WO2009037481A1 - Procédés permettant de diagnostiquer et de traiter la démence - Google Patents

Procédés permettant de diagnostiquer et de traiter la démence Download PDF

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WO2009037481A1
WO2009037481A1 PCT/GB2008/003196 GB2008003196W WO2009037481A1 WO 2009037481 A1 WO2009037481 A1 WO 2009037481A1 GB 2008003196 W GB2008003196 W GB 2008003196W WO 2009037481 A1 WO2009037481 A1 WO 2009037481A1
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ubapl
polypeptide
dementia
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gene
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PCT/GB2008/003196
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Stuart Pickering-Brown
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The University Of Manchester
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Priority claimed from GB0718462A external-priority patent/GB0718462D0/en
Priority claimed from GB0803731A external-priority patent/GB0803731D0/en
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Publication of WO2009037481A1 publication Critical patent/WO2009037481A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders

Definitions

  • the present invention related to methods of use in diagnosing neurological conditions and products that can be used to treat such conditions.
  • Dementia is the progressive decline in cognitive function due to damage or disease in the brain beyond what might be expected from normal aging. Particularly affected areas may be memory, attention, language, and problem solving. Especially in the later stages of the condition, affected persons may be disoriented in time (not knowing what day of the week, day of the month, month, or even what year it is), in place (not knowing where they are), and in person (not knowing who they are). The prevalence of dementia is rising as the global life expectancy is rising. Particularly in Western countries, there is increasing concern about the economic impact that dementia will have in future, older populaces. Dementia is a non-specific term encompassing many disease processes. At present there is no cure for many types of dementia.
  • FTLD frontotemporal lobar degeneration
  • FTLD is a pathologic process involving degeneration of gray matter in the frontal lobe and anterior portion of the temporal lobe of the cerebrum, with sparing of the parietal and occipital lobes.
  • FTLD is the second most common form of dementia after Alzheimer's disease and is therefore a major cause of neurological problems in the elderly.
  • the syndrome of FTLD encompasses the clinical subgroups of frontotemporal dementia (FTD), FTD with motor neuron disease, semantic dementia and primary progressive aphasia, and is characterized by changes in behaviour, personality and language with relative preservation of memory and perception.
  • FTLD-U neuronal cytoplasmic inclusions and neurites that are immunoreactive for ubiquitin (ub-ir) but not for tau.
  • FTLD pathology of this type was first described in patients with motor neuron disease (MND) and dementia but has subsequently been recognized as a common neuropathological feature of FTLD in patients without motor symptoms. This ub-ir pathology is characteristically found in granule cells of dentate fascia of the hippocampus and in neurons of layer 2 of the frontal and temporal neocortex.
  • TDP-43 the major ubiquitinated protein in the pathological lesions in FTLD-U. It was demonstrated that this protein is cleaved and phosphorylated in disease and importantly, is also the basis of the pathological features of MND suggesting a disease spectrum between these two conditions.
  • FTLD FTLD-associated protein tau.
  • 7 disease loci reported to date, these being on chromosomes 3, 9p (2 loci), 9q, 17q21 (2 loci) and 17q24. Only 4 of the genes within these loci are known. It has been reported that a mutation in the splice acceptor site of exon 6 of CHMP2B on chromosome 3 causes in a large Danish family with DLDH-type of histology. However, the inventors have shown that this is a rare genetic cause of FTLD. 15-20% of familial FTLD results from mutations in the MAPT gene on chromosome 17q21 , encoding the microtubule associated protein tau.
  • FTLD is the second most common form of dementia in individuals under the age of 65 where approximately half of all patients with FTLD present with a family history of a similar disorder indicating a significant genetic contribution to the etiology of this disease.
  • Existing methods of diagnosing FTLD are based on a combination of neuropsychological test results, brain imaging studies, and physical findings. However there remains a clear need for developing further methods of diagnosing FTLD as well as assessing the likelihood that a subject will develop this disorder. Furthermore, at present there is no treatment for FTLD.
  • the inventors have undertaken further studies of the genetics of FTLD. Using a linkage disequilibrium approach the inventors genotyped one SNP per every haplotype block of all genes in the linkage region on chromosome 9q21 locus using a FTLD cohort
  • SNPs/haplotypes were consistently associated with a maximum sliding window P value of 0.00025.
  • the inventors have also examined the association of UBAPl polypeptide with the tau protein. As can be seen in the accompanying examples and figures, they have demonstrated that tau protein can be immunoprecipitated from neural cells using a UBAPl antibody. Moreover, tau and UBAPl polypeptide are colocalised in the parenchyma of neurons of subjects having FTLD. This suggests that UBAPl and tau polypeptides interact and are present in a cellular complex.
  • UBAPl is involved in the ubiquitin proteomsome system (UPS), facilitating degradation of proteins. Impairment of the functions of UBAPl is likely to be detrimental to a neurons health.
  • Tau protein is the main component of neurofibrillary tangles, one of the pathological hallmarks of dementias such as FTLD and Alzheimer's disease.
  • the inventors have demonstrated that tau and UBAPl interact using both immunoprecipitation and colocalisation with confocal microscopy (see accompanying examples). While not wishing to be bound to any particular theory, it is possible that a reduction in the function or amount of UBAPl would slow down the degradation of proteins, such as tau or other disease-related proteins (e.g. amyloid precursor protein), by the UPS. This would likely encourage the aggregation of these proteins and, in the case of tau, would probably lead to the formation of neurofibrillary tangles which could precipitate or accelerate disease.
  • proteins such as tau or other disease-related proteins (e.g. am
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • Alzheimer's disease has been identified as a protein misfolding disease, or proteopathy, due to the accumulation of abnormally folded A-beta and tau proteins in the brains of AD patients.
  • A-beta also written AB, is a short peptide that is a proteolytic byproduct of the transmembrane protein amyloid precursor protein (APP), whose function is unclear but thought to be involved in neuronal development.
  • the presenilins are components of a proteolytic complex involved in APP processing and degradation.
  • amyloid beta monomers are soluble and harmless, they undergo a dramatic conformational change at sufficiently high concentration to form a beta sheet-rich tertiary structure that aggregates to form amyloid fibrils that deposit outside neurons in dense formations known as senile plaques or neuritic plaques, in less dense aggregates as diffuse plaques, and sometimes in the walls of small blood vessels in the brain in a process called amyloid angiopathy or congophilic angiopathy.
  • AD is also considered a tauopathy due to abnormal aggregation of the tau protein, a microtubule-associated protein expressed in neurons that normally acts to stabilize microtubules in the cell cytoskeleton.
  • tau is normally regulated by phosphorylation; however, in AD patients, hyperphosphorylated tau accumulates as paired helical filaments that in turn aggregate into masses inside nerve cell bodies known as neurofibrillary tangles and as dystrophic neurites associated with amyloid plaques.
  • Alzheimer's disease Clinical signs of Alzheimer's disease are characterized by progressive cognitive deterioration, together with declining activities of daily living and by neuropsychiatric symptoms or behavioural changes. It is the most common type of dementia. Plaques which contain misfolded peptides called amyloid beta (AB) are formed in the brain many years before the clinical signs of Alzheimer's are observed. Together, these plaques and neurofibrillary tangles form the pathological hallmarks of the disease. These features can only be discovered at autopsy and help to confirm the clinical diagnosis.
  • AB amyloid beta
  • the inventors assessed whether there is a linkage between the UBAPl and AD. As set out in the accompanying example, they identified a positive association between UBAPl and AD in a cohort of 360 AD patients. This argues that UBAPl is a risk factor for Alzheimer's disease. Furthermore, as discussed above tau protein is the main component of neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, and the inventors have demonstrated that tau and UBAPl polypeptides interact using both immunoprecipitation and colocalisation with confocal microscopy.
  • UBAPl a likely component of the ubiquitin proteomsome system, as having a key and previously unrecognised role in the development of dementias, including Alzheimer's disease and FTLD. This finding can be the basis for the development of new medicaments and prognostic markers of use in combating these debilitating diseases.
  • a first aspect of the invention provides a method of screening for compounds of use in preventing or treating dementia wherein a cell having UBAPl is treated with a test compound and the effect of the test compound on the amount and/or function of UBAPl is assessed.
  • Ubiquitin Associated Protein 1 (UBAPl) encodes a protein of 502 residues, predicted to have a molecular weight of 55KDa. The gene is a member of the Ubiquitin-activated enzymes (UBA) family whose members include proteins having connections to ubiquitin and the ubiquitination pathway.
  • the protein itself has two Ubiquitin- associated domains (UBA), between residues 389-430, and 451-498, and an Ubiquitin System Cue domain between residues 459-499, believed to be involved in the binding of ubiquitin-conjugating enzymes.
  • UBA domains are found in various proteins involved in the ubiquitin/proteosome pathway, growth control, receptor function, stress responses, DNA excision-repair and cell signalling via protein kinases suggesting a variety of possible roles for UBAPl .
  • UBAPl originates from a gene locus in a refined region on chromosome 9 undergoing loss of heterozygosity in nasopharyngeal carcinoma (NPC) and is presently considered to be an effective diagnosis candidate for NPC. Furthermore, decreased expression of UBAPl protein is a possible point of dysfunction along the pathogenesis pathway for NPC that may contribute to malignant transformation. Therefore UBAPl is thought in the prior art to be associated with NPC and until the present invention was not considered to be associated with the development of any form of dementia. However, it is important to point out that UBA domains are found in various proteins involved in the ubiquitin/proteosome pathway and therefore UBAPl can be considered an excellent candidate gene for FTLD pathology. In addition, as demonstrated herein UBAPl polypeptide interacts with tau protein, and the gene is genetically associated with both FTLD and Alzheimer's disease, suggesting a broader role for UBAPl in dementia.
  • Human UBAPl genomic DNA sequence can be located from a number of publicly available databases.
  • GenBank contains UBAPl DNA sequence as part of the sequence information from human Chromosome 9.
  • GenBank accession number NM O 16525 provides the UBAPl mRNA and polypeptide sequences set out below, as well as a link to the genomic DNA for the UBAPl gene.
  • An example of the genomic DNA sequence for UBAPl is provided at the end of the examples section of the description below.
  • the sequence of human mRNA encoding the UBAPl protein can be located from a number of different GenBank accessions, for example, NM016525, and is provided below.
  • the sequence of the human UBAPl protein can be located from a number of different GenBank accessions, for example, NM O16525, and is provided below.
  • UBAPl polypeptide interacts with tau protein and the gene is genetically associated with dementia. They propose that a modulation, in particular reduction, in the function and/or amount of UBAPl would slow down the degradation of proteins, such as tau or other disease-related proteins (e.g. amyloid precursor protein), by the UPS, encouraging the aggregation of these proteins. In the case of tau, this would probably lead to the formation of neurofibrillary tangles, and this could precipitate or accelerate disease. In light of this, it is clear that compounds that modulate the function and/or amount of UBAPl would have particular utility in the prevention or treatment of dementia, particularly FTLD and Alzheimer's disease.
  • proteins such as tau or other disease-related proteins (e.g. amyloid precursor protein)
  • the first aspect of the invention is a "screening method" to identify compounds of use in preventing or treating dementia.
  • a compound that affects the amount and/or function of UBAPl is considered a compound that could be of use in preventing or treating dementia.
  • a cell having UBAPl set out in the first aspect of the invention, we include cells including nucleic acid sequence encoding the UBAPl polypeptide.
  • Such a nucleic acid sequence may be a "native" gene present in the genome of that cell, or it may be an extrachromosomal nucleic acid molecule. Examples of nucleic acid sequence encoding the UBAPl polypeptide are set out above.
  • the cell has a UBAPl polypeptide and the effect of a test compound on the amount and/or function of UBAPl polypeptide is assessed.
  • the cell could be any cell having UBAPl. However, it is preferred that the cell is a mammalian cell, most preferably a human cell. The cell could be any type. However, it is preferred that the cell is a neural cell, such as a neuroblastoma cell. Examples of such cells include SH-SY5Y and H4 neuroblastoma cells, which are further discussed below.
  • the step of assessing the "amount and/or function of UBAPl" may be performed using a number of different methods.
  • a method of assessing the effect of the test compound on the amount of UBAPl polypeptide is to quantify the amount of said polypeptide.
  • the effect of the test compound in the first aspect of the invention can be determined by quantifying the amount of nucleic acid, preferably mRNA, encoding the UBAPl polypeptide.
  • Methods of assessing the amount of UBAPl polypeptide may be performed using a number of different methods, which are discussed below.
  • the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in the cell can be compared to that of a "reference sample", i.e. a sample of protein or nucleic acid taken from a cell not exposed to the test compound.
