA3 or POLG1 cDNA, and/or at least one pair of specific oligonucleotide primers specific for hybridization with HTRA3 RNA and/or POLG RNA or a combination of primer pairs specific for each of said HTRA3 or POLG1 cDNA or RNA.
A “marker specific for POLG1 protein” is a marker suitable for specifically targeting and optionally revealing the qualitative and/or quantitative presence of POLG1 protein when further performing, in particular, immunofluorescence, Western Blotting or ELISA detection methods.
In the case of POLG1 protein level measurement, such a marker may be an antibody specific for POLG1 protein or a combination of several antibodies specific for POLG1 protein, and, optionally, one or several secondary antibody(ies) or reagent(s) to reveal a complex between specific antibody(ies) recited above and its(their) target, as in particular described herein within the Materials and Methods section. Other markers may include detectable molecules having a binding capacity or interaction capacity with POLG1.
By “conditions enabling said marker(s) to react with their respective targets”, it is meant either “conditions enabling a marker to react with its respective target RNA and/or cDNA”, or “conditions enabling a marker to react with its respective target protein”.
By “conditions enabling a marker to react with its respective target RNA and/or cDNA”, it is meant conditions enabling hybridization of primers to their nucleic acid target(s) for further performing an amplification method, as known by a person skilled in the art and/or described in notices provided by manufacturers when commercial kits are used, as in particular described herein within the Materials and Methods section.
By “conditions enabling said marker to react with a protein”, in particular the POLG1 protein, it is meant, in particular, but not exclusively, conditions enabling an immunological reaction to take place, as known by a person skilled in the art and/or described in notices provided by manufacturers when commercial kits and/or reagents are used, as in particular described herein within the Materials and Methods section.
By “measurement of the marker(s) that has(have) reacted with its(their) respective species it is therefore in particular made reference to the process of measurement of level(s) of protein(s), as described above, in particular by Immunofluorescence (IF), Western Blot or ELISA detection methods.
By “measurement of the reaction product(s) obtained after reacting the marker(s) with its(their) respective species”, it is therefore in particular made reference to the process of measurement of level(s) of RNA(s), as described above, in particular as reflected by the amplification product obtained after performing a PCR-based amplification method, such as RT-qPCR, on said RNA present in the assayed sample.
By “determining the level of at least one species selected in step a) from the group of: POLG1 protein, POLG1 RNA, POLG2 protein, protease(s) which have POLG as a target, in particular serine protease(s) such as HTRA3 protein, HTRA2 protein, and HTRA3 RNA, HTRA2 RNA, or any combination of these species in said biological sample”, it is meant obtaining an absolute or relative value representative of the amount of target species in the assayed sample, in particular by either:
In particular, by “determining the level of POLG1 protein”, it is meant obtaining an absolute or relative value, in particular by interpretation of the results (raw data or transformed data) obtained through the immunofluorescence, Western Blotting or ELISA detection methods mentioned above, or as described herein within the Materials and Methods section, which is suitable for evaluating the amount of POLG1 protein present in the assayed sample.
It will be understood from the above that marker(s) used within the present invention to obtain level(s) of specific species(s) as disclosed herein, are means suitable for revealing, directly or not, said level(s), if necessary after further steps based on the formation of an immunological complex between a particular marker and its target (e.g. when antibodies or labeled antibodies or set of antibodies are used), and/or further steps based on the hybridization between a particular marker and a nucleic acid target (e.g. when primers are used), and subsequent nucleic acid amplification and counting, according to methods known in the art and/or disclosed herein.
According to a particular embodiment, the level of POLG1 protein determined in step b) is decreased with respect to the normal threshold value introduced in step c) by at least 10%, in particular when the detection method that is used is not immunofluorescence.
In a general manner, percentages are evaluated with respect to the unit value(s) appropriate in the method carried out for the described measurement(s).
According to a particular embodiment, the level of POLG1 protein determined in step b) is decreased with respect to the normal threshold value introduced in step c) by at least 20%, or at least 30% or at least 40%, and up to 80% or up to 90%, in particular when the detection method that is used is immunofluorescence staining.
According to a particular embodiment, the level of POLG2 protein determined in step b) is increased with respect to the normal threshold value introduced in step c) by at least 15%, in particular when the detection method that is used is not immunofluorescence.
According to a particular embodiment, the level of POLG2 protein determined in step b) is increased with respect to the normal threshold value introduced in step c) by at least 25%, or at least 35% or at least 45%, in particular when the detection method that is used is immunofluorescence staining.
According to a particular embodiment, the level of HTRA3 protein, in particular the long isoform of HTRA3 protein, and/or HTRA3 RNA determined in step b) is increased with respect to the normal threshold value introduced in step c) by at least 2 folds, in particular when the detection method that is used is not immunofluorescence staining.
The level of increase with respect to a normal threshold value may depend on the quantification sensitivity of the detection method that is used.
According to a particular embodiment, the level of HTRA3 protein, in particular the long isoform of HTRA3 protein, and/or HTRA3 RNA determined in step b) is increased with respect to the normal threshold value introduced in step c) by at least 5 folds, or at least 10 folds, in particular when the detection method that is used for detecting the level of HTRA3 protein is immunofluorescence staining. In a specific embodiment, said level of HTRA3 is increased by at least 50 or at least 60 or at least 70 folds when the detection method that is used is immunofluorescence staining. In a particular embodiment, the inventors found huge increase of HTRA3 RNA (2 to 160-fold) and HTRA3 protein by IF (60 to 3000-fold). However, when RT-qPCR is used for detecting RNA, obtained values may be lower, which is consistent with the fact that as one single RNA molecule can be used to produce multiple proteins. Therefore, according to a particular embodiment wherein the level of HTRA3 RNA is determined, if necessary in all combination(s) with other parameter(s) (in particular, species) as disclosed herein, said level of HTRA3 RNA determined in step b) is increased with respect to the normal threshold value introduced in step c) by at least 2 folds.
According to particular embodiments, all the levels indicated above can be used in combination, in particular when meeting the above-mentioned thresholds, for concluding according to the explanations provided herein and/or deducible by a person skilled in the art.
By “normal threshold value(s) determined for healthy subject(s) for POLG” it is meant level(s) of POLG protein found by assaying one biological sample from an healthy subject or alternatively found by assaying several biological samples from several distinct healthy subjects, the resulting normal threshold value being then determined as the mathematical mean of the levels of POLG protein values of all the assayed healthy subjects biological samples, or alternatively found by assaying a pool of biological samples from several distinct healthy subjects.