  • Non-exhaustive examples of methods of determining the amount and/or function of polypeptide, or the amount of nucleic acid that encodes the polypeptide, are provided below. Further information regarding some of the experimental procedures set out below are described further in Sambrook et al. (2000) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  • Assaying protein levels in a sample can be performed using any art-known method. Total protein levels within a sample can be measured using Bradford reagent, fluorescamine dye or by using the Lo wry method: these techniques are standard laboratory procedures.
  • the amount of a polypeptide may be measured by labelling a compound having affinity for that particular polypeptide.
  • antibodies, aptamers and the like may be labelled and used in an assay.
  • Preferred for assaying protein levels in a biological sample are antibody-based techniques.
  • immunoassays include immunofluorescence techniques known to the skilled technician, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay analyses.
  • the effect of a test compound on the amount of UBAPl expression can be measured using an antibody to this polypeptide, as part of techniques such as western blotting, immunohistochemistry and ELISA. Such an antibody is used in the accompanying examples.
  • Levels of mRNA encoding particular polypeptides may be performed using the RT- PCR method. Briefly, this method involves converting mRNA isolated from a sample to cDNA using a reverse transcriptase enzyme. The cDNA products are then subject to PCR according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction product corresponding to the mRNA encoding the particular polypeptide is quantified. Variations on the RT-PCR method will be apparent to the skilled artisan. Any set of oligonucleotide primers which will amplify reverse transcribed target mRNA can be used and can be designed as will be well known to those skilled in the art.
  • Levels of mRNA encoding the particular polypeptide can also be assayed using northern blotting, a method well known to those skilled in the art.
  • RNA levels include in situ hybridisation, in situ amplification, nuclease protection, probe arrays and amplification based systems.
  • microarray analysis a technique well known to those skilled in the art, may also be used to assess the amount of mRNA encoding a particular polypeptide.
  • the expression of a certain gene can be measured using promoter-reporter constructs, a technique well known to the skilled person.
  • the screening methods of the invention may also include assessing the effect of the test compound on the function of UBAPl polypeptide.
  • an assay can be devised to measure the effect of a test compound on the ubiquitin and the ubiquitination pathway activities using UBAPl.
  • the inventors have demonstrated that UBAPl polypeptide interacts with tau protein in vivo, and propose that this interaction is important for modulating cellular tau protein levels. Therefore, an assay for the function of UBAPl polypeptide could be based on measuring the effects of a test compound on UBAPl and tau polypeptide levels. That is, a test compound that modulates both UBAPl and tau polypeptide levels is considered to be a compound that affects UBAPl function.
  • an appropriate cell line expressing tau and UBAPl e.g. SH-SY5Y or H4 neuroblastoma cells, both of which are well known in the art and are available from, for example, the ATCC
  • SH-SY5Y or H4 neuroblastoma cells both of which are well known in the art and are available from, for example, the ATCC
  • Cells would be treated with an appropriate test compound or small molecule library for an appropriate period on time.
  • Levels of tau and UBAPl would then be assayed using standard methods such as western blot or ELISA.
  • Test compound that affect both UBAPl levels and tau levels would be selected as compounds that could be of use in treating dementia.
  • Tau protein is well known in the art. Reagents that can be used to measure tau protein levels are well known and are readily available; for example, the accompanying experimental data uses an antibody to tau which is readily commercially available.
  • a fluorescent in vivo cellular assay could be developed.
  • Dickey et al (2005) Curr Alzheimer Res. Apr ;2(2) :231 -8 describe a fluorescent in cell western assay that can be used to simultaneously measure tau protein levels in a sample along with another protein, for use in a high throughput drug screening assay for the detection of changes in tau levels.
  • This assay could be readily adapted to simultaneously measure tau protein levels in a sample along with UBAPl polypeptide levels.
  • a compound that modulates both UBAPl and tau polypeptide levels is considered to be a compound that affects UBAPl function.
  • a possible function UBAPl polypeptide is to promote the degradation of proteins such as tau via the UPS.
  • a reduction in the amount and/or function of UBAPl polypeptide may lead to an accumulation of such protein which, in the case of tau, could precipitate or accelerate disease.
  • a test compound that increases the amount of UBAPl polypeptide and decreases the amount of tau protein would be an example of a compound that could be of use in preventing or treating dementia, in particular FTLD or Alzheimer's disease.
  • UBAPl and tau polypeptide are known to interact in the cell. Therefore one measure of UBAPl function is the ability of the polypeptide to interact with tau. Again this function could be used as a basis for an assay.
  • an assay can be devised to measure the effect of a test compound on UBAPl /tau interaction using an immunoprecipitation protocol.
  • an appropriate cell line expressing tau and UBAPl would be grown in an appropriate medium.
  • Cells would be treated with an appropriate test compound or small molecule library for an appropriate period on time.
  • UBAPl /tau interaction would then be assayed using standard methods such as the immunoprecipitation protocol outlined in the accompanying examples.
  • Test compound that affect the amount of UBAPl /tau interaction would be selected as compounds that could be of use in treating dementia.
  • UBAPl and TDP-43 polypeptides have been shown to co-localise together in neuronal cytoplasmic inclusions in a case of familial FTLD. Therefore one function of UBAPl could be in the metabolism of TDP-43. Therefore one measure of UBAPl function is the ability of the polypeptide to co-localise with UBAPl, or to regulate the metabolism of TDP-43. Again this function could be used as a basis for an assay.
  • an assay can be devised to measure the effect of a test compound on UBAPl /TDP-43 co-localistion using an immunoprecipitation protocol.
  • an appropriate cell line expressing TDP-43 and UBAPl would be grown in an appropriate medium. Cells would be treated with an appropriate test compound or small molecule library for an appropriate period on time.
  • UBAPl /TDP-43 co-localistion would then be assayed using standard methods such as the immunoprecipitation protocol outlined in the accompanying examples.
  • Test compound that affect the amount of UBAPl /TDP-43 co-localistion interaction would be selected as compounds that could be of use in treating dementia.
  • FTLD frontotemporal lobar degeneration
  • AD frontotemporal lobar degeneration
  • the inventors consider that the UBAPl gene may be used to identify therapeutic agents and as a diagnostic marker for the disorders within this broad category. Therefore the aspects of the invention provided herein are applicable to a wide range of dementias, including, FTLD, Alzheimer's disease, motor neuron disease, Parkinson's disease, dementia with Lewy bodies, prion diseases, progressive supranuclear palsy or multisystem atrophy.
  • An embodiment of the first aspect of the invention is wherein the dementia is characterised by tauopathy.
  • Tauopathy is characterised by the accumulation of hyperphosphorylated tau in neurons and occasionally in glia.
  • a preferred embodiment of the first aspect of the invention is wherein the dementia is frontotemporal lobar degeneration (FTLD).
  • the syndrome of FTLD encompasses the clinical subgroups of frontotemporal dementia (FTD), FTD with motor neuron disease, semantic dementia and primary progressive aphasia, and is characterized by changes in behaviour, personality and language with relative preservation of memory and perception.
  • An embodiment of the first aspect of the invention is wherein the frontotemporal lobar degeneration is characterised by ub-ir.
  • FTLD-U neuronal cytoplasmic inclusions and neurites that are immunoreactive for ubiquitin (ub-ir) but not for tau.
  • FTLD pathology of this type was first described in patients with motor neuron disease (MND) and dementia but has subsequently been recognized as a common neuropathological feature of FTLD in patients without motor symptoms. This ub-ir pathology is characteristically found in granule cells of dentate fascia of the hippocampus and in neurons of layer 2 of the frontal and temporal neocortex.
  • TDP-43 the major ubiquitinated protein in the pathological lesions in FTLD-U. It was demonstrated that this protein is cleaved and phosphorylated in disease and importantly, is also the basis of the pathological features of MND suggesting a disease spectrum between these two conditions.
  • a preferred embodiment of the first aspect of the invention is wherein the dementia is Alzheimer's disease.
  • the method of the first aspect of the invention relates to screening methods for drugs or lead compounds.
  • the test compound may be a drug-like compound or lead compound for the development of a drug-like compound.
  • drug-like compound is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament.
  • a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble.
  • a drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.
  • lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • test compound as used in the first aspect of the invention, we include where a cell is exposed to more than one compound at the same time, as is commonly performed in high throughput screening assays well known in the art.
  • An embodiment of the first aspect of the invention is wherein the method further comprises the step of selecting a compound that increases the amount and/or function of UBAPl; preferably UBAPl polypeptide.
  • An embodiment of the first aspect of the invention is wherein the method further comprises the step of selecting a compound that decreases the amount and/or function of UBAPl; preferably UBAPl polypeptide.
  • a further embodiment of the first aspect of the invention is wherein the selected compound is formulated into a pharmaceutically acceptable composition.
  • a discussion of pharmaceutical compositions is provided below.
  • a second aspect of the invention provides a method of screening for compounds of use in preventing or treating dementia wherein a non-human animal is administered a test compound and the effect of the test compound on the amount and/or function of UBAPl is assessed; preferably UBAPl polypeptide.
  • the second aspect of the invention is also a "screening method".
  • the non-human animal may be any non-human animal, including non-human primates such as baboons, chimpanzees and gorillas, new and old world monkeys as well as other mammals such as cats, dogs, rodents, pigs or sheep, or other animals such as poultry, for example chickens, fish such as zebrafish, or amphibians such as frogs.
  • the animal is a rodent such as a mouse, rat, hamster, guinea pig or squirrel.
  • the animal is mouse.
  • the non-human animal has a nucleic acid sequence encoding UBAPl.
  • a third aspect of the present invention there is provided the use of an agent that modulates the amount or activation of UBAPl for the prevention or treatment of dementia
  • a fourth aspect of the present invention there is provided the use of an agent that modulates the amount and/or activation of UBAPl in the manufacture of a medicament for the prevention or treatment of dementia.
  • a method of preventing or treating dementia comprising administering to a subject a therapeutically effective quantity of an agent that modulates the amount and/or activation of UBAPl .
  • the inventors as explained above and in the Examples, have demonstrated that the UBAPl gene is genetically linked to dementia. This lead them to realise that agents of use in the third, fourth or fifth aspects of the invention, which modulate the amount or activation of UBAPl, are useful for preventing or treating dementia. The inventors were surprised to make these correlations because until the present application UBAPl has not been associated with any dementia disorders.
  • agent is effective for modulating the amount and/or activation of UBAPl such that the amount and/or activation of the UBAPl gene or gene product (mRNA and protein) is significantly increased or reduced in the subject when compared to that in a subject not administered the agent.
  • the agent increases the amount and/or activation of UBAPl .
  • the agents may be used in the treatment of a number of different dementias, preferably frontotemporal lobar degeneration (FTLD) or Alzheimer's disease (AD); preferably the dementia is characterised by tauopathy.
  • FTLD frontotemporal lobar degeneration
  • AD Alzheimer's disease
  • agents which may be used according to the invention include where the agent may bind to the UBAPl polypeptide and increase or prevent UBAPl functional activity, e.g. antibodies and fragments and derivatives thereof (e.g. domain antibodies or
  • the agent may act as a competitive inhibitor to UBAPl by acting as an antagonist UBAPl .
  • the agent may be an activator of UBAPl by acting as an agonist of UBAPl .
  • the agent may inhibit or activate enzymes or other molecules in the UBAPl pathway.
  • the agent may bind to mRNA encoding UBAPl polypeptide in such a manner as to lead to an increase or reduction in that mRNA and hence a modulation in the amount of UBAPl polypeptide.
  • the agent may bind to a nucleic sequence encoding UBAPl in such a manner that it leads to an increase or reduction in the amount of transcribed mRNA encoding UBAPl polypeptide.
  • the agent may bind to coding or non-coding regions of the UBAPl gene or to DNA 5' or 3' of the UBAPl and thereby reduce or increase expression of the protein.
  • the agent may have been identified from the method of the first or second aspects of the invention as being of use in the prevention or treatment of dementia.
  • An embodiment of the third, fourth or fifth aspects of the invention is wherein the agent increases the amount and/or activation of UBAPl .
  • a further embodiment of the third, fourth or fifth aspects of the invention is wherein the agent is UBAPl polypeptide.
  • the UBAPl polypeptide may be administered directly to the subject.
  • this may consists of administering a nucleic acid sequence encoding UBAPl to the subject, for example, by gene therapy.
  • Gene therapy consists of the insertion or the introduction of a gene or genes into a subject in need of treatment.
  • the gene UBAPl encoding the UBAPl polypeptide is used. Accordingly, it is preferred that at least one, and preferably, more than one, copy of the UBAPl gene will be introduced in to a subject to be treated.
  • sequence of the UBAPl gene used in the therapeutic aspects of the invention is from the same genus as that of the subject being treated.
  • the methods according to the invention will use mammalian UBAPl gene, and hence mammalian UBAPl enzyme. It is especially preferred that the UBAPl gene used is from the same species as that of the subject being treated.
  • the method according to the invention will use the human UBAPl gene, and hence human UBAPl polypeptide, and so on.
  • the UBAPl gene used in the methods according to the invention is substantially homologous to the subject's native UBAPl gene, or a functional fragment thereof.
  • the degree of homology between the sequence of the UBAPl gene used in the method and the sequence of the subject's native UBAPl gene is at least 60% sequence identity, preferably, at least 75% sequence identity, preferably at least 85% identity; at least 90% identity; at least 95% identity; at least 97% identity; and most preferably, at least 99% identity.
  • Calculation of percentage identities between different amino acid/polypeptide/nucleic acid sequences may be carried out as follows.
  • a multiple alignment is first generated by the ClustalX program (pairwise parameters: gap opening 10.0, gap extension 0.1, protein matrix Gonnet 250, DNA matrix IUB; multiple parameters: gap opening 10.0, gap extension 0.2, delay divergent sequences 30%, DNA transition weight 0.5, negative matrix off, protein matrix gonnet series, DNA weight IUB; Protein gap parameters, residue-specific penalties on, hydrophilic penalties on, hydrophilic residues GPSNDQERK, gap separation distance 4, end gap separation off).
  • the percentage identity is then calculated from the multiple alignment as (N/T)*100, where N is the number of positions at which the two sequences share an identical residue, and T is the total number of positions compared.
  • percentage identity can be calculated as (N/S)*100 where S is the length of the shorter sequence being compared.
  • the amino acid/polypeptide/nucleic acid sequences may be synthesised de novo, or may be native amino acid/polypeptide/nucleic acid sequence, or a derivative thereof.
  • UBAPl polypeptide for provision as a therapeutic agent may be produced by known techniques.
  • the protein may be purified from naturally occurring sources of UBAPl polypeptide.
  • such naturally occurring sources of UBAPl polypeptide may be induced to express increased levels of the protein, which may then be purified using well-known conventional techniques.
  • cells that do not naturally express UBAPl polypeptide may be induced to express such proteins.
  • One suitable technique involves cellular expression of an UBAPl polypeptide/A/s construct. The expressed construct may subsequently be highly purified by virtue of the his "tag".
  • Polynucleotide sequences encoding the UBAPl polypeptide are provided herein.
  • UBAPl represents a favourable agent to be administered by techniques involving cellular expression of polynucleotide sequence encoding UBAPl. Such methods of cellular expression are particularly suitable for medical use in which the therapeutic effects of UBAPl are required over a prolonged period of time.
  • the UBAPl gene may further comprise elements capable of controlling and/or enhancing its expression in the cell being treated.
  • the UBAPl gene may be contained within a suitable vector to form a recombinant vector and preferably adapted to produce UBAPl polypeptide.
  • the vector may for example be a plasmid, cosmid or phage.
  • Such recombinant vectors are highly useful in the delivery systems of the invention for transforming cells with the nucleic acid molecule.
  • suitable vectors include pCMV6-XL5 (OriGene Technologies Inc), NTC retroviral vectors (Nature Technology Corporation), adeno-associated viral vectors (Avigen Technology).
  • vectors will be used to introduce genes coding for products with at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity with the UBAPl protein sequence provided herein.
  • State of the art vectors containing DNA coding for UBAPl protein may be introduced into the blood stream.
  • Any state of the art advantages of gene therapy for example, considerably improved viral vectors derived from adeno-associated viruses, retroviruses, particularly lentiviruses) may be used to introduce DNA sequences coding for UBAPl and homologies.
  • At least 2 administrations of 1-1000 million units/ml is given at certain intervals, depending on vectors used (the vectors will influence the stability of expression and persistence of UBAPl in organisms, from only several weeks to permanent expression) and individual requirements of the organism to be treated.
  • Recombinant vectors may comprise other functional elements to improve the gene therapy.
  • recombinant vectors can be designed such that they will autonomously replicate in the cell in which they are introduced. In this case, elements that induce nucleic acid replication may be required in the recombinant vector.
  • the recombinant vector may comprise a promoter or regulator to control expression of the UBAPl gene as required.
  • the recombinant vector may be designed such that the vector and UPABl gene integrates into the genome of the cell, hi this case nucleic acid sequences, which favour targeted integration (e.g. by homologous recombination) may be desirable.
  • Recombinant vectors may also have DNA coding for genes that may be used as selectable markers in the cloning process.
  • the UBAPl gene may (but not necessarily) be one, which becomes incorporated in the DNA of cells of the subject being treated.
  • the delivery system may provide the UBAPl gene the subject without it being incorporated in a vector.
  • the nucleic acid molecule may be incorporated within a liposome or virus particle.
  • a "naked" nucleic acid molecule may be inserted into a subject's cells by a suitable means e.g. direct endocytotic uptake.
  • the UBAPl nucleic acid molecule may be transferred to the cells of a subject to be treated by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment.
  • transfer may be by ballistic transfection with coated gold particles, liposomes containing the nucleic acid molecule, viral vectors (e.g. adenovirus) and means of providing direct nucleic acid uptake (e.g. endocytosis) by application of the gene directly.
  • UBAPl polypeptide or UBAPl gene may be combined in compositions having a number of different forms depending, in particular on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, transdermal patch, liposome or any other suitable form that may be administered to a person or animal.
  • the vehicle of the composition of the invention should be one which is well tolerated by the subject to whom it is given, and preferably enables delivery of the UBAPl polypeptide or UBAPl gene to the target cell, tissue, or organ.
  • UBAPl polypeptide is delivered by means of a suitably protected carrier particle, for example, a micelle.
  • compositions comprising UBAPl polypeptide or UBAPl gene according to the invention may be used in a number of ways. For instance, systemic administration may be required in which case the compound may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Alternatively, the composition may be administered by injection into the blood stream. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion). The compounds may be administered by inhalation (e.g. intranasally).
  • UBAPl polypeptide or UBAPl gene may also be incorporated within a slow or delayed release device. Such devices may, for example, be inserted on or under the skin, and the compound may be released over weeks or even months. Such devices may be particularly advantageous when long term treatment with a UBAPl polypeptide or UBAPl gene according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
  • the amount of a UBAPl polypeptide or UBAPl gene that is required is determined by its biological activity and bioavailability which in turn depends on the mode of administration, the physicochemical properties of the UBAPl polypeptide or UBAPl gene employed, and whether the UBAPl polypeptide or UBAPl gene is being used as a monotherapy or in a combined therapy. Also, the amount will be determined by the number and state of target cells to be treated. The frequency of administration will also be influenced by the above-mentioned factors and particularly the half-life of the UBAPl polypeptide or UBAPl gene within the subject being treated.
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular UBAPl polypeptide or UBAPl gene in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
  • UBAPl polypeptide or UBAPl gene may be used to establish specific formulations of UBAPl polypeptide or UBAPl gene according to the invention and precise therapeutic regimes (such as daily doses of the UBAPl polypeptide or UBAPl gene and the frequency of administration).
  • a daily dose of between 0.01 ⁇ g/kg of body weight and 0.5 g/kg of body weight of UBAPl polypeptide or UBAPl gene according to the invention may be used for the prevention and/or treatment of dementia, depending upon which specific UBAPl polypeptide or UBAPl gene is used. More preferably, the daily dose is between 0.01 mg/kg of body weight and 200 mg/kg of body weight, and most preferably, between approximately 1 mg/kg and 100 mg/kg.
  • Daily doses may be given as a single administration (e.g. a single daily injection).
  • the UBAPl polypeptide or UBAPl gene used may require administration twice or more times during a day.
  • UBAPl polypeptide or UBAPl gene according to the invention may be administered as two (or more depending upon the severity of the condition) daily doses of between 25 mg and 7000 mg (i.e. assuming a body weight of 70kg).
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3 or 4 hourly intervals thereafter.
  • a slow release device may be used to provide optimal doses to a patient without the need to administer repeated doses.
  • This invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a UBAPl polypeptide or UBAPl gene according to the invention and optionally a pharmaceutically acceptable vehicle.
  • the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.01 mg to about
  • the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.01 mg to about 500 mg. In another embodiment, the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.01 mg to about
  • the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.1 mg to about 60 mg. In another embodiment, the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.1 mg to about 20 mg.
  • This invention provides a process for making a pharmaceutical composition
  • a pharmaceutical composition comprising combining a therapeutically effective amount of an UBAPl polypeptide or UBAPl gene according to the invention and a pharmaceutically acceptable vehicle.
  • a "therapeutically effective amount” is any amount of an UBAPl polypeptide or UBAPl gene according to the invention which, when administered to a subject provides prevention and/or treatment of dementia.
  • a "subject” is a vertebrate, mammal, domestic animal or human being.
  • a "pharmaceutically acceptable vehicle” as referred to herein is any physiological vehicle known to those of ordinary skill in the art useful in formulating pharmaceutical compositions.
  • a further embodiment of the third, fourth or fifth aspects of the invention is where the agent decreases the amount and/or activation of UBAP 1.
  • Agent for use in the third, fourth or fifth aspects of the invention may bind to UBAPl polypeptide or to a nucleic acid encoding UBAPl polypeptide. Examples of nucleic acid and polypeptide sequences for UBAPl are shown above and at the end of the example section of the description.
  • the agent When the agents binds to UBAPl polypeptide, it is preferred that the agent binds to an epitope defined by the protein that has been correctly folded into its native form. It will be appreciated, that there can be some sequence variability between species and also between genotypes. Accordingly other preferred epitopes will comprise equivalent regions from variants of the gene. Equivalent regions from further UBAPl polypeptides can be identified using sequence similarity and identity tools, and database searching methods, outlined herein. It is most preferred that the agent binds to a conserved region of the UBAPl polypeptide or a fragment thereof.
  • An embodiment of the third, fourth or fifth aspects of the invention is wherein the agent is an antibody or fragment thereof.
  • antibodies as agents to modulate polypeptide activity is well known. Indeed, therapeutic agents based on antibodies are increasingly being used in medicine. It is therefore apparent that such agents have great utility as medicaments for the improving the prevention or treatment of dementia. Moreover, such antibodies can be used in the prognostic methods set out below in further aspects of the invention.
  • Antibodies, for use in treating human subjects may be raised against UBAPl polypeptide per se or a number of peptides derived from the UBAPl polypeptide, or peptides comprising amino acid sequences corresponding to those found in the UBAPl polypeptide.
  • the antibodies are raised against antigenic structures from human UBAPl polypeptide, and peptide derivatives and fragments thereof.
  • Antibodies may be produced as polyclonal sera by injecting antigen into animals.
  • Preferred polyclonal antibodies may be raised by inoculating an animal (e.g. a rabbit) with antigen (e.g. all or a fragment of the UBAPl polypeptide) using techniques known to the art.
  • the antibody may be monoclonal. Conventional hybridoma techniques may be used to raise such antibodies.
  • the antigen used to generate monoclonal antibodies for use in the present invention may be the same as would be used to generate polyclonal sera.
  • antibodies or immunoglobulin proteins are Y-shaped molecules usually exemplified by the ⁇ -immunoglobulin (IgG) class of antibodies.
  • the molecule consists of four polypeptide chains two identical heavy (H) chains and two identical (L) chains of approximately 5OkD and 25kD each respectively. Each light chain is bound to a heavy chain (H-L) by disulphide and non-covalent bonds.
  • H-L heavy chain
  • Two identical H-L chain combinations are linked to each other by similar non-covalent and disulphide bonds between the two H chains to form the basic four chain immunoglobulin structure (H-
  • Light chain immunoglobulins are made up of one V-domain (V L ) and one constant domain (C L ) whereas heavy chains consist of one V-domain and, depending on H chain isotype, three or four C-domains (C H I , C H 2, C H 3 and C H 4).
  • V domain At the N-terminal region of each light or heavy chain is a variable (V) domain that varies greatly in sequence, and is responsible for specific binding to antigen.
  • Antibody specificity for antigen is actually determined by amino acid sequences within the V- regions known as hypervariable loops or Complementarity Determining Regions (CDRs).
  • CDRs Complementarity Determining Regions
  • Each H and L chain V regions possess 3 such CDRs, and it is the combination of all 6 that forms the antibody's antigen binding site.
  • the remaining V-region amino acids which exhibit less variation and which support the hypervariable loops are called frameworks regions (FRs).
  • variable domains The regions beyond the variable domains (C-domains) are relatively constant in sequence.
  • the characterising feature of antibodies according to the invention is the V H and V L domains.