According to another embodiment, an in vitro method for investigating mtDNA replication dysfunction of the invention also encompasses determination of the level(s) of other markers (and consequently, species), which are: POLG2 protein, protease(s) which have POLG1 as a target in a sample provided in conditions enabling said marker to react with said protease(s), in particular serine protease(s) such as HTRA3 protein, HTRA2 protein, or a marker specific for HTRA3 RNA or HTRA2 RN A in a sample provided in conditions enabling said marker to react with their respective RNA target, as an additional parameter (species) to POLG1 level determination or as an alternative to a determination of the level of POLG1 protein.
Such markers may be an antibody specific for a protein selected amongst: POLG2 and/or HTRA3 and/or HTRA2 or a combination of several antibodies specific for POLG2 and/or HTRA3 and/or HTRA2, and, optionally, one or several of secondary antibody(ies) or reagent(s) to reveal a complex between specific antibody(ies) recited above and its(their) target.
In case of HTRA3 RNA determination, such a marker may be at least one pair of specific oligonucleotide primers specific for hybridization with HTRA3 cDNA or at least one pair of specific oligonucleotide primers specific for hybridization with HTRA3 RNA, and, optionally, at least one label or marker for detection of nucleic acids, in particular a dye detectable in a real-time PCR equipment, for revealing the HTRA3 RNA or cDNA marker.
According to a particular embodiment, the in vitro method for investigating mtDNA replication dysfunction of the invention encompasses a determination of any combination of the above-mentioned species, in particular a combination of determination of POLG1 and POLG2 or a combination of determination of POLG1 and HTRA3 or a combination of determination of determination of POLG1, POLG2 and HTRA3.
According to a particular embodiment, the in vitro method for investigating mtDNA replication dysfunction of the invention encompasses a determination of any combination of the above-mentioned species, in particular a combination of determination of POLG1 and HTRA3 and optionally HTRA2 or a combination of determination of POLG1, POLG2 and HTRA3 and optionally HTRA2.
In a specific embodiment, the method of the invention comprises, in addition to a determination if the level(s) of POLG1 protein, the steps of:
POLG2 protein or HTRA3, or HTRA2 protein level(s) may be determined by immunofluorescence, by Western Blotting or by ELISA testing. The level(s) of HTRA3 or HTRA2 RNA or POLG transcripts may be determined by reverse transcription polymerase chain reaction (RT-qPCR).
According to another particular embodiment, the conclusion of steps d) above of the existence of a mtDNA replication dysfunction is made if the level of each species selected to carry out the above disclosed steps is as follows:
at least two of the above measurements meet the above thresholds.
In another aspect of the method of the invention, the level of POLG1 transcripts (synonym for POLG1 RNAs) is also determined and compared with a normal threshold value determined for healthy subject(s), conclusion being made of the existence of a mtDNA replication dysfunction if the level of POLG1 protein is decreased with respect to the normal threshold value by at least 10%, in particular at least 20%, especially when immunofluorescence is used for detection, and the level of POLG1 transcripts is within the range of normal threshold value determined for this species on normal (in particular non CS) cells, in combination with another species or not.
Indeed, the inventors particularly found out that, in cells of CS patients, the levels of POLG1 RNA do not change compared to controls, in contrast to POLG1 protein levels. This indicates that variation in POLG1 protein level results from degradation of the protein (likely due to the action of proteases, in particular HTRA3) rather than its insufficient expression.
Moreover, it is surprising that in CS cells, where POLG1 protein levels are low, the corresponding RNAs level is normal. Therefore, a double check with these two species enables to be quite confident that variation in POLG1 protein level does not result from mutation(s) affecting PolG1 gene but rather POLG1 protein degradation after it is expressed.
As a consequence, the invention also relates to an in vitro method for investigating mtDNA replication dysfunction in a biological sample removed from a subject susceptible of suffering or suffering from Cockayne syndrome (CS), or symptoms thereof, said method comprising the steps of:
According to an aspect of the invention, a marker for revealing the level of POLG1 transcripts (synonym for POLG1 RNAs) may be at least one pair of specific oligonucleotide primers specific for hybridization with POLG1 cDNA, or at least one pair of specific oligonucleotide primers specific for hybridization with POLG1 RNA, and, optionally, at least one label or marker for detection of nucleic acids, in particular a dye detectable in a real-time PCR equipment. In this respect, the Material and Methods section provides examples of such markers or dyes that may suitably be used.
Performing the above method involving a marker specific for POLG1 RNA also enables performing a cross check that can be used, if necessary, in combination with other parameters (in particular, species) enabling to partially investigate mtDNA replication dysfunction, in particular in a biological sample removed from a subject susceptible of suffering or suffering from Cockayne syndrome (CS), or symptoms thereof, and ultimately investigate the occurrence of CS in a subject. Other parameters may be the protein level(s)) disclosed herein. The above method involving a marker specific for POLG1 RNA may be performed according to all the features disclosed herein.
The invention also encompasses a method, for monitoring or diagnosing the health status of a subject susceptible of suffering from physiological ageing, or physiopathological or accelerated ageing or a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof, said method comprising performing the method of determination of defective mtDNA replication as disclosed above encompassing the determination of POLG1 and alternatively or optionally one or several other species as disclosed herein and further comprising the following step:
According to a particular embodiment, the conclusion is the presence or a risk of occurrence or of a presence of physiological or accelerated ageing or a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof if conclusion is made of the existence of a mtDNA replication dysfunction.
In particular embodiments of the invention, the assayed biological sample is from a subject in need of being and/or diagnosed with physiological or accelerated ageing or a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof, and/or a subject having a family history of physiological or accelerated ageing or progeroid syndrome(s), such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof.
Progeroid syndromes referred to above may be selected amongst: Hutchinson-Guilford progeria syndrome (HGPS), Werner syndrome (WS), Bloom syndrome (BS), Rothmund-Thomson syndrome (RTS), Fanconi anemia (FA), Ataxia telangiectasia (A-T), Cockayne syndrome (CS), Xeroderma pigmentosum (XP) and trichothiodystropy (TTD), and the neurodegenerative disorder is selected amongst Alzheimer and Parkinson diseases.
According to another embodiment, the assayed biological sample is from a subject known to bear a mutation in the CSB or CSA gene associated with a risk of Cockayne syndrome (CS), in particular is known to be homozygous for a mutation in the CSB or CSA gene associated with a risk of Cockayne syndrome (CS). Accordingly, the present invention may reveal the extent of mitochondrial impairment on the basis of the assayed sample.
The invention also relates to a protease inhibitor which interacts with protease(s) degrading POLG (i.e., the POLG entity as defined herein) for use in restoring POLG levels (i.e., the POLG entity as defined herein) in a patient in need thereof to treat or delay physiological or physiopathological ageing, in particular premature ageing, or accelerated ageing or a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof, as defined above.