  • the precise nature of the C H and C L domains is not, on the whole, critical to the invention.
  • preferred antibodies for use in the invention may have very different C H and C L domains.
  • preferred antibody functional derivatives may comprise the Variable domains without a C-domain (e.g. scFV antibodies).
  • Preferred antibodies considered to be agents of use in the third, fourth or fifth aspects of the invention may have the V L (first domain) and V H (second domain) domains.
  • a derivative thereof may have 75% sequence identity, more preferably 90% sequence identity and most preferably has at least 95% sequence identity. It will be appreciated that most sequence variation may occur in the framework regions (FRs) whereas the sequence of the CDRs of the antibodies, and functional derivatives thereof, should be most conserved.
  • a number of preferred embodiments of the agent of the third, fourth or fifth aspects of the invention relate to molecules with both Variable and Constant domains.
  • antibody fragments e.g. scFV antibodies or FAbs
  • scFV antibodies or FAbs are also encompassed by the invention that comprise essentially the Variable region of an antibody without any Constant region.
  • a scFV antibody fragment considered to be an agent of the third, fourth or fifth aspects of the invention may comprise the whole of the V H and V L domains of an antibody raised against IFN polypeptide.
  • the V H and V L domains may be separated by a suitable linker peptide.
  • Antibodies, and particularly mAbs, generated in one species are known to have several serious drawbacks when used to treat a different species. For instance when murine antibodies are used in humans they tend to have a short circulating half-life in serum and may be recognised as foreign proteins by the immune system of a patient being treated. This may lead to the development of an unwanted human anti-mouse antibody (HAMA) response. This is particularly troublesome when frequent administration of an antibody is required as it can enhance its clearance, block its therapeutic effect, and induce hypersensitivity reactions. These factors limit the use of mouse monoclonal antibodies in human therapy and have prompted the development of antibody engineering technology to generate humanised antibodies.
  • HAMA human anti-mouse antibody
  • the antibody capable of modulating the amount or activation of UBAPl is to be used as a therapeutic agent for preventing or treating dementia in a human subject, then it is preferred that antibodies and fragments thereof of non-human source are humanised.
  • Humanisation may be achieved by splicing V region sequences (e.g. from a monoclonal antibody generated in a non-human hybridoma) with C region (and ideally FRs from V region) sequences from human antibodies.
  • V region sequences e.g. from a monoclonal antibody generated in a non-human hybridoma
  • C region and ideally FRs from V region sequences from human antibodies.
  • the resulting 'engineered' antibodies are less immunogenic in humans than the non-human antibodies from which they were derived and so are better suited for clinical use.
  • Humanised antibodies may be chimaeric monoclonal antibodies, in which, using recombinant DNA technology, rodent immunoglobulin constant regions are replaced by the constant regions of human antibodies.
  • the chimaeric H chain and L chain genes may then be cloned into expression vectors containing suitable regulatory elements and induced into mammalian cells in order to produce fully glycosylated antibodies.
  • the biological activity of the antibody may be pre-determined.
  • Such chimaeric molecules may be used to treat or prevent dementia.
  • Such antibodies may involve CDR-grafting or reshaping of antibodies.
  • Such antibodies are produced by transplanting the heavy and light chain CDRs of a non-human antibody (which form the antibody's antigen binding site) into the corresponding framework regions of a human antibody.
  • Humanised antibody fragments represent preferred agents for use according to the invention.
  • Human FAbs recognising an epitope on UBAPl polypeptide may be identified through screening a phage library of variable chain human antibodies. Techniques known to the art (e.g as developed by Morphosys or Cambridge Antibody Technology) may be employed to generate Fabs that may be used as agents according to the invention.
  • a human combinatorial Fab antibody library may be generated by transferring the heavy and light chain variable regions from a single-chain Fv library into a Fab display vector. This library may yield 2.1 x 10 10 different antibody fragments. The peptide may then be used as "bait" to identify antibody fragments from then library that have the desired binding properties.
  • dAbs represent another preferred agent that may be used according to this embodiment of the invention.
  • dAbs are the smallest functional binding unit of antibodies and correspond to the variable regions of either the heavy or light chains of human antibodies.
  • Such dAbs may have a molecule weight of around 13kDa (corresponding to about 1/10 (or less) the size of a full antibody).
  • peptides may be used to modulate the amount or activation of UBAPl.
  • Such peptides represent other preferred agents for use according to the invention.
  • These peptides may be isolated, for example, from libraries of peptides by identifying which members of the library are able to modulate the amount or activation of UBAPl polypeptide. Suitable libraries may be generated using phage display techniques.
  • Aptamers represent another preferred agent of the third, fourth or fifth aspects of the invention.
  • Aptamers are nucleic acid molecules that assume a specific, sequence- dependent shape and bind to specific target ligands based on a lock-and-key fit between the aptamer and ligand.
  • aptamers may comprise either single- or double- stranded DNA molecules (ssDNA or dsDNA) or single-stranded RNA molecules (ssRNA).
  • ssDNA or dsDNA single-stranded RNA molecules
  • Aptamers may be used to bind both nucleic acid and non-nucleic acid targets. Accordingly aptamers may be generated that recognise and so modulate the activity or amount of UBAPl.
  • Suitable aptamers may be selected from random sequence pools, from which specific aptamers may be identified which bind to the selected target molecules with high affinity.
  • Methods for the production and selection of aptamers having desired specificity are well known to those skilled in the art, and include the SELEX (systematic evolution of ligands by exponential enrichment) process. Briefly, large libraries of oligonucleotides are produced, allowing the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction.
  • Antisense molecules represent another preferred agent for use according to the third, fourth or fifth aspects of the invention.
  • Antisense molecules are typically single- stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence produced by a gene and inactivate it, effectively turning that gene "off.
  • the molecule is termed "antisense” as it is complementary to the gene's mRNA, which is called the “sense” sequence, as appreciated by the skilled person.
  • Antisense molecules are typically are 15 to 35 bases in length of DNA, RNA or a chemical analogue. Antisense nucleic acids have been used experimentally to bind to mRNA and prevent the expression of specific genes.
  • Antisense therapies as drugs for the treatment of cancer, diabetes and inflammatory diseases.
  • Antisense drugs have recently been approved by the US FDA for human therapeutic use. Accordingly, by designing an antisense molecule to polynucleotide sequence encoding UBAPl polypeptide it would be possible to reduce the expression of UBAPl polypeptide in a cell and thereby reduce in UBAPl activity.
  • siRNA Small interfering RNA
  • siRNA molecules that can reduce UBAPl expression may have utility in the preparation of medicaments for the prevention or treatment of dementia.
  • siRNA are a class of 20-25 nucleotide-long RNA molecules are involved in the RNA interference pathway (RNAi), by which the siRNA can lead to a reduction in expression of a specific gene, or specifically interfere with the translation of such mRNA thereby inhibiting expression of protein encoded by the mRNA.
  • RNAi RNA interference pathway
  • siRNAs have a well defined structure: a short (usually 21-nt) double- strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. In vivo this structure is the result of processing by Dicer, an enzyme that converts either long dsRNAs or hairpin RNAs into siRNAs.
  • siRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA.
  • RNAi via siRNAs has generated a great deal of interest in both basic and applied biology.
  • RNAi screens that are designed to identify the important genes in various biological pathways.
  • disease processes also depend on the activity of multiple genes, it is expected that in some situations turning off the activity of a gene with a siRNA could produce a therapeutic benefit.
  • RNAi for biomedical research and drug development.
  • Recent phase I results of therapeutic RNAi trials demonstrate that siRNAs are well tolerated and have suitable pharmacokinetic properties. siRNAs and related RNAi induction methods therefore stand to become an important new class of drugs in the foreseeable future.
  • siRNA molecules designed to nucleic acid encoding UBAPl polypeptide can be used to reduce the expression of UBAPl.
  • the agent is a siRNA molecule having complementary sequence to UBAPl polynucleotide.
  • a polynucleotide sequence encoding an UBAPl polypeptide is provided above.
  • siRNA molecules having complementary sequence to UBAPl polynucleotide For example, a simple internet search yields many websites that can be used to design siRNA molecules.
  • siRNA molecule we include a double stranded 20 to 25 nucleotide-long RNA molecule, as well as each of the two single RNA strands that make up a siRNA molecule.
  • siRNA is used in the form of hair pin RNA (shRNA).
  • shRNA hair pin RNA
  • Such shRNA may comprise two complementary siRNA molecules that are linked by a spacer sequence (e.g. of about 9 nucleotides).
  • the complementary siRNA molecules may fold such that they bind together.
  • a ribozyme capable of cleaving RNA or DNA encoding UBAPl polypeptide represent another preferred agent of the third, fourth or fifth aspect of the invention.
  • An sixth aspect of the invention provides a method of making a pharmaceutical composition comprising mixing a compound identified from the screening methods of the invention with a pharmaceutically acceptable carrier.
  • the amount of an agent needed according to the invention is determined by biological activity and bioavailability which in turn depends on the mode of administration and the physicochemical properties of the agent.
  • the frequency of administration will also be influenced by the abovementioned factors and particularly the half-life of the agent within the target tissue or subject being treated.
  • Known procedures such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials etc), may be used to establish specific formulations of the agents and precise therapeutic regimes (such as daily doses and the frequency of administration).
  • a daily dose of between O.Ol ⁇ g/kg of body weight and O.lg/kg of body weight of an agent may be used; more preferably the daily dose is between O.Olmg/kg of body weight and lOOmg/kg of body weight.
  • a suitable dose of an antibody according to the invention is lO ⁇ g/kg of body weight - 100mg/kg of body weight, more preferably about Olmg/kg of body weight - lOmg/kg of body weight and most preferably about 6mg/kg of body weight.
  • Daily doses may be given as a single administration (e.g. a single daily injection or a single dose from an inhaler).
  • the agent e.g. an antibody or aptamer
  • Medicaments according to the invention should comprise a therapeutically effective amount of the agent and a pharmaceutically acceptable vehicle.
  • a “therapeutically effective amount” is any amount of an agent according to the invention which, when administered to a subject inhibits or prevents cancer growth or metastasis.
  • a "subject” may be a vertebrate, mammal, domestic animal or human being. It is preferred that the subject to be treated is human. When this is the case the agents may be designed such that they are most suited for human therapy (e.g. humanisation of antibodies as discussed above). However it will also be appreciated that the agents may also be used to treat other animals of veterinary interest (e.g. horses, dogs or cats).
  • a "pharmaceutically acceptable vehicle” as referred to herein is any physiological vehicle known to those skilled in the art as useful in formulating pharmaceutical compositions.
  • the medicament may comprise about 0.01 ⁇ g and 0.5 g of the agent. More preferably, the amount of the agent in the composition is between 0.01 mg and 200 mg, and more preferably, between approximately 0.1 mg and 100 mg, and even more preferably, between about lmg and lOmg. Most preferably, the composition comprises between approximately 2mg and 5mg of the agent.
  • the medicament comprises approximately 0.1% (w/w) to 90% (w/w) of the agent, and more preferably, 1% (w/w) to 10% (w/w).
  • the rest of the composition may comprise the vehicle.
  • Nucleic acid agents can be delivered to a subject by incorporation within liposomes.
  • the "naked" DNA molecules may be inserted into a subject's cells by a suitable means e.g. direct endocytotic uptake.
  • Nucleic acid molecules may be transferred to the cells of a subject to be treated by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment.
  • transfer may be by ballistic transfection with coated gold particles, liposomes containing the DNA molecules, viral vectors (e.g. adenovirus) and means of providing direct DNA uptake (e.g. endocytosis) by application of the DNA molecules directly to the target tissue topically or by injection.
  • the antibodies, or functional derivatives thereof may be used in a number of ways. For instance, systemic administration may be required in which case the antibodies or derivatives thereof may be contained within a composition which may, for example, be ingested orally in the form of a tablet, capsule or liquid. It is preferred that the antibodies, or derivatives thereof, are administered by injection into the blood stream.
  • Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion). Alternatively the antibodies may be injected directly to the liver.
  • Nucleic acid or polypeptide therapeutic entities may be combined in pharmaceutical compositions having a number of different forms depending, in particular on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, transdermal patch, liposome or any other suitable form that may be administered to a person or animal.
  • the vehicle of the composition of the invention should be one which is well tolerated by the subject to whom it is given, and preferably enables delivery of the therapeutic to the target cell, tissue, or organ.
  • the pharmaceutical vehicle is a liquid and the pharmaceutical composition is in the form of a solution.
  • the pharmaceutical vehicle is a gel and the composition is in the form of a cream or the like.
  • compositions comprising such therapeutic entities may be used in a number of ways.
  • systemic administration may be required in which case the entities may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid.
  • the composition may be administered by injection into the blood stream. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion).
  • the entities may be administered by inhalation (e.g. intranasally).
  • Therapeutic entities may also be incorporated within a slow or delayed release device. Such devices may, for example, be inserted on or under the skin, and the compound may be released over weeks or even months. Such devices may be particularly advantageous when long term treatment with an entity is required and which would normally require frequent administration (e.g. at least daily injection).
  • the inventors also identified a positive association between UBAPl and AD in a cohort of 360 AD patients. This argues that UBAPl is a risk factor for Alzheimer's disease.
  • tau protein is the main component of neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, and the inventors have demonstrated that tau and UBAPl polypeptides interact using both immunoprecipitation and colocalisation with confocal microscopy.
  • UBAPl presence of one or more mutations in UBAPl indicates that a subject has or is likely to develop a dementia, i.e. they have a higher than average likelihood of having or developing a dementia.
  • a seventh aspect of the invention provides a method of assessing whether a subject has or is likely to develop a dementia comprising determining whether the subject has a mutation in the UBAPl gene.
  • the method of this aspect of the invention includes determining whether a subject has a mutation in the UBAPl gene. If the subject has a mutation in the UBAPl gene, this indicates that subject has or is likely to develop dementia.
  • a mutant UBAPl nucleic acid is any UBAPl nucleic acid containing a mutation as compared to a wild type UBAPl nucleic acid.
  • a mutant human UBAPl nucleic acid can be a nucleic acid having the nucleotide sequence above having at least one mutation.
  • mutation as used herein with respect to nucleic acid, we include insertions of one or more nucleotides, deletions of one or more nucleotides, nucleotide substitutions, and combinations thereof, including mutations that occur in coding and non-coding regions (e.g., exons, introns, untranslated sequences, sequences upstream of the transcription start site of UBAPl mRNA, and sequences downstream of the transcription termination site of UBAPl mRNA).
  • coding and non-coding regions e.g., exons, introns, untranslated sequences, sequences upstream of the transcription start site of UBAPl mRNA, and sequences downstream of the transcription termination site of UBAPl mRNA.
  • UBAPl nucleic acid examples include those provided below in relation to this aspect of the invention.
  • a mutant UBAPl polypeptide is any UBAPl polypeptide containing an alteration to the amino acid sequence as compared to a wild type UBAPl polypeptide.
  • a mutant human UBAPl polypeptide can be a polypeptide having the amino acid sequence above having at least one alteration; for example this could be a substitution of one or more amino acid residues with other amino acid residues; this could be an insertion of one or more amino acid residues; this could be a deletion of one or more amino acid residues, and possibly a truncation of a large region of the UBAPl polypeptide.
  • UBAPl gene we include the nucleic acid sequence set out above that encodes the UBAPl polypeptide or any fragment of that sequence. This can be genomic DNA sequence, mRNA sequence and cDNA sequence. UBAPl gene nucleic acid sequences include the untranslated regions extending both upstream of the transcription start site of UBAPl mRNA and downstream of the transcription termination site of UBAPImRNA by, for example, 5Kb. UBAPl gene nucleic acid sequences include all exon and intron sequences. We also include polymorphisms or variations in that nucleotide sequence that are naturally found between individuals of different ethnic backgrounds or from different geographical areas and which do not affect the function of the gene.
  • UBAPl gene we also include “regulatory elements”, including the 5' and 3' of the gene which is involved in regulating gene transcription. For instance, transcription factor binding sequences, the TATA box, the 5' promoter and 5' and 3' untranslated regions (UTRs). This definition also encompasses the DNA 5' of the first codon of the first exon of UBAPl. At least some of this sequence information is provided at the end of the example section of the description
  • the method according to the present invention is an in vitro method and can be performed on a sample containing nucleic acid and/or polypeptide derived from the subject.
  • Various different approaches can be used to determine whether a subject has a mutation in the UBAPl gene. These include haplotype analysis of genomic DNA of the subject; determining the nucleic acid sequence of the UBAPl gene; determining the nucleic acid sequence of mRNA encoding the UBAPl polypeptide; determining whether the subject has a mutant UBAPl polypeptide.
  • a preferred method of determining whether a subject has a mutation in the UBAPl gene is to use haplotype analysis.
  • haplotype analysis is used when the method of the seventh aspect of the invention relates to FTLD.
  • Haplotype analysis is a powerful technique that can be used to determine whether a subject has, or is likely to have, a mutation in a specific gene.
  • a haplotype is a set of genetic markers on a single chromatid that are statistically associated. It is thought that these associations, and the identification of a few alleles of a haplotype block, can unambiguously identify all other polymorphic sites in its region. Such information is very valuable for investigating the genetics behind common diseases and is collected by the International HapMap Project. Haplotypes of the UBAPl gene are individually associated with specific mutations in that gene.
  • haplotype analysis In contrast to genotyping methods, a significant advantage of haplotype analysis is that it is not necessary to determine the sequence of the gene under investigation, e.g. UBAPl. Rather, certain haplotypes are statistically associated with a mutated version of the gene, i.e. a marker allele to disease gene association. Therefore, where a subject has a certain haplotype, then it can be concluded that the subject also has a mutated version of the gene; in the present case UBAPl.
  • a further significant advantage of haplotype analysis is that the presence of a mutated version of a gene can be detected in a subject even when the precise nucleotide mutation within that gene has not been established. Therefore it is possible to determine whether a subject has a mutation within the UBAPl gene without first having to determine the exact genetic mutation present.
  • the haplotype of the subject is determined, and from that an assessment can be made as to the likelihood of that subject having a mutation in UBAP 1.
  • haplotype analysis uses haplotype blocks.
  • haplotype blocks we mean a set of genetic markers within particular region of a chromosome that are statistically linked. Various different genetic markers can be located within the haplotype block, for example: nucleotide deletions, nucleotide insertions, nucleotide repeat sequences, nucleotide rearrangements, and single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • the haplotype block has two or more SNPs markers.
  • the inventors have surprisingly found that the presence of a mutation in the UBAPl gene of a subject can be determined using a haplotype block, preferably comprising two or more SNPs.
  • a haplotype block preferably comprising two or more SNPs.
  • set out below are details of a number of SNPs that can included in a haplotype block which can form part of a haplotype analysis of UBAPl .
  • Further information from each of the SNPs can be obtained from, for example, the webpage of the International HapMap Project: http://www.hapmap.org/. By searching the database on that website information regarding the nucleotide sequence of the SNP and the distribution of the haplotype can be obtained.
  • the SNPs in the haplotype block are selected from the following group of SNPs: rsl3283064, rsl3283069, rs7018487, rslO971977, rsl2375731, rsl0814079, rs2380925, rsl7258783, rs4574933, rsl0814083.
  • An embodiment of the method of the invention is wherein the step of determining whether the subject has a mutation in the UBAPl gene comprises genotyping the UBAPl gene.
  • the step of genotyping UBAPl includes examining the nucleotide sequence of that gene to identify whether one or more genetic variations in the sequence are present.
  • Such genetic variations can include mutations which, as discussed above, insertions of one or more nucleotides, deletions of one or more nucleotides, nucleotide substitutions, and combinations thereof, including mutations that occur in coding and non-coding regions.
  • SNPs are also genetic variations, and hence this method of the invention can be used to identify whether any SNPs are present in UBAPl.
  • SNPs rsl3283064, rslO971969, rs7018487, rslO971977, rsl2375731, rsl0814079, rs2380925, rsl7258783, rs4574933 and rsl0814083 are associated with FTLD; rslO971977, rsl2375731, rs2380925 and rs 10972030 are associated with Alzheimer's disease.
  • the method can comprise genotyping UBAPl to detect the presence of SNPs rsl3283064, rslO971969, rs7018487, rslO971977, rsl2375731, rsl0814079, rs2380925, rsl7258783, rs4574933 and rsl0814083.
  • the method can comprise genotyping UBAPl to detect the presence of SNPs rslO971977, rsl2375731, rs2380925 and rsl0972030.
  • genotype may preferably be determined by testing a sample from the subject.
  • sample contains genomic DNA.
  • Methods of providing samples of genomic DNA from a subject are discussed above and can be routinely performed by the skilled person.
  • the nucleic acid sequence for UBAPl is provided herein and as part of the GenBank accession entries given above. This information can be used to design materials, such as oligonucleotide primers or probes specific for each allele that can be used when determining the genotype of the UBAPl gene of a subject.
  • design materials such as oligonucleotide primers or probes specific for each allele that can be used when determining the genotype of the UBAPl gene of a subject.
  • the design of such oligonucleotide primers is routine in the art and can be performed by the skilled person with reference to the information provided herein without any inventive contribution. If required, the primer(s) or probe(s) may be labelled to facilitate detection.
  • Techniques that may be used to detect mutations include:- (1) Direct sequencing of the polymorphic region of interest (e. g. using commercially available kits such as the Cysts Thermo Sequence dye terminator kit-Amersham Pharmacia Biotech); (2) Sequence Specific Oligonucleotide Hybridization (SSO) (involving dot or slot blotting of amplified DNA molecules comprising the polymorphic region; hybridisation with labelled probes which are designed to be specific for each polymorphic variant; and detection of said labels); (3) Heteroduplex and single-stranded conformation polymorphism (SSCP) Analysis (involving analysis of electrophoresis band patterns of denatured amplified DNA molecules comprising the polymorphic region); (4) Sequence Specific Priming (SSP) [also described as Amplification Refractory Mutation System (ARMS)]; (5) Mutation Scanning [e.
  • Direct sequencing of the polymorphic region of interest e. g. using commercially available kits such as the Cysts Thermo Sequ
  • Reference Strand mediated Conformational Analysis can also be used for UBAPl genotyping.
  • a PCR reaction is performed on a sample of DNA isolated from a subject using primers that flank a region of the UBAPl gene.
  • the amplified product is then hybridized with fluorescent-labeled reference DNA molecules at a temperature that permits annealing to occur, even when mismatches are present. Mismatches between the reference strand and the sample DNA result in the formation of bulges or "bubbles" in the heteroduplex that is formed.
  • the number and location of the bulges give the heteroduplex a unique mobility on a polyacrylamide gel, and can be used to determine whether there is a mutation in the UBAPl gene.
  • a further method is sequence based typing (SBT).
  • SBT sequence based typing
  • DNA isolated from a subject is used as a template for a PCR reaction that amplifies a region of the UBAPl gene to create a primary amplification product. That product is then purified to remove excess reaction reagents, though there are single-tube reactions available in which this purification step is not required.
  • the primary amplification product is then used as a template for sequencing reactions. Once complete, the sequence reactions are analysed by a sequencer, and the products analysed to determine whether there is a mutation in the UBAPl gene.
  • PCR primers may be designed such that they are suitable for amplifying a region of the UBAPl gene.
  • the design of suitable PCR primers is a routine laboratory technique.
  • genomic rearrangements can lead to mutations in the UBAPl gene.
  • Methods of determining genomic rearrangements include Southern blotting (essentially as performed as set out in Sambrook et al (1989). Molecular cloning, a laboratory manual, 2 nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York) or quantitative PCR.
  • a further embodiment of this aspect of the invention is wherein the method comprises determining the nucleic acid sequence of mRNA encoding the UBAPl polypeptide.
  • nucleotide sequence of the mRNA molecule can be determined, preferably from a cDNA sample prepared from mRNA isolated from the subject.
  • sequence of cDNA molecules can be determined according to the genotyping methods set out above.
  • a further embodiment of this aspect of the invention is wherein the method comprises determining whether the subject has a mutant UBAPl polypeptide. That is, if a sample from a subject has a mutant UBAPl polypeptide, then that subject has a mutation in the UBAPl gene.
  • polypeptide sequence for UBAPl is provided herein. This information can be used to design materials, such as antibodies or further specific binding molecules, that may be required for the methods set out below.
  • Determining whether a subject has a mutant UBAPl polypeptide may be conducted by isolating then sequencing UBAPl protein from a sample derived from that subject.
  • Methods of purifying proteins are well known in the art and can be readily applied to the method of the invention.
  • a molecule that selectively binds to the UBAPl protein e.g. an antibody or a fragment of an antibody, can be used to purify the UBAPl protein from the sample from the subject.
  • peptide sequencing methods such as N-terminal sequencing, the amino acid sequence of the isolated UBAPl protein can be determined and compared to that of the UBAPl protein provided herein.
  • the presence mutant UBAPl polypeptide in the sample can be detected using immunological methods.
  • the presence of a mutant UBAPl polypeptide in the sample can be detected using an antibody that selectively binds to a mutant UBAPl polypeptide.
  • Antibodies which can selectively bind to mutant UBAPl polypeptides can be made, for example, using peptides that include amino acid sequences particular to that mutation.