According to a particular embodiment, the invention also relates to a protease inhibitor which interacts with protease(s) degrading POLG1 for use in a patient in need thereof to treat or delay physiological or physiopathological ageing, in particular premature ageing, or accelerated ageing or a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof, as defined above.
By “protease inhibitor which interacts with protease(s) degrading POLG1” it is meant a protease inhibitor having as target protease(s) degrading POLG1 (global proteasome inhibitors which target cysteine and serine proteases, or more specific serine protease inhibitors). Such a protease inhibitor may be specific of a particular protease degrading POLG1 or having a broad range of specificity, i.e., specificity for several proteases degrading POLG1. Such a protease inhibitor may also target cysteine and serine protease(s).
According to a particular embodiment, the protease inhibitor which interacts with protease(s) degrading POLG1 of the invention is a protease inhibitor targeting the HTRA3 protein as defined herein.
The experiments carried out by the inventors have indeed demonstrated that the HTRA3 protein has a key role when it comes to the levels of the POLG1 protein in cells, including in cells representative of the conditions disclosed herein.
Since the inventors also found that the level(s) of HTRA2 protein increase(s) in CS patients, and according to a particular embodiment, the invention also relates to a protease inhibitor targeting HTRA2 and/or HTRA3 for use in restoring POLG1 levels in a patient in need thereof to treat or delay physiological or physiopathological ageing, in particular premature ageing, or accelerated ageing or a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof, as defined above.
The sequence of human HTRA2 protein (HTRA2 protein NCBI Reference Sequence: NP_037379.1 http://www.ncbi.nlm.nih.gov/protein/NP_037379.1), is provided herein under SEQ ID NO:7. The DNA sequence of human HTRA2 is provided under SEQ ID NO:8. As shown on page http://www.ensembl.org/Homo_sapiens/Transcript/Summary?db=core; g=E NSG00000115317; r=2:74757117-74760459; t=EN5T00000352222, there are several protein transcripts (splicing variants) corresponding to human HTRA2. Accordingly, by HTRA2 protein as referred to herein, it is meant the native form of the protein having a sequence as disclosed in databases and/or literature and/or herein, but also isoforms or variants thereof having a polypeptidic sequence showing 60% or 70% or 80% or 90% or 95% and up to 99% identity with the polypeptidic sequence of the native HTRA2 protein.
According to another particular embodiment, the protease inhibitor which interacts with protease(s) degrading POLG1 of the invention is a protease inhibitor targeting the HTRA2 protein as defined herein.
According to a further particular embodiment, the protease inhibitor which interacts with protease(s) degrading POLG1 of the invention is a protease inhibitor targeting both the HTRA3 and the HTRA2 proteins as defined herein.
HTRA proteins including HTRA2 and HTRA3 are Trypsin-like serine proteases, by contrast to other proteases classified as Elastase-like or Chymotrypsin-like proteases.
Therefore, according to a particular embodiment, the protease inhibitor(s) suitable for use within the present invention are inhibitors of Trypsin-like serine proteases, which encompass the family of Kunitz-type trypsin inhibitors.
According to a particular embodiment, the protease inhibitor which interacts with protease(s) degrading POLG1 of the invention is a protease inhibitor pertaining to the family of Kunitz-type trypsin inhibitors.
Natural trypsin inhibitors, also known as serine protease inhibitors (serpins), control the activation and catabolism of proteins by the inhibition of serine proteases in vivo. There are four natural sources of trypsin inhibitors: bovine pancreas, ovomucoid, soybean, and lima bean. All these sources are suitable sources for the natural trypsin inhibitors encompassed herein.
Molecules part of the family of Kunitz-type trypsin inhibitors include: BPTI (Basic Pancreatic Trypsin Inhibitor), Ovomucoid (also named Trypsin Inhibitor from chicken egg white (Type II-O or Type III-O)), Kunitz Soybean Protease Inhibitor, BBI (Bowman-Birk (BBI) Soybean Protease Inhibitor), LBTI, STI, Tia1, Trypsin inhibitor Type I-S/Type II-S, which are, according to particular embodiments, suitable for use as protease inhibitor(s) within the present invention, taken alone or in all combinations thereof.
In specific embodiments of the invention, the protease inhibitor to be administered to a patient in need thereof is a serine protease inhibitor, for example Soybean trypsin inhibitor (KSTI).
By “restoring POLG1 levels in a patient in need thereof” it is meant obtaining POLG1 levels values equal to or close to the normal threshold value(s) determined for healthy subject(s) for POLG1, as defined above.
According to a particular embodiment, the physiopathological ageing, in particular premature ageing, or accelerated ageing or a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof recited above are associated with mtDNA replication dysfunction, and mtDNA replication dysfunction is in particular determined according to the method for investigating mtDNA replication dysfunction of the invention as defined herein.
In a particular embodiment, the physiopathological ageing, in particular premature ageing, or accelerated ageing or a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof are associated with an abnormal expression of a functional protease, in particular an abnormal expression of functional POLG, more particularly functional POLG1, an abnormal expression being defined by reference to a normal expression value determined for healthy subject(s), as introduced above, said activity value(s) corresponding to level(s) of expressed functional protease(s), for example determined by immunofluorescence or Western Blotting or ELISA testing.
By “functional protease”, it is meant a protease that has the capability to perform its function with the same performances than in a healthy cell. The capability to function with the same performances than in a healthy cell may be tested in living cells with mutants or knock down/silenced or knock out gene(s) coding for the protease of interest, and assessing the levels of POLG1 or any other protein of interest by Western Blot (WB) or Immunofluorescence (IF). Functional POLG1 may also be detected by sequencing the corresponding gene or checking that there is no large mtDNA depletion (as it is the case for pathological POLG1 mutations). For such a testing in protein extract(s), the protease of interest should be biochemically isolated and tested in vitro with a labeled substrate, the activity of which may be monitored to check its disappearance after contact with the protease of interest. Isolation of the protease of interest may encompass the use of sample containing several proteins, or the isolated protein of interest alone.
In a particular embodiment, an abnormal expression of a functional protease is an expression that is increased by reference to a predetermined normal expression value (e.g. at least a 2-fold increase for HTRA3 RNA; and/or at least a 10-fold of immunofluorescence signal for HTRA3 protein). Similarly to the determination of levels of proteins in healthy subjects referred to above, one skilled in the art can use common methods to determine said predetermined normal expression value by retrieving data from experiments carried on sample(s) containing healthy cells, or pools of such samples.
In a specific embodiment of the invention, a protease inhibitor to be administered to a patient in need thereof is a proteasome inhibitor, for example MG132 or is a serine protease inhibitor, for example Soybean trypsin inhibitor (KSTI).