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982). Such methods include the use of hybridomas, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
  • antibody-like molecules may be used in the method of the inventions including, for example, antibody fragments or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody- like antigen binding motifs.
  • Sequence analysis of the coding region of UBAPl has identified three mutations in that sequence. Two of these lead to amino acid substitutions: a P to L change at position 96 (P96L); and a E to K change at position 87 (E87K).
  • a further mutation leads to a stop codon being introduced after the M at position 413 (M413X), and therefore results in a truncated UBAPl polypeptide; this mutation is also known as S391Afs21X .
  • a further mutation in UBAPl identified from sequence analysis is a P to L change at position 256 (P256L).
  • the P96L mutation is caused by a C to T change at position 522 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM 016525.
  • the E87K mutation is caused by a G to A change at position 494 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM 016525.
  • the M413X (S391Afs21X) mutation is caused by a stop codon being introduced at amino acid position 413 (M413X), and therefore results in a truncated UBAPl polypeptide.
  • the stop codon is introduced as a result of a single nucleotide deletion in the codon encoding the proline at position 390.
  • the deletion is of a C nucleotide at position 1404 or 1405 according to the numbering of the UBAPl nucleic acid set out above; preferably nucleotide 1404.
  • This deletion causes a "frame shift" in the open reading frame, such that the methionine residue at position 413 is altered to a stop codon.
  • the amino acid sequence of the M413X mutation is provided below.
  • a preferred embodiment of the invention is wherein the method determines whether the subject has a P96L, E87K or M413X (S391Afs21X) mutation in UBAPl.
  • this embodiment is used when the method of the seventh aspect of the invention relates to FTLD.
  • a further preferred embodiment of the invention is wherein the method determines whether the subject has C to T change at position 522, a G to A change at position 494 or a deletion of a C nucleotide at position 1404.
  • this embodiment is used when the method of the seventh aspect of the invention relates to FTLD.
  • the presence of UBAPl nucleic acid containing one or more mutations e.g., one or more mutations listed above
  • the presence of UBAPl nucleic acid containing one or more mutations in a subject can indicate that that subject has dementia or is likely to develop dementia.
  • the presence of UBAPl nucleic acid containing one or more mutations in a human can indicate that that human has dementia, especially when that human is between the ages of 35 and 75, has a family history of dementia, and/or presents symptoms of dementia.
  • Symptoms of dementia can include changes in behaviour such as changes that result in impulsive, repetitive, compulsive, or even criminal behaviour. For example, changes in dietary habits and personal hygiene can be symptoms of dementia.
  • Symptoms of dementia also can include language dysfunction, which can present as problems in expression of language, such as problems using the correct words, naming objects, or expressing oneself.
  • Difficulties reading and writing can also develop, hi some cases, the presence of UBAPl nucleic acid containing one or more mutations in a subject, together with positive results of other diagnostic tests, can indicate that the subject has dementia.
  • the presence of a mutation in UBAPl nucleic acid together with results from a neurological exam, neurophysical testing, cognitive testing, and/or brain imaging can indicate that a mammal has dementia.
  • Other diagnostic tests can include, without limitation, tests for mutations in MAPT and/or apolipoprotein E (APOE) nucleic acid.
  • any subject containing a mutation in UBAPl nucleic acid can be classified as having an elevated risk of developing dementia.
  • a subject having one or more than one mutation in UBAPl nucleic acid e.g., one or more than one mutation set out above
  • a human having one or more mutations in UBAPl nucleic acid can be classified as having an elevated risk of developing dementia when the human also has one or more mutations in MAPT or APOE nucleic acid and is less than, for example, 35 years old or does not appear to have symptoms of dementia.
  • FTLD frontotemporal lobar degeneration
  • AD frontotemporal lobar degeneration
  • the inventors consider that the UBAPl gene may be used as a diagnostic marker and a therapeutic agent for the disorders within this broad category. Therefore the aspects of the invention provided herein are applicable to a wide range of dementias, including Alzheimer's disease, motor neuron disease, Parkinson's disease, dementia with Lewy bodies, prion diseases, progressive supranuclear palsy or multisystem atrophy.
  • a preferred embodiment of the seventh aspect of the invention is wherein the dementia is frontotemporal lobar degeneration (FTLD).
  • FTLD frontotemporal lobar degeneration
  • the syndrome of FTLD encompasses the clinical subgroups of frontotemporal dementia (FTD), FTD with motor neuron disease, semantic dementia and primary progressive aphasia, and is characterized by changes in behaviour, personality and language with relative preservation of memory and perception
  • An embodiment of the seventh aspect of the invention is wherein the dementia, including frontotemporal lobar degeneration and Alzheimer's disease, is characterised by tauopathy.
  • Tauopathy is characterised by the accumulation of hyperphosphorylated tau in neurons and occasionally in glia.
  • An embodiment of the seventh aspect of the invention is wherein the frontotemporal lobar degeneration is characterised by ub-ir.
  • An embodiment of the seventh aspect of the invention is wherein the dementia is Alzheimer's disease.
  • the method of the invention can be applied to animal subjects of veterinary interest, it is preferred that the subject to be tested is a human subject.
  • the method according to the present invention is an in vitro method and can be performed on a sample containing nucleic acid and/or polypeptide derived from the subject.
  • the method of the invention is particularly suitable for being carried out on genomic DNA, particularly on isolated genomic DNA.
  • genomic DNA may be isolated from blood or tissue samples (e. g. hair, oral buccal swabs, nail or skin, blood, plasma, bronchoalveolar lavage fluid, saliva, sputum, cheek-swab or other body fluid or tissue), or from other suitable sources, using conventional methods.
  • the nucleic acid containing sample that is to be analysed can either be a treated or untreated biological sample isolated from the individual.
  • a treated sample may be for example, one in which the nucleic acid contained in the original biological sample has been isolated or purified from other components in the sample (tissues, cells, proteins etc), or one where the nucleic acid in the original sample has first been amplified, for example by polymerase chain reaction.
  • the sample may equally be a nucleic acid sequence corresponding to the sequence in the sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR, before analysis of allelic variation.
  • the method of the invention can also be carried out on protein samples obtained from a subject.
  • protein samples obtained from a subject may be isolated from blood or tissue samples as specified above, or from other suitable sources using conventional methods.
  • an embodiment of this aspect of the invention is wherein the UBAPl gene or polypeptide is derived from a sample of genomic DNA or polypeptide from the subject.
  • the sample is derived from blood or tissue samples.
  • the identification of one or more UBAPl mutations (e.g., one or more mutations listed in below) in an allele can be used to determine whether a subject has or is likely to develop a dementia.
  • Such a method may be performed when a subject has already exhibited clinical symptoms of dementia, i.e. as an adjuvant to existing techniques for diagnosing such neurological disorders.
  • the method may be performed as a means of assessing whether the subject has a predisposition towards developing dementia. This enables a medical practitioner to take appropriate action to prevent or lessen the likelihood of onset of the disease or disorder or to allow appropriate treatment of the disease or disorder.
  • the inventors have determined that the UBAPl gene is associated with dementia, specifically FTLD, and Alzheimer's disease. As set out above, this finding is the basis for the method of the seventh aspect of the invention in which the presence of a mutation is the UBAPl gene is indicative of a subject having, or being predisposed to developing, a dementia.
  • the linkage of the UBAPl gene to dementia suggests that, as well as mutations in that gene being linked to dementia, the amount of UBAPl polypeptide present in a subject can be used to determine if a subject has is predisposed to developing, a dementia.
  • a method of assessing whether a subject has or is likely to develop a dementia comprising determining whether the subject has an altered amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide.
  • the method of this aspect of the invention includes determining whether a subject has a modified amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide. If the subject has an altered amount of said polypeptide and/or nucleic acid, this indicates that the subject has or is likely to develop dementia.
  • Suitable samples that may be used in this method of the invention include those which contain representative samples of the patient's polypeptide and/or nucleic acid as set out above.
  • the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in the subject is compared to that of a "reference sample", i.e. a sample of protein or nucleic acid taken from a subject that does not have dementia.
  • the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in a sample of protein or nucleic acid taken from a control subject By comparing the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in a sample of protein or nucleic acid taken from the subject, it is possible to determine whether the subject has an altered amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide. In one embodiment of this aspect of the invention, the amount of UBAPl polypeptide or nucleic acid encoding UBAPl polypeptide is elevated.
  • “elevated” we include where the subject has, for example, 150%, 200%, 250%, 500%, 1000%, or 10000% of the amount of the polypeptide or nucleic acid in the control subject.
  • the amount of UBAPl polypeptide or nucleic acid encoding UBAPl polypeptide is reduced.
  • This method may be useful in the diagnosis of dementia or as a basis of genetic counseling.
  • the method of the invention can be applied to animal subjects of veterinary interest, it is preferred that the subject to be tested is a human subject.
  • the method of the invention can also be carried out on protein samples obtained from a subject.
  • protein may be isolated from blood or tissue samples as specified above, or from other suitable sources using conventional methods. Therefore an embodiment of this aspect of the invention is wherein the amount of UBAPl polypeptide or nucleic acid encoding said polypeptide is derived from a sample of genomic DNA or polypeptide from the subject.
  • the sample is derived from blood or tissue samples.
  • a preferred embodiment of the eighth aspect of the invention is wherein the dementia is characterised by tauopathy.
  • a preferred embodiment of the eighth aspect of the invention is wherein the dementia is frontotemporal lobar degeneration (FTLD) or
  • Alzheimer's disease As discussed above, FTLD and Alzheimer's disease are types of dementia, and the inventors consider that the UBAPl gene may be used as a diagnostic marker and a therapeutic agent for the disorders within this broad category.
  • a preferred embodiment of the eighth aspect of the invention is wherein the subject is a human subject.
  • the inventors have determined that the UBAPl gene is associated with dementia, specifically FTLD and Alzheimer's disease. As set out above, this finding is the basis for the method of the eighth aspect of the invention in which the presence of a mutation is the UBAPl gene is indicative of a subject having, or being predisposed to developing, a dementia.
  • a ninth aspect of the invention provides a non-human genetically modified animal having or predisposed to develop dementia, wherein the dementia results from an altered amount and/or function of UBAPl polypeptide.
  • UBAPl is associated with dementia.
  • Animals with an altered amount and/or function of UBAPl polypeptide can be expected to display dementia and may therefore be useful in screening for potential therapeutic agents for preventing or treating dementia.
  • sequence analysis of the coding region of UBAPl has identified three mutations in that sequence. Two of these lead to amino acid substitutions: a P to L change at position 96 (P96L); and a E to K change at position 87 (E87K).
  • a further mutation leads to a stop codon being introduced after the M at position 413 (M413X), and therefore results in a truncated UBAPl polypeptide; this mutation is also known as S391Afs21X .
  • a further mutation in UBAPl identified from sequence analysis is a P to L change at position 256 (P256L).
  • the P96L mutation is caused by a C to T change at position 522 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM Ol 6525.
  • the E87K mutation is caused by a G to A change at position 494 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM 016525.
  • the M413X is caused by a C to T change at position 522 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM Ol 6525.
  • the E87K mutation is caused by a G to A change at position 494 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM 016525.
  • (S391Afs21X) mutation is caused by a stop codon being introduced at amino acid position 413 (M413X), and therefore results in a truncated UBAPl polypeptide.
  • the stop codon is introduced as a result of a single nucleotide deletion at position 1404 or
  • nucleotide 1405 according to the numbering of the UBAPl nucleic acid set out above; preferably nucleotide 1404.
  • a preferred embodiment of this aspect of the invention is wherein the non- human genetically modified animal has a mutation(s) in UBAPl equivalent to the P96L, E87K and/or M413X (S391Afs21X) mutation in human UBAPl described above.
  • the non-human genetically modified animal may have been specifically genetically engineered to have a copy of the human UBAPl gene, but having one or more mutations in that gene. Therefore a further preferred embodiment of this aspect of the invention is wherein the non-human genetically modified animal has a human UBAPl nucleic acid sequence having a P96L, E87K and/or M413X mutation as described above.
  • the native homologue of human UBAPl has been removed or otherwise mutated such that no native UBAPl polypeptide is produced.
  • the non-human animal may be any non-human animal, including non-human primates such as baboons, chimpanzees and gorillas, new and old world monkeys as well as other mammals such as cats, dogs, rodents, pigs or sheep, or other animals such as poultry, for example chickens, fish such as zebrafish, or amphibians such as frogs.
  • the animal is a rodent such as a mouse, rat, hamster, guinea pig or squirrel.
  • the animal is mouse.
  • ementia we include those disorders discussed above in relation to the first aspect of the invention.
  • a preferred embodiment is wherein the dementia is characterised by tauopathy.
  • the dementia is FTLD or Alzheimer's disease.
  • altered amount and/or function we include that, in comparison to a normal animal of the same species or strain, the animal of the ninth aspect of the invention has a reduced or elevated amount of UBAPl polypeptide and/or a reduced or elevated amount of the UBAPl polypeptide can function in the way that the same polypeptide operates in the comparative animal.
  • the animal of this aspect of the invention may have the same amount of polypeptide per se, but the polypeptide is in a non-functional state.
  • the animal of the ninth aspect of the invention has a reduced amount and/or function of UBAPl polypeptide.
  • the altered amount of UBAPl polypeptide may be due to an altered amount of nucleic acid encoding the UBAPl polypeptide.
  • altered amount and/or function of includes where the animal has a reduced amount, i.e. 50%, 25%, 10%, 5%, 1%, 0.1% or 0% of the amount and/or function of the polypeptide, or nucleic acid, in the normal animal.
  • the non-human animal has no functional UBAPl polypeptide.
  • the animal may have an elevated amount, i.e. 150%, 200%, 250%, 500%, 1000%, or 10000% of the amount and/or function of the polypeptide, or nucleic acid, in the normal animal.
  • the non-human animal of this aspect of the invention may have an altered amount and/or function of UBAPl polypeptide due to the animal being genetically modified so as to have an agent which can modify said polypeptide function.
  • the animal could be genetically modified to express a peptide or antibody which can bind to the UBAPl and prevent function or sub-cellular localisation.
  • the non-human animal of this aspect of the invention may have an altered amount of nucleic acid encoding UBAPl polypeptide due to the animal being genetically modified so as to have an agent which can cause or induce degradation of said nucleic acid, for example a ribozyme which can target the nucleic acid, or an antisense molecule which can bind to the UBAPl nucleic acid.
  • antisense we include RNA interference (RNAi) technologies.
  • the animal may be genetically modified in such a manner as to alter the native UBAPl gene.
  • the term "genetically modified” is well known to those skilled in the art.
  • the term includes animals having introduced native or foreign nucleic acid. The animal may have had a modification made to its genome.
  • non-human genetically modified animal There are a number of different methods that can be employed to generate a non-human genetically modified animal according to this aspect of the invention. These will be discussed in turn below. Preferred methods include those in which the gene encoding the said polypeptide is altered or removed so as to produce little or none of said polypeptide. Other methods include inhibiting the transcription of the said gene or preventing any mRNA encoded by said gene from being translated due to the animal being genetically modified so as to have an agent which can modify said polypeptide transcription, translation and/or function. Preferably, the methods set out below are employed to generate a non-human genetically modified animal according to this aspect of the invention in which the function of the UBAPl polypeptide altered.
  • “Homologous recombination” is a technique well known to those skilled in the art. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout" animals. Hence this aspect of the invention includes wherein the amount and/or function of UBAPl polypeptide is altered by mutated one or more gene(s) encoding UBAPl by homologous recombination.
  • “Insertional mutagenesis” is also a term well known to those skilled in the art.
  • Examples of such mutagenesis include transposon-tagging, homing endonuclease genes (HEGs).
  • HEGs homing endonuclease genes
  • a region of DNA is introduced into a gene such that the controlling or coding region of the gene is disrupted.
  • Such methods can be used to disrupt one or more genes encoding UBAPl polypeptide. As a result the animal will no longer be able to synthesise UBAPl polypeptide, i.e. there will be a reduction in the amount of this polypeptide.
  • Chemical or physical mutagenesis can also be used in the method of this aspect of the invention.
  • a gene is mutated by exposing the genome to a chemical mutagen, for example ethyl methylsulphate (EMS) or ethyl Nitrosurea (ENU), or a physical mutagen, for example X-rays.
  • EMS ethyl methylsulphate
  • ENU ethyl Nitrosurea
  • X-rays X-rays.
  • agents can act to alter the nucleotide sequence of a gene or, in the case of some physical mutagens, can rearrange the order of sequences in a gene.
  • Homologous recombination, insertional mutagenesis and chemical or physical mutagenesis can be used to generate a non-human animal which is heterozygous for the target gene, e.g. UBAPl gene ( + ⁇ ). Such animals may be of particular use if the homozygous non-human animal has too severe a phenotype.
  • the non-human animal of this aspect of the invention could be genetically modified to include an antisense molecule or siRNA molecule that can affect the expression of UBAPl.
  • Antisense oligonucleotides are single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed "antisense” because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise sequence-specific molecules which specifically bind double-stranded DNA via appropriate formation of major groove hydrogen bonds.
  • the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A)addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.
  • antisense we also include all methods of RNA interference, which are regarded for the purposes of this invention as a type of antisense technology.
  • a further method of generating a non-human animal of this aspect of the invention is wherein the animal is genetically modified so as to have a ribozyme capable of cleaving RNA or DNA encoding UBAPl polypeptide.
  • a further method of generating a non-human animal of this aspect of the invention is wherein the animal is genetically modified so as to have an agent that acts as antagonist to UBAPl amount and/or function.
  • antagonist is well known to those skilled in the art. By “antagonist” we include in this definition any agent that acts to alter the level and/or functional ability of UBAPl polypeptide.
  • An example of an antagonist would include a chemical ligand that binds to and affects UBAPl function, and in broader terms this could also include an antibody, or antibody fragment, that binds to one of the said polypeptides such that the polypeptide cannot effect its normal function.
  • the antagonist may also alter the subcellular localisation of UBAPl polypeptide. In this way, the amount of functional polypeptide is reduced.
  • a further method of generating a non-human animal of this aspect of the invention is wherein the animal is genetically modified so as to have a dominant inactive form of a UBAPl polypeptide.
  • UBAPl polypeptides we include the human and mouse UBAPl polypeptides as well as further homologues, orthologues or paralogies of UBAPl polypeptides.
  • UBAPl polypeptides are disclosed in GenBank. Methods by which homologues, orthologues or paralogues of polypeptides can be identified are well known to those skilled in the art: for example, in silico screening or database mining.
  • polypeptides have at least 40% sequence identity, preferably at least
  • nucleic acid encoding UBAPl polypeptide includes both DNA and RNA molecules, including mRNA. By encode we mean that the sequence of bases in the nucleic acid molecule is such that, on transcription and/or translation, it encodes a polypeptide having the sequence of a UBAPl polypeptide.
  • kits for assessing whether a subject has or is likely to develop a dementia comprising means for determining whether the subject has a mutation in the UBAPl gene.
  • the molecules given in the seventh aspect of the invention that can be used to determine whether the subject has a mutation in the UBAPl gene.
  • the molecule is an oligonucleotide probe or antibody.
  • kit of the tenth aspect of the invention may also comprise relevant buffers and regents for conducting such methods.
  • the buffers and regents provided with the kit may be in liquid form and preferably provided as pre-measured aliquots.
  • the buffers and regents may be in concentrated (or even powder form) for dilution.
  • kits for assessing whether a subject has or is likely to develop a dementia comprising means for determining whether the subject has an altered amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide.
  • ⁇ we include the molecules given in the eighth aspect of the invention that can be used to determine the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide.
  • the molecule is an oligonucleotide probe or antibody.
  • the kit of the eleventh aspect of the invention may also comprise relevant buffers and regents for conducting such methods.
  • the buffers and regents provided with the kit may be in liquid form and preferably provided as pre-measured aliquots.
  • the buffers and regents may be in concentrated (or even powder form) for dilution.
  • a further aspect of the invention provides an isolated polypeptide having the amino acid sequence:
  • the polypeptide of this aspect of the invention corresponds to the UBAPl polypeptide having a P96L mutation.
  • the invention also includes a nucleic acid molecule encoding the polypeptide of this aspect of the invention. Such a polypeptide or nucleic acid molecule would have utility, for example, in preparing agents of use in the diagnostic methods of the invention.
  • a further aspect of the invention provides an isolated polypeptide having the amino acid sequence:
  • the polypeptide of this aspect of the invention corresponds to the UBAPl polypeptide having a E87K mutation.
  • the invention also includes a nucleic acid molecule encoding the polypeptide of this aspect of the invention. Such a polypeptide or nucleic acid molecule would have utility, for example, in preparing agents of use in the diagnostic methods of the invention.
  • a further aspect of the invention provides an isolated polypeptide having the amino acid sequence:
  • the polypeptide of this aspect of the invention corresponds to the UBAPl polypeptide having a M413X (S391Afs2 IX) mutation.
  • the invention also includes a nucleic acid molecule encoding the polypeptide of this aspect of the invention. Such a polypeptide or nucleic acid molecule would have utility, for example, in preparing agents of use in the diagnostic methods of the invention.
  • Figure 1 Sliding Window Haplotype analysis for the combined Dutch and Manchester UBAPl data. Green line corresponds to a p value of 0.05.
  • Figure 2 Confocal images of neuronal staining of tau-2 (a) and UBAPl (b) and colocalisation (d) in hippocampus in FTLD.
  • Figure 3 Immunoprecipitation of UBAPl pulling down. Progranulin (arrow). A - total protein input; B - no antibody IP control; C - IP of progranulin.
  • Figure 6 Sliding window analysis with a maximal global P value of 0.0002 using a sliding window size of 7.
  • Example 1 Genetic variation of the gene Ubiquitin Associated protein 1 increases the risk of developing frontotemporal lobar degeneration or other neurodegenerative diseases.
  • UBAPl Ubiquitin Associated Protein 1
  • UBAPl originally cloned from a tumour suppressor locus (Qian et al, supra), has two Ubiquitin-associated domains (UBA), between residues 389-430, and 451-498, and an Ubiquitin System Cue domain between residues 459-499 and is likely involved in the binding of ubiquitin-conjugating enzymes.
  • UBA domains are found in various proteins involved in the ubiquitin/proteosome pathway and is therefore an excellent candidate gene for FTLD. Sequence analysis of the open reading frames has identified 3 mutations (P96L, E87K, M413X, all in fully conserved residues in other species), however, we do not have access to other family members to demonstrate segregation but these are absent in 280 controls.
  • UBAPl staining is present in neurons and astrocytes in grey and white matter, and appear as discrete granules or larger globules usually clustered into larger conglomerates within the perikaryon (Fig 2).
  • Example 2 Genetic variation of the gene Ubiquitin Associated protein 1 increases the risk of developing Alzheimer's disease.
  • Example 3 UBAPl association with tau protein.
  • the UBAPl antibody can immunoprecipitate cellular tau protein. Colocalisation oftau and UBAPl
  • Sections were then incubated in primary antibodies (rabbit anti-UBAPl-359 (diluted 1:200 in PBS); mouse anti-Tau2 (diluted 1:2000 in PBS)) overnight at 4 0 C then rinsed 3 times with PBS containing 0.01% Tween-20 (PBS-TT). Sections were incubated in secondary antibodies (Alexa- 488 conjugated goat anti-mouse antibody; Alexa-546 conjugated goat anti-rabbit antibody (diluted 1:200 in PBS)) for lhr at RT then rinsed 3 times with PBS-TT and mounted in Vectashield a fluorescent mounting medium (Vectorlabs, UK) and coveslips sealed.
  • primary antibodies rabbit anti-UBAPl-359 (diluted 1:200 in PBS); mouse anti-Tau2 (diluted 1:2000 in PBS)
  • PBS-TT PBS containing 0.01% Tween-20
  • Frontotemporal lobar degeneration is the term used for the clinical syndrome characterized by changes in behaviour, personality and language with relative preservation of memory and perception 1 .
  • FTLD is a common form of dementia in individuals under the age of 65 and around half of all patients present with a family history of a similar disease .
  • the genetic aetiology of FTLD is complex with 7 loci identified to date on chromosomes 3, 9p (two loci), 9q, 17q,21 (two locii) and 17q24 3 .
  • FTLD 4"8 There are two common neuropathological subgroups observed in FTLD being tauopathy, the accumulation of abnormally phosphorylated and insoluble aggregates of tau protein in neurons and sometimes glia, the other is referred to as FTLD-U where neuronal inclusions containing TAR DNA binding protein (TDP-43) are present in varying quantities 9 .
  • FTLD-U has been further divided into three main subgroups determined by the distribution of the TDP-43 pathology 9 ' 10 .
  • Familial tauopathy is often associated with mutations in MAPT whereas null-mutations of progranulin have been shown to lead to FTLD-U 6 ' 7 . Cases with mutations in progranulin have been designated type 3 FTLD-U whereas those cases from families linked to chromosome 9p have been identified as having type 2 pathology 9 .