According to a particular embodiment of the invention, use is made of a proteasome inhibitor as a protease inhibitor, which pertains to the family of benzyloxycarbonyl analogues or carbobenzoxy analogues.
MG132 is a molecule also known under its IUPAC name: benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate. Other names for MG132 are N-(benzyloxycarbonyl)leucinylleucinylleucinal or Z-Leu-Leu-Leu-al or carbobenzoxy-Leu-Leu-leucinal.
According to another particular embodiment, use is made of a protease inhibitor, which is not a proteasome inhibitor as defined herein, or more particularly of a protease inhibitor to the exclusion of the MG132 described above.
According to a more specific embodiment, use is made of a protease inhibitor as defined herein, to the exclusion of the proteasome inhibitors or of the MG132 when the physiopathological ageing condition to be treated is a neurodegenerative disorder.
It has been indeed observed that proteasome inhibitors such as MG132 have toxic effects on their cellular targets that may be regarded as counterproductive in the context of use according to the invention.
KSTI, as discussed above, is one of the two major trypsin inhibitors in soybeans, as disclosed in Reza Roosta et al, 2011 Advances in Environmental Biology, 5(1): 145-153, which describes its isolation and characterization. KSTI is in particular known to target, at least, both HTRA2 and HTRA3 proteases amongst the family of serine proteases.
In a particular embodiment of the invention, the protease inhibitor used for increasing POLG levels in a patient in need thereof is administered to a patient diagnosed with Cockayne syndrome (CS).
Since management of oxidative stress is altered in CS defective cells and involved in the new mechanism described herein explaining defects in CS cells, the invention also relates to a nitroso-redox stress scavenger compound or a composition comprising or consisting essentially of such a compound for use in a patient in need thereof:
By “nitroso-redox stress scavenger compound(s)”, it is meant a compound having for functional achievement(s) to act on the nitroso-redox balance by scavenging one or, preferably both, amongst reactive oxygen species (ROS) and reactive nitrogen species (RNS). Such compounds may be antioxidant(s), in particular antioxidant(s) with a porphyrine core or moiety.
According to a particular embodiment, said “nitroso-redox stress scavenger compound(s)” pertain to the category of SOD Mimetic and Peroxynitrite Scavenger(s).
According to another particular embodiment, said “nitroso-redox stress scavenger compound(s)” is/are a cell permeable superoxide dismutase mimetic(s) (against ROS) and peroxynitrite scavenger(s) (against RNS).
According to another particular embodiment, said “nitroso-redox stress scavenger compound(s)” pertain to the family of porphyrin chloride analogs.
According to another particular embodiment, said “nitroso-redox stress scavenger compound(s)” is/are metalloporphyrin(s), in particular manganese porphyrin(s).
According to a particular embodiment, the nitroso-redox stress scavenger compound is selected amongst:
and a mixture of (I) and (II), or analogs thereof.
By “restoring the level(s) of protein(s) selected in the group of: HTRA2, HTRA3 and POLG, or combinations thereof” it is meant obtaining HTRA2 and/or HTRA3 and/or POLG (i.e., the POLG entity as defined herein) levels values, taken alone or in all combinations of these species, equal to or close to the normal threshold value(s) determined for healthy subject(s) for HTRA2 and/or HTRA3 and/or POLG (i.e., the POLG entity as defined herein), respectively or in combination, as defined above and also illustrated in the Examples.
A nitroso-redox stress scavenger compound according to a particular embodiment of the present invention, as illustrated by formulas (I) and (II) above, is known in the literature under the common name MnTBAP.
According to this embodiment of the invention, and as disclosed herein, MnTBAP has the molecular formula C48H28ClMnN4O8 and encompasses compounds corresponding to the IUPAC name Chloro[4,4′,4″,4′″-(5,10,15,20-porphyrintetrayl-K2N21,N23)tetrabenzoato(2-)]manganese or systematic names Chloro [4,4′,4″,4′″-(5,10,15,20-porphyrintetrayl-K2N21,N23) tetrabenzoato(2-)] Manganese or Manganese (3+) chloride 5,10,15,20-tetrakis(4-carboxyphenyl)porphine-21,23-diide (1:1:1) or any one of the names Mn(III) meso-Tetra (4-carboxyphenyl) porphine chloride or Manganese(III)-tetrakis(4-benzoic acid)porphyrin or Mn(III) tetrakis(4-benzoic acid)porphyrin, or manganese [III] tetrakis (5, 10, 15, 20 benzoic acid) porphyrin, or Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride or their equivalents depending on the nomenclature referred to for the name used.
MnTBAP can be found under its salt form (II) or as a complex (I) or mixtures thereof. Commercial preparations of MnTBAP exist that may contain MnTBAP with amounts of Mn-free ligand. Such preparations are also suitable for carrying out the present invention.
Analogs of (I) or (II) may include a manganese porphyrin related to MnTBAP, for example, Mn [III] tetra(4-pyridyl) porphyrin (MnTyP). Analogs may further include copper containing porphyrins, such as copper(II) (3, 5-diisopropyl salicylate)2 (CuDIPS) and its derivatives.
According to another particular embodiment, suitable “nitroso-redox stress scavenger compound(s)” are MNTEPyP and its analogs, or MNTMPyP and its analogs.
By “nitroso-redox stress”, it is meant the alteration of the nitroso-redox balance. The nitroso-redox balance consists in the interaction between nitric oxide (NO) and reactive oxygen species (ROS) production. The nitroso-redox balance has relevant signaling function in the organism and its impairment may result in dysfunctions.
According to a particular embodiment, a nitroso-redox stress scavenger compound according to the present disclosure is used for treating or delaying Cockayne syndrome (CS) or symptoms thereof, and/or restoring the level(s) of protein(s) selected in the group of: HTRA2, HTRA3 and POLG, or combinations thereof, in particular to treat or delay Cockayne syndrome (CS) or symptoms thereof, wherein the Cockayne syndrome (CS) or symptoms thereof are associated with mtDNA replication dysfunction, in particular mtDNA replication dysfunction that is determined according to the method for investigating mtDNA replication dysfunction of the invention as defined herein.
According to a particular embodiment, a nitroso-redox stress scavenger compound according to the present disclosure is used for restoring the level(s) of protein(s) selected in the group of: HTRA2, HTRA3 and POLG, or combinations thereof, in order to treat or delay Cockayne syndrome (CS) or symptoms thereof.
In a particular embodiment, the Cockayne syndrome (CS), or symptoms thereof, are associated with an abnormal expression of a functional protease, in particular an abnormal expression of functional POLG, more particularly functional POLG1, an abnormal expression being defined by reference to a normal expression value determined for healthy subject(s), as introduced above, said activity value(s) corresponding to level(s) of expressed functional protease(s), for example determined by immunofluorescence or Western Blotting or ELISA testing. Definitions of “functional protease” and “abnormal expression of a functional protease” are the same than provided above.
According to a particular embodiment of the invention, the use of a protease inhibitor interacting with protease(s) degrading POLG1, for increasing POLG1 levels in a patient in need thereof and/or treating or delaying physiological or physiopathological ageing, or neurodegenerative disorders or symptoms thereof, as defined above, is performed on individuals previously subjected to the method for investigating mtDNA replication dysfunction according to any one of the embodiments described herein.
According to a particular embodiment, the use of a nitroso-redox stress scavenger compound according to the present disclosure for treating or delaying Cockayne syndrome (CS) or symptoms thereof, and/or restoring the level(s) of protein(s) selected in the group of: HTRA2, HTRA3 and POLG, or combinations thereof, is performed on individuals previously subjected to the method for investigating mtDNA replication dysfunction according to any one of the embodiments described herein.
It is another object of the present invention to use a protease inhibitor which interacts with protease(s) degrading POLG1 as defined herein in combination with a nitroso-redox stress scavenger compound as defined herein, or to use a nitroso-redox stress scavenger compound as defined herein in combination with a protease inhibitor which interacts with protease(s) degrading POLG1 as defined herein, in a patient in need thereof:
According to particular embodiments, the administration in combination of said protease inhibitor and nitroso-redox stress scavenger compound can be made concomitantly, separately or in a sequential regime, with the protease inhibitor being administered before or after the nitroso-redox stress scavenger to the patient in need thereof.
When active compounds are administered, to either cells or patients, the appropriate dosage regimen may be determined by the person skilled in the art. In particular, it is not unusual to severely decrease the dose administered to patients with respect to the dose administered to cells, without loss of effect.
The invention also relates to a kit suitable for carrying out a method of the invention, comprising:
In another aspect, a kit of the invention a kit suitable for carrying out a method of the invention, comprises:
The invention also relates to the use of a kit as defined above for investigating mtDNA replication dysfunction, and/or monitoring or diagnosing the health status of a subject susceptible of suffering from physiological or accelerated ageing or a progeroid syndrome, such as Cockayne syndrome (CS), or neurodegenerative disorders or symptoms thereof.
Examples of reagents suitable for being used within kits according to the present invention are also described in the Material and Methods section herein.
Another aspect of the invention is an in vitro process for screening protease inhibitor(s) for identifying protease inhibitor(s) capable of restoring POLG1 level in a cell, and/or for screening nitroso-redox stress scavenger compound(s) for identifying nitroso-redox stress scavenger compound(s) capable of restoring POLG1 level in a cell, comprising the steps of:
i. contacting a cell or a cell culture, in particular a fibroblast or a culture thereof, having a decreased level of POLG1 by at least 10%, in particular at least 20% with respect to a normal threshold value determined for cells characteristic of healthy subject(s) with a marker specifically recognizing POLG1,
ii. contacting said cell(s) with protease inhibitor(s) and/or nitroso-redox stress scavenger compound(s) to assay, wherein steps i. and ii. can be inverted,
iii. measuring and/or visualizing the change(s) in properties, in particular the level of POLG1 of the cell(s) contacted in steps i. and ii., and,
iv. optionally, recording or quantifying the change(s) in properties, in particular the level of POLG1, of the cell(s) contacted in steps i) and ii).
A definition of “nitroso-redox stress scavenger compound(s)” is provided above. The functional achievement(s) of such compound(s) is also disclosed herein. Nitroso-redox stress scavenger compound(s) may be ROS and/or peroxynitrite (or nitro-oxidative molecules) scavengers, as disclosed herein. Administration of such nitroso-redox stress scavenger compound(s) to a cell may ultimately lead to reduce ROS levels in said cell.
The change(s) in properties referred to in step iii) above are also mentioned in the present disclosure.
The invention will be further described herein, referring to the following figures, material and methods section, and experimental section.
1. Materials and Methods
Cells plated on slides were fixed with 2% PFA and permeabilized with 0.5% Triton X-100. The slides were incubated in blocking buffer (BSA 5% in PBS) for 1 h then with the primary antibody (POLγ and HTRA3 purchased from SantaCruz Biotechnology, or as available from any other provider) for 1 h at room temperature. A second, fluorescent antibody (goat anti-mouse and goat anti-rabbit Alexa® Fluor 488, Alexa® Fluor 555, conjugated secondary antibodies from Invitrogen or by any other provider), was incubated for 1-2 h at room temperature, and DNA was stained with 10 μg/ml Hoechst.
Image analysis was carried out using Perkin-Elmer Ultraview RS Nipkow-spinning disk confocal microscope. Three-dimensional reconstruction of all the z-stacks was achieved using the 3D-volume rendering of IMARIS software (Bitplane). A regular fluorescence microscope can also be used, including for fluorescence quantification, although in this last case it will quantify one section of the cell only and not the entire volume; this quantification may be sufficient for comparative studies (normal versus patient cells). Confocal acquisition (even in the absence of spinning disk) in 3D allows quantification of the entire volume, and differences among samples are therefore more robust.
Total RNA was isolated from HeLa cells using the RNAeasy Mini kit (Qiagen), treated with DNasel (Qiagen), then reverse-transcribed using Superscript®III Reverse transcriptase (Invitrogen). Real-time quantitative PCR was performed using Power Sybr Green PCR Master Mix (Applied Biosystems) and the rate of dye incorporation was monitored using the StepOne™ Plus RealTime PCR system (Applied Biosystems). Three biological replicates were used for each condition. Data were analyzed by StepOne Plus RT PCR software v2.1 and Microsoft excel. TBP transcript levels were used for normalisation of each target (=ACT). Real-time PCR CT values were analyzed using the 2−ΔΔCt method to calculate the fold expression (Schmittgen and Livak, 2008). Custom primers were designed using the Primer3Plus online software (http://www.bioinformatics.nl/cgi-bin/primer3plus.cgi). Customs primers used by the inventors (SEQ ID NO: 9 to 18) are listed in the table below.
Cells were lysed by lysis solution (20 mM Tris, 18 mM NaCl, 0.5% Lauryl β Maltoside, 1 mM MgCl2, 200 mM Na4P2O7, 1 mM EGTA, 20 mM NaF, 2 mM NaVO4, 1 mM Pefabloc (Sigma), 1 mM Aprotinin (Sigma), 1 mM Leupeptin (Sigma). Protein content was determined with the Bradford reagent (Sigma) and 30 μg of protein were loaded for SDS-PAGE. After blotting, Hybond ECL nitrocellulose filters were probed with anti-POLγ or anti-HTRA3 antibodies. Detection was performed using Odyssey Infrared Imaging system scanner and Odyssey application software v 3.0 (LI-COR Biosciences).