  • One characteristic that unites the two differing histological subtypes in FTLD is that both tau and TDP-43 inclusions are ubiquitinated to varying degrees. The observation that many proteins that accumulate within neurons and/or glia in neurodegenerative diseases and are ubiquitinated has lead to the suggestion that dysfunction of the ubiquitin proteosome system could be an aetiological factor in this group of conditions ⁇ .
  • the series comprised of 116 men (mean age at onset 59.1 years, range 35 to 79), and 98 women (mean age at onset 60.1 years, range 23 to 83).
  • 44 patients had come to postmortem.
  • 119 patients 64 men and 55 women, mean age at onset 58.0 years (standard deviation 9.7 years), range 23 to 82) were diagnosed clinically with frontotemporal dementia (FTD).
  • FTD frontotemporal dementia
  • 33 patients (20 men, 13 women; mean age at onset 60.1 years (7.7 years), range 43 to 74) were diagnosed with FTD and motor neurone disease (MND).
  • Dutch cohort 214 patients with FTLD comprising of 118 females and 96 males with a mean age at onset 57.9 +/- 9.0 (30-76) and a disease duration of illness 8.3 +/- 3.9 with age at death being 65.0+/- 9.6 where known. 138 of these had a clinical diagnosis of FTD, 38 had semantic dementia, 23 progressive non-fluent aphasia and 15 had FTD + MND. Dutch controls consisted of 149 males and 149 females with an average age of 61.06 +/-2.89 years at the time of collection.
  • a total of 133 genes (as of genome build 35) were found in the region, Linkage disequilibrium (LD) maps were obtained from HapMap, where multiple genes fell within a single haplotype block, tag SNP 's were selected to reduce the total number of SNPs required for genotyping using the tagger application 16 .
  • a minimum of one SNP per gene or two SNPs per haplotype block were prioritized to give as complete coverage as possible of the region. Any SNPs also in coding regions or with a potential biological function were prioritized for genotyping selection, as were SNPs with a minor allele frequency of 0.2 or greater.
  • DNA samples were randomly assigned across 2 genotyping plates with 5 cell line samples duplicated across the plates, and an additional 7 duplicate samples on each plate to test for genotyping consistency. 15ng of each DNA was genotyped using the Sequenom MassArray genotyping technology according to manufactures instructions.
  • Genotyping quality control for each SNP assay included concordance of genotypes between replicate samples both internally per genotyping plate and externally between plates. An equal call of genotypes had to be observed in both the case and control groups of samples (the average ration of genotyping calls between the cases and controls being 1.002), with a minimum of 75% samples with an assigned genotype. All SNPs were required to be in Hardy- Weinberg in the control population with a minimum P value of 0.05, while cases could be out of equilibrium for inclusion into the study, no SNP was out of equilibrium in the cases and not in the controls.
  • Haploview 17 Statistical analysis Simple ⁇ 2 and Hardy- Weinberg Equilibrium P values for each SNP variant were calculated using Haploview 17 .
  • Haplotype block structure was examined using haploview 1? , a block defined using the confidence intervals option, where 95% confidence bounds on D' are generated, a block defined if 95% of informative comparisons are in strong linkage disequilibrium .
  • data corresponding to genomic positions 26856230 to 37152282 on chromosome 9 were downloaded from the international haplotype mapping project (http://www.hapmap.org/).
  • the CEPH samples comprise of Utah (USA) residents with ancestry from northern and western Europe.
  • Haplo.Stats (version 1.2.2) was used to carry out sliding-window haplotype analysis, used to estimate haplotype effects under the generalized linear model (http://mayoresearch.mayo.edu/mayo/research/biostat/schaid.cfm).
  • UBAP 1-359 An affinity purified polyclonal rabbit anti-UBAPl peptide antibody (UBAP 1-359) was generated by Eurogentec raised against peptides matching the C-terminal coding sequence of UBAPl (CQDNALEDLMARAGAS). Also, rabbit polyclonal UBAPl antibody from Proteintech was used. For TDP-43 immunohistochemistry mouse anti- TARDBP monoclonal antibody (Clone MOl; Abnova) was employed.. Alexa-488 conjugated goat anti-mouse and Alexa-546 conjugated goat anti-rabbit secondary antibodies were obtained from Invitrogen Molecular Probes, UK. Insoluble protein fractions from human brain were generated using the TDP-43 protocol 2 .
  • Sections were then incubated in primary antibodies (rabbit anti- UBAP1-359 (diluted 1 :200 in PBS); mouse anti-TDP-43 (diluted 1 :2000 in PBS)) overnight at 4°C then rinsed 3 times with PBS containing 0.01% Tween-20 (PBS-TT). Sections were incubated in secondary antibodies (Alexa-488 conjugated goat anti- mouse antibody; Alexa-546 conjugated goat anti-rabbit antibody (diluted 1 :200 in PBS)) for lhr at RT then rinsed 3 times with PBS-TT and mounted in VectashieldTM fluorescent mounting medium (Vectorlabs, UK) and coveslips sealed.
  • primary antibodies rabbit anti- UBAP1-359 (diluted 1 :200 in PBS); mouse anti-TDP-43 (diluted 1 :2000 in PBS)
  • PBS-TT 0.01% Tween-20
  • haplotype block structure of both TEK and RECK showed concordance with that seen in the CEPH HAPMAP data, however neither gene displayed a haplotype that was associated with a significant effect for FTLD (data not shown), hi contrast, in addition to the first round rs4574933 SNP, and additional 6 SNPs were found to have an allele association with FTLD (table 1). Sliding window analysis confirmed this with a maximal global P value of 0.0002 using a sliding window size of 7 (figure 6).
  • This 7 SNP haplotype includes 6 of the 7 significant SNP assays (rslO971977 to rsl0814083), corresponding to the genomic region that contains and surrounds the UBAPl coding sequence.
  • Haploview analysis confirmed that the haplotype structure around UBAPl agreed with that from by the CEPH hapmap data, with a 10 SNP haplotype block that spans all of the UBAPl genomic sequence (Fig 6). Indeed inheritance of the TATTAGATGC block was associated with an increased risk of FTLD of OR 1.71 (95% CI 1.20 - 2.44) in the Manchester cohort.
  • UBAPl sequence analysis The entire open reading frame of UBAPl was sequenced on both the Manchester and Dutch cohorts. Five variants were identified in separate familial cases (E87K, P96L, S391Afs21X and H149Q in the Manchester samples and a P256L in the Dutch cohort). Four of these were absent from 450 controls and are possibly mutations, however, the H149Q was found in controls (frequency X) and is therefore likely to be a polymorphism. Unfortunately, samples from other family members were unavailable to test for segregation. Nevertheless, the E87K, P96L, P256L and S391Afs21X are all in fully conserved regions of UBAPl (data not shown) suggesting functional significance.
  • UBAPl immunoreactivity was present in pyramidal and non-pyramidal neurons throughout the cerebral cortex in the form of small granules or larger globules or a mixture of both. These were evenly scattered throughout the perikaryon and showed no focal accumulation at any particular region of their neurons. Occasional glial cells, probably astrocytes, also showed similar but smaller accumulations of UBAP-I immunoreactivity. In all but one case the UBAPl failed to stain any pathological inclusions i.e. TDP-43 cytoplasmic or intranulcear inclusions, tangles or Pick bodies.
  • UBAPl encodes a protein of 502 residues, predicted to have a molecular weight of 55KDa and was originally cloned from a tumour suppressor locus 24 . While little is known of the actual function of the protein, the gene is likely a member of the Ubiquitin-activated enzymes family whose members include proteins having connections to ubiquitin and the ubiquitination pathway.
  • the protein itself has two Ubiquitin-associated domains (UBA), between residues 389-430, and 451-498, and an Ubiquitin System Cue domain between residues 459-499, believed to be involved in the binding of ubiquitin-conjugating enzymes.
  • the UBA domains are found in various proteins, including p62 which is found in certain TDP-43 positive inclusions in FTLD 25 , and are involved numerous processes including the ubiquitin/proteosome pathway, growth control, receptor function, stress responses, DNA excision-repair and cell signalling via protein kinases 26 . It is believed that the ubiquitin proteomsome system (UPS) plays a vital role in protecting the CNS from the accumulation of toxic proteins u . Furthermore, it has been demonstrated that mutations of certain genes involved in the UPS can lead to Parkinson's disease and clearly link a dysfunctional UPS to neurodegeneration 27 . Therefore UBAPl is an excellent candidate gene for FTLD.
  • UPS ubiquitin proteomsome system
  • UBAPl and TDP-43 proteins co-localise in together in neuronal cytoplasmic inclusions in a case of familial FTLD is important as it directly implicates UBAPl in the metabolism of TDP-43. It also suggests that UBAPl related FTLD will likely be of the FTLD-U neuropathological subtype, it is a very common feature that the protein products of genetic risk factors for neurodegenerative disease are found in the cytoplasmic pathological inclusions of these diseases and our current data are supportive of this hypothesis in at least some cases of FTLD .
  • the family with UBAPl positive neuropathology is the same family recently reported to have a mutation in the IFT74 gene 29 .
  • UBAPl as a risk factor for FTLD is an important discovery because for the first time it provides evidence for a link between the UPS and this group of conditions. This observation, therefore, suggests the UPS is a potential future therapeutic target for FTLD. It will be interesting to establish whether UBAPl mutations lead to a TDP-43 or tauopathy based histology and to establish whether this protein has a wider role in neurodegenerative disease. Moreover, it will be important to investigate UBAPl in further populations and in families with FTLD definitively linked to chr9p. Finally, if pathogenic variations are absent these latter families this does not refute our finding of UBAPl being an independent risk factor for FTLD in this region. If this scenario was true it would be analogous to situation on chromosome 17 where the two genes, MAPT and PGRN, causing FTLD are only 1.7Mb apart ' 7 .
  • TATTAGATGC 194 20.27 91 20.04 1.18 0.86-1.63 0.288

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Abstract

Cette invention concerne un procédé de criblage permettant de rechercher des composés utilisés pour prévenir ou traiter la démence; lequel procédé consiste à traiter une cellule comprenant UBAPI avec un composé d'essai, puis à évaluer l'effet du composé d'essai sur la quantité et/ou la fonction de UBAPI. De préférence, les composés utilisés pour prévenir ou traiter la démence augmentent la quantité et/ou l'activité de UBAPI. L'invention concerne également des procédés consistant à déterminer si un sujet est atteint de démence ou s'il est susceptible d'en être atteint, lesquels procédés consistent à déterminer si le sujet présente une mutation du gène UBAPI. Cette invention concerne également un animal génétiquement modifié non humain présentant une démence ou une prédisposition à la démence, laquelle démence découle d'une quantité modifiée et/ou d'une fonction modifiée du polypeptide UBAPI.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012085217A1 (fr) * 2010-12-22 2012-06-28 Universite De La Mediterranee (Aix-Marseille Ii) Méthode de diagnostic de démences
WO2020190752A1 (fr) * 2019-03-15 2020-09-24 University Of Miami Procédés de détection et de traitement de la paraplégie spastique héréditaire

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Title
MACKENZIE IAN R A ET AL: "Heterogeneity of ubiquitin pathology in frontotemporal lobar degeneration: classification and relation to clinical phenotype", ACTA NEUROPATHOLOGICA, vol. 112, no. 5, November 2006 (2006-11-01), pages 539 - 549, XP002511867, ISSN: 0001-6322 *
QIAN JUN ET AL: "Isolation and characterization of a novel cDNA, UBAP1, derived from the tumor suppressor locus in human chromosome 9p21-22", JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY, SPRINGER INTERNATIONAL, BERLIN, DE, vol. 127, no. 10, 1 October 2001 (2001-10-01), pages 613 - 618, XP002257948, ISSN: 0171-5216 *
ROLLINSON S. J. ET AL: "UBAP1 is a risk factor for frontotemporal lobal degeneration", ALZHEIMER'S AND DEMENTIA, vol. 4, no. 4, July 2008 (2008-07-01), pages T402, XP002511868 *
XIAO BINGYI ET AL: "Purification of novel UBAP1 protein and its decreased expression on nasopharyngeal carcinoma tissue microarray", PROTEIN EXPRESSION AND PURIFICATION, vol. 47, no. 1, May 2006 (2006-05-01), pages 60 - 67, XP002511866, ISSN: 1046-5928 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012085217A1 (fr) * 2010-12-22 2012-06-28 Universite De La Mediterranee (Aix-Marseille Ii) Méthode de diagnostic de démences
WO2020190752A1 (fr) * 2019-03-15 2020-09-24 University Of Miami Procédés de détection et de traitement de la paraplégie spastique héréditaire
US20220177527A1 (en) * 2019-03-15 2022-06-09 University Of Miami Methods of detecting and treating hereditary spastic paraplegia

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