ELISA methods have been performed according to standard methods as known by the person skilled in the field and according to the recommendations of the fabricant(s) when standard kits were used.
2. Experimental Section
A. Diagnosis
The inventors found that POLG1 protein levels, measured by immunofluorescence and Western blot, are reduced in fibroblasts from CS patients (either from type I or type II, the latter displaying the most severe phenotype) compared to fibroblasts from healthy individual and from UVSS patients (
Silencing of CSB in HeLa cells using replicative pEBVsiRNA plasmids19 resulted in decreased polG levels (tested by RT-qPCR and immunofluorescence) at levels compatible with those observed in CS patients (
POLG2 is an accessory protein that increases the processivity of the catalytic subunit of POLG120. The inventors observed that POLG2 levels essentially increased in cells from CS patients compared to healthy individuals and to UVSS, although the effect was particularly strong for CSB mutations than for CSA mutations (
Reduced levels of POLG1 in CSB altered cells (in patients and by gene silencing) in the presence of regular levels of POLG1 transcripts indicate that either translation is impaired or protein degradation is enhanced. HTRA3 (high-temperature requirement factor A3) is a nuclear-encoded mitochondrial serine protease that degrades damaged proteins, and has a function during development, and possibly as tumor suppressor21. The inventors observed dramatically high levels of HTRA3 protein, assessed by immunofluorescence, in fibroblasts from CS patients, compared to controls and UVSS fibroblasts (
In Experiment 4, it has been shown association between increase of HtrA3 and depletion of PolG1, but not directly shown that increase of HtrA3 induced depletion of PolG1. This experiment provides direct evidence that this is the case. It is shown that overexpression of HtrA3 protein in HeLa cells decreases PolG1 levels. Both overexpression of HtrA3 (on a logarithmic scale) and PolG1 depletion appear comparable to levels detected in CS patient cells (
HtrA3 is a serine protease, which could target and degrade HtrA2 or a protein involved in the regulation of HtrA2. The inventors assessed HtrA2 levels in HtrA3 overexpressing HeLa cells, and observed that HtrA2 protein immunofluorescence was not altered by a few thousand-fold increase in HtrA3 (
B. Therapy Strategy
For the reasons developed above, the precocious ageing phenotype can be ascribed to the mismanagement of oxidative stress in CS and cells affected by ageing, let it be physiological or physiopathological, as described herein.
Through the preceding experiments, the inventors showed that cells from CS patients, compared to cells from healthy individuals and from UVSS, are characterized by lower levels (e.g. at least a 20% decrease, in particular when tested by immunofluorescence) of the nuclear encoded mitochondrial DNA polymerase gamma (POLG1), by higher levels of the accessory factor POLG2 (e.g. at least a 25% increase, in particular when tested by immunofluorescence), and by dramatically higher levels of the serine protease HTRA3 (e.g. at least a 10-folds increase, when tested by immunofluorescence). They also showed that alterations in the levels of these proteins are linked to impairment of CSB.
Thus, CSB impairment directly or indirectly increases Htra3 levels, and this serine protease in turn degrades its targets, which include POLG1. In spite of increased levels of the accessory protein POLG2, likely to compensate for the impaired mitochondrial DNA replication complex, replication of mitochondrial DNA is affected when CSB is not operating, leading to a decline in the mitochondrial function and thereby to enhanced production of oxidative stress. Increased oxidative stress and affected mitochondrial function, which cumulate with time, contribute to leading to precocious ageing phenotype.
HTRA3 is a serine protease. The inventors hypothesized that inhibition of proteases should decrease HTRA3 levels and help restoring correct POLG1 levels. They thus tested two protease inhibitors, MG132 that is a specific proteasome inhibitor, and Soybean trypsin inhibitor (KSTI), a natural serine protease inhibitor.
MG132 is a potent, reversible, and cell-permeable proteasome inhibitor that reduces the degradation of ubiquitin-conjugated proteins in mammalian cells. MG132 is known for its induction of apoptosis and to specifically target cancer cells versus normal cells, although the reasons for this specificity have not been elucidated23,24. Soybean trypsin inhibitor is a natural serine protease inhibitor25,26. It is mentioned as Kunitz soybean trypsin inhibitor (KSTI).
The inventors treated fibroblasts from healthy individuals, from UVSS and from Cockayne syndrome of type I and II (the last being the more severe), with protease inhibitors MG132 (5 μM) and KSTI (100 μg/ml) for 5 hours and then tested for POLG1 levels. The inventors found that treatment with either MG132 or KSTI resulted in increased levels of POLG1 immunofluorescence in fibroblasts of healthy individuals and of UVSS (
Thus, by treatment with protease inhibitors in CS cells it is possible to restore POLG1 levels at least as high as in normal cells. The fact that in two cases the inventors did not observe increase in POLG1 levels suggests that other proteases could be targeted using additional protease inhibitors. HTRA3 may therefore not be the only protease interacting with POLG1, suggesting that treatment aimed at increasing POLG1 levels can be effective through protease inhibitors having a different specificity than only specificity for HTRA3 as a target, in particular protease inhibitors having a large-range specificity. Alternatively, the POLG1 substrate could be improperly modified by other enzymatic activities, or be poorly modified by these activities, so that the protein becomes a poor target for being degraded by HTRA3 or other proteases.
In addition, the inventors also carried out experiments aimed at assessing the relative levels of oxidative stress in Cockayne syndrome (CS) fibroblasts, thereby revealing a preminent nitroso-redox imbalance in said fibroblasts.
The inventors assessed the relative levels of oxidative stress using the fluorescent probe dichlorofluorescein diacetate (DCFHDA), which prevalently detects reactive oxygen species (ROS)29. They reported that whereas UVSS cells display moderate (25%) increase of signal compared to controls, all CS cells are characterized by higher levels (1.6 to 2-fold) of oxidative stress (
High levels of ROS react with nitric oxide (NO), thereby quenching NO and promoting the formation of peroxynitrite (ONOO−), which is a powerful oxidant and nitration agent31. Using the fluorescent probe dihydrorhodamine 123 (DHR123), which selectively detects peroxynitrite29, the inventors observed that UVSS and CS cells significantly accumulate peroxynitrite compared to normal fibroblasts (
CSB-dependent accumulation of ROS and peroxynitrite was confirmed in CSB-silenced and CSB deficient cells, as well as restoration of CSB-proficient values in cells CSB overexpressing revertants (
In another experiment, the inventors also showed that ROS and peroxynitrite scavenging rescues altered mitochondrial parameters in fibroblasts from Cockayne Syndrome (CS) patients. By altered mitochondrial parameters, it is in particular meant HTRA2 and/or HTRA3 and/or POLG protein(s) level(s), as illustrated below and in
The inventors quantified fluorescence intensities on cells as summarized in Table 1 before and after treatment with MnTBAP (purchased from Millipore) of DCF and of DHR123 per cell (
As KSTI (serine protease inhibitor) and MnTBAP (ROS/RNS scavenger) rescue the CS phenotype by 24 h treatment, the inventors assessed cell survival in culture at longer incubation times with either drug. They used doses of the drugs as those affecting CS cells: 100 μg/ml of KSTI and 100 μM MnTBAP on adult primary fibroblasts IMR-90 and BJ (from ATCC repository). KSTI did not show effect on cell survival (cell number and cell shape) after continuous treatment by 6 days, and MnTBAP by 3 days. Additional experiments are ongoing to test the survival at longer incubation times and lower doses of the drug.
3. Conclusions and Discussion: Originality of the Tested Approach and Extent of the Investigations Carried Out
Inventors' data point to a completely new mechanism to explain defects in CS cells, which are also relevant for the process of precocious ageing in other diseases, and also for the process of physiological ageing. Inventors' data do not exclude that DNA repairs alterations take place in these cells, and that these alterations may lead to the symptoms of precocious ageing and tumours. Inventors' data show that cells from patients with CS display dramatically reduced POLG1, the nuclear-coded DNA polymerase that replicates mitochondrial DNA, compared to cells from normal individuals and UVSS patients (these patients carry a mutation in CSA, as it is the case for several CS patients, the other being mutated in CSB). The inventors thus assume that as a consequence the mtDNA is not properly replicated and thereby mitochondria are dysfunctional, in spite of no apparent modifications in shape and network, compared to cells from normal individuals. Such dysfunction will lead to an increase of oxidative stress, which is essentially produced by mitochondria, likely leading to dysfunctions observed in CS cells. The inventors showed that POLG1 decrease is associated with the CSB mutation as silenced CSB in HeLa cells behave as CS cells in this aspect, and restoration of the regular levels of CSB results in returning (at least) to POLG1 values as in non-silenced cells.
The decrease in POLG1 levels in CS cells is associated with the increase in POLG2, a co-factor of POLG1 that does not contain the catalytic subunit. The inventors also showed that reduced levels of POLG1 protein, in particular by at least 20% (but not of POLG transcripts that are not affected) are due to increased levels (in particular by at least a 10-folds increase) of HTRA3 (transcript and protein), a serine protease that has POLG1 and other proteins as potential target. Furthermore, by inhibiting HTRA3 with specific (e.g. Soybean trypsin inhibitor (KSTI)), or large-range (e.g. MG-132) inhibitors of proteases the inventors restored normal POLG1 levels in CS cells (from patients) as well as in CSB-silenced HeLa cells.
A. Diagnosis
Inventors' data show that CSA and CSB mutations in cells from Cockayne syndrome patients, and CSB-silencing are associated with decrease of the mitochondrial DNA polymerase POLG1 and with the increase in the accessory factor POLG2. In turn, decrease of POLG1 is associated with dramatic increase in the serine protease HTRA3. POLG1, POLG2 and HTRA3 levels should be considered as markers of Cockayne syndrome and possibly of events of precocious ageing or physiological ageing in general, the symptoms of which are described here above. Indeed, increased levels of HTRA3 or another protease that targets POLG1, generated from misregulation of this protease, which could be also age-driven (in normal subjects), results in decreased levels of POLG1, which in turn induce increased levels of POLG2 to compensate the impairment of the mtDNA replication complex. MtDNA replication is thereby impaired and mitochondria cannot fully ensure their function, leading to progressive dysfunction of the organelle, with reduced ATP production by mitochondria, decreased antioxidative response, and thereby increased oxidative stress, and finally altering global cellular activity, which together leads to the aged cellular and organism phenotype. Moreover, POLG1 mutations that affect the exonuclease activity of the polymerase, which corrects errors produced during DNA synthesis, have also been correlated to ageing phenotype in the literature. Reduced efficiency of mtDNA replication, as in the presence of altered levels of POLG1 and POLG2 could also lead to inefficient accuracy of the mtDNA copy, and therefore contribute to the ageing phenotype.
Mitochondrial dysfunction has been also associated with neurodegenerative diseases as overproduction of oxidative stress is a central feature of all neurodegenerative disorders (Lin et al, 2006, Nature 443: 787-95). Due to their high energy demand, muscle and nerve are the most affected tissue when mitochondrial function is impaired.
The inventors found that cells from CS patients are associated with very low levels of mitochondrial DNA polymerase gamma (POLG), which is responsible for replication of the mitochondrial genome. Levels of POLG1 are not altered in cells of healthy individuals and UVSS patients. Low levels concern the protein POLG1 and not its transcript, which is produced at regular levels in CS cells. The inventors demonstrated that silencing CSB resulted in low levels of POLG1, thereby linking the levels of this polymerase to CSA and CSB impairment.
They also found that in CS patients lower levels of POLG1 are associated with higher levels of the associated protein POLG2, compared to healthy individuals.
Moreover, they found that decreased levels of POLG1 in CS cells (from patients and after silencing) are associated with higher levels of HTRA3, a serine-protease that has POLG1 as potential target.
Therefore, POLG1, POLG2 protein and HTRA3 protein and transcript levels can be used as distinct markers for the diagnosis of Cockayne syndrome, as well as markers of mitochondrial dysfunctions associated with ageing in general, as described herein, and in neurodegenerative disorders.
B. Therapy Strategy
In the experiments provided herein, the inventors showed that anti-proteases rescue low POLG1 levels. Administration of anti-proteases is therefore a possible therapy for CS patients. It is important to recall here that absolutely no treatment is proposed to these patients, whom maximum life expectancy is around 20 years.
Although functional POLG1 can be detected by sequencing the gene or checking that there is no large mtDNA depletion (as it is the case for pathological POLG1 mutations), POLG1 mutations are associated with severe pathological phenotypes in the child, characterized by various levels of muscle and nerve impairment, but not with precocious ageing. In addition, it is not necessary to be sure that POLG1 is functional for providing a treatment within the context of the invention. Indeed the treatment of the invention can only improve mitochondrial function if POLG1 is functional. Would POLG1 not be functional, a treatment according to the invention would not be efficient.
The inventors found that treating primary fibroblasts with either inhibitor increases the levels of POLG1 in healthy individuals, demonstrating that POLG1 is indeed degraded by a protease that is targeted by MG132 or KSTI.
Importantly, the inventors found that POLG1 levels increase in most of CS cells in the presence of at least one protease inhibitor resulting in POLG1 levels at least as high as in untreated healthy cells.
It is therefore proposed to use protease inhibitors to increase POLG1 levels, whose reduction is a major indication of the CS phenotype, for treating Cockayne syndrome patients, and in particular for targeting the precocious ageing phenotype.
Regarding the ROS imbalance in Cockayne syndrome (CS) fibroblasts, the inventors showed that cells from Cockayne syndrome patients, mutated in CSA or CSB, and CSB-deficient immortalized fibroblasts (CsiAN), as well as CSB-silenced HeLa cells (siCSBA and si CSBb) display high levels of the serine proteases HTRA2 and HTRA3, and in turn low levels of the mitochondrial DNA polymerase POLG. These alterations seem at the base of the mitochondrial impairment observed in CS cells. HTRA3 and POLG levels are not altered in cells from a UVSS patient (no precocious ageing), which are mutated in CSA. Although it is not clearly elucidated what modulates HTRA3 levels, the inventors postulate, by analogy with HTRA2, whose expression increases in tissues undergoing oxidative stress27, that HTRA3 expression is also promoted in the presence of stress. CS cells have been reported to accumulate oxidative stress28. Alteration of ROS levels may also affect the nitroso-redox balance, as ROS and NO are linked. Nitroso-redox imbalance plays a key role in cell and organ failure, and this could also be the case for the aetiology of CS (Nediani et al, 2011 Antioxidants & Redox signaling 14 (2) 289-331; Takahashi, 2012 J. of Reproduction and Development 58 (1):1-9; Taverne et al, 2012 J. Appl. Physiol 112: 1644-1652)
Regarding the fact that ROS and peroxynitrite scavenging rescues altered mitochondrial parameters in fibroblasts from Cockayne Syndrome (CS) patients, the inventors reasoned that if Reactive Oxygen Species (ROS) and peroxynitrite induce serine proteases accumulation thereby resulting in POLG depletion, original parameters would be restored in CS fibroblasts treated with ROS and peroxynitrite scavengers.
Manganase(III)tetrakis(4-benzoic acid)porphyrin (MNTBAP) is a synthetic metalloporphyrin which mimics superoxide dismutase and scavenges ROS and peroxynitrite32. Treatment with MNTBAP for 24 h decreased by two thirds the levels of ROS, measured by DCFHDA, in control and UVSS fibroblasts, confirming the ROS scavenger effect of this molecule (
The inventors observed that the ROS and peroxynitrite scavenging action of MNTBAP was able to reduce by one half the levels of serine protease HTRA2 already in control cells. HTRA2 reduction was dramatically higher in UVSS and all CS cells, which originally displayed elevate levels of this protein (
Importantly, MNTBAP increased the levels of HTRA3 in control cells, but did reduce in UVSS and, to a largest extent in CS cells, where it restored control levels of HTRA3 (
Scavenging of nitro-oxidative molecules by MNTBAP resulted in increased levels of POLG1 in normal fibroblasts and, to a larger extent UVSS and CS cells, indicating that patient cells are particularly sensitive to ROS and peroxynitrite levels in the context of POLG1 regulation (
The inventors also observed that the glycolytic shift reported in CS cells is attenuated after 24 h treatment with MNTBAP, increasing the fraction of ATP produced by mitochondria (
As a consequence, it is therefore proposed to use MnTBAP to rescue altered mitochondrial parameters, in particular POLG levels, whose reduction is a major indication of the CS phenotype, and/or use MnTBAP for treating Cockayne syndrome patients.
As a short summary of the inventors' findings and conclusions disclosed herein, it is reminded that data in the literature30 and inventors' data show that CS cells display increased oxidative stress, and thereby display alteration of the oxidative stress management. Increased oxidative stress results in altered ROS levels, which in turn affects the nitroso-redox balance since these two parameters are linked (Nediani et al, 2011 Antioxidants & Redox signaling 14 (2) 289-331; Takahashi, 2012 J. of Reproduction and Development 58 (1):1-9; Taverne et al, 2012 J. Appl. Physiol 112: 1644-1652). Also, CSB mutation(s) result(s) in increased ROS levels and POLG1/POLG2/HTRA3 alterations. According to inventors' data, POLG1/POLG2 alterations induce mitochondrial impairment, revealed notably by altered mitochondrial DNA content and altered mitochondrial mass, reduced mitochondrial respiration, which in turn can generate more ROS. Moreover, the inventors consider that there is a relationship between CSB and the regulation of the levels of ROS. Ultimately, CSB mutation(s) increase(s) ROS levels by affecting the expression of ROS-regulating factors. ROS levels also increase because of dysfunctional mitochondria (due to POLG1/POLG2/HTRA3 alterations, as shown by the inventors), which is a fact also dependent of the CSB mutation(s). The combination of these two aspects results in unbalanced ROS levels (and thereby nitroso-redox levels), which act by promoting ageing. Promoting ageing by ROS is part of the largely accepted free radical and mitochondrial theories of ageing, well discussed in the literature18 (Cui, H., Kong, Y. & Zhang, H. Oxidative stress, mitochondrial dysfunction, and aging. Journal of signal transduction 2012, 646354, doi: 10.1155/2012/646354 (2012)). Therefore, either blocking HTRA3 degradation or scavenging ROS by MnTBAP results in reducing ROS levels, thereby restoring <<normal>> conditions (restoring mitochondrial function).
Since it is possible that the defects observed and discussed herein with respect to the CS phenotype may appear, although at a minor extent, also during normal (physiological) ageing, the treatment(s) described herein may also be considered in the context of preventive or prophylactic therapies. According to a particular embodiment, all the methods described herein are used for preventive or prophylactic purpose.
Acronyms used within the present disclosure: Ataxia telangiectasia (A-T); Base excision repair (BER); Bloom syndrome (BS); Cockayne syndrome (CS); DNA polymerase gamma (POLG1); DNA polymerase subunit gamma-2 (POLG2); Double strand break repair (DSB); Global genome NER (GG-NER); Fanconi anemia (FA); High-temperature requirement factor A2 (HTRA2); High-temperature requirement factor A3 (HTRA3); Hutchinson-Guilford progeria syndrome (HGPS); Kunitz Soybean trypsin inhibitor (KSTI); Manganese (III) tetrakis (4-benzoic acid)porphyrin (MnaTBP); Mitochondrial transcription factor A (TFAM); Nucleotide excision repair (NER); Rothmund-Thomson syndrome (RTS); Reactive oxygen species (ROS); Spontaneous sister chromatid exchanges (SCE); Transcription-coupled NER (TC-NER also called TCR); Trichothiodystropy (TTD); Xerodema Pigmentosum (XP); Werner syndrome (WS)
| Number | Date | Country | Kind |
|---|---|---|---|
| 14305203.3 | Feb 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/053167 | 2/13/2015 | WO | 00 |
