WO2016059166A1

WO2016059166A1 - Humanized neuromelanin-containing rodent

Info

Publication number: WO2016059166A1
Application number: PCT/EP2015/073909
Authority: WO
Inventors: Miquel Vila Bover; Iria CARBALLO CARBAJAL; Jordi BOVÉ BADELL; Thais CUADROS ARASA; Ariadna LAGUNA TUSET
Original assignee: Institucio Catalana de Recerca i Estudis Avancats ICREA; Centro de Investigacion Biomedica en Red de Enfermedades Neurodegenerativas CIBERNED; Fundacio Institut de Recerca Hospital Universitari Vall dHebron
Current assignee: Institucio Catalana de Recerca i Estudis Avancats ICREA; Centro de Investigacion Biomedica en Red de Enfermedades Neurodegenerativas CIBERNED; Fundacio Institut de Recerca Hospital Universitari Vall dHebron
Priority date: 2014-10-15
Filing date: 2015-10-15
Publication date: 2016-04-21
Anticipated expiration: 2017-04-15

Abstract

The invention provides a humanized rodent exhibiting age-dependent production and intracellular accumulation of human-like neuromelanin, up to levels reached in elder humans, within Parkinson's disease-vulnerable neuronal groups. The humanized neuromelanin-containing rodent stably expresses the human tyrosinase gene in at least the dopaminergic neurons of the substantia nigra pars compacta and can be a wild-type rodent to which the human tyrosinase gene has been transduced via a viral vector, or a transgenic rodent that expresses the human gene in a constitutive or inducible form. In any of these cases, the rodent develops features of Parkinson's disease such as nigro-striatal dopaminergic neurodegeneration and motor impairment, making it a valuable tool for the (i) search of biomarkers for the early detection, diagnosis or progression of the disease, and (ii) the screening of new therapies with disease-modifying potential to combat PD.

Description

Humanized neuromelanin-containing rodent

The present invention provides a rodent that expresses the human tyrosinase gene in the brain, an enzyme which might be involved in the production of neuromelanin, and accumulates the latter pigment in dopaminergic neurons. This rodent has potential applications in the pre-clinical research of

Parkinson's disease (PD), enabling: (i) the search of potential neuromelanin- related biomarkers for early detection, diagnosis and/or progression of PD, and (ii) the screening for treatments aimed at modulating neuromelanin accumulation to eventually slow down or halt the progression of PD.

BACKGROUND ART

Parkinson's disease is one of the main degenerative diseases of the central nervous system, second only to Alzheimer's. It is estimated to affect around 7 million people worldwide, and its prevalence is estimated to be around 0.3% in the industrialized nations. It is characterized by a progressive and sustained neurodegeneration in different brain regions, mostly of dopaminergic neurons in the substantia nigra pars compacta (SNPC), located in the midbrain. This neuronal death causes a variety of symptoms which become more severe with time, such as motor impairment, dementia and depression. PD is treated with Levodopa, dopamine agonists, anticholinergics and MAO inhibitors, among others. However, there is currently no cure for the disease. Therefore, new disease-modifying treatments are badly needed.

The exact mechanism by which the dopamine-producing neurons die in PD is not known. However it is widely accepted that there exists a selective vulnerability of the groups of neurons that contain the pigment neuromelanin (NM), which are basically those of the SNPC, and to a lesser extent the noradrenergic neurons of the locus coeruleus (LC) and dorsal motor nucleus of the vagus (DMNV). The mechanisms of synthesis, accumulation pattern and function of NM are currently a matter of debate, as there are conflicting disclosures on whether NM is a by-product resulting from catecholamines auto-oxidation or whether its synthesis is enzymatically regulated (Ikemoto, K. et.al. "Does tyrosinase exist in neuromelanin-pigmented neurons in the human substantia nigra?" Neurosc. Lett. 1998, vol. 1 1 , pp. 198-200; Tief, K., et.al. "Tyrosinase, the key enzyme in melanin synthesis, is expressed in murine brain" Eur. J. Biochem. 1996, vol. 241 , pp. 12-16; Xu, Y., et.al. "Tyrosinase mRNA is expressed in human substantia nigra" Brain Res. Mol. Brain Res. 1997, vol. 45, pp.159-162; Greggio E., et. al. "Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease" J. of Neurochem. 2005, vol. 93, pp. 246-256). Furthermore, dying neurons in PD are also characterized by the presence of cytoplasmic protein inclusions known as Lewy bodies (LB), whose main component is the protein a- synuclein. The latter has been extensively studied, and its precipitation, misfolding and aggregation is now considered to be one of the main culprits in the development of at least some forms of PD (Olanow, W., et. al.

"Parkinson's disease and alpha synuclein: Is Parkinson's disease a prion-like disorder?" Movement Disorders 2013, vol. 28, pp. 31 -40). Furthermore, NM has been shown to associate to lipid content, oxidation or iron loading, reactions known to precipitate a-synuclein. In fact, several studies have reported that a-synuclein accumulation occurs around the NM granules (Fasano, M, et al. "Residual substantia nigra neuromelanin in Parkinson's disease is cross-linked to a-synuclein". Neurochem Int 2003, vol. 42, pp. 603- 606); Halliday, G. M. et al. "a-Synuclein redistributes to neuromelanin lipid in the substantia nigra early in Parkinson's disease". Brain 2005, vol. 128, pp. 2654-2664).

Aging is one of the main risk factors associated to the development of PD. In humans, NM progressively accumulates with age since it cannot be degraded or eliminated by neurons. However, the potential contribution of the

accumulation of NM to PD is not known mainly due to the fact that, unlike humans, common experimental animal species used in pre-clinical studies (mainly rodents) lack NM. In fact, the great abundance of NM in the SNPC seems to be characteristic of humans, as a macroscopic dark pigmentation of the midbrain is not observed in other species or NM is only present at moderate levels in some species of monkeys, horses or sheep.

Many animal models used for studying PD are based on the exposure to neurotoxic compounds such as 6-OHDA and MPTP (Blum, D., et.al.

"Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease" Prog. In Neurobiol. 2001 , vol. 65, pp. 135-172) whose effects mimic, at least partially, clinical or neuropathological aspects of the disease. Several transgenic rodent models targeting PD-causative genes have also been described, including transgenics for DJ-1 , LRRK2, Parkin, PINK1 and a-synuclein (Harvey, B.K. et. al. "Transgenic rodent models of Parkinson's disease" 2008, vol. 101 , pp. 89- 92), as well as for other PD-related non-causative genes. All these models have enormously contributed to getting a deeper insight into the familial as well as the sporadic forms of the disease.

In spite of these efforts, so far there is no animal model for studying the potential contribution of NM accumulation in the development and progression of PD. Therefore, a factor so intimately linked to PD such as NM is not taken into account in current experimental paradigms of PD. Thus, there is a long felt need to develop rodent models which recapitulate NM synthesis and accumulation over time within catecholaminergic PD-vulnerable neuronal regions, as it occurs in humans. Besides throwing light into the potential role of NM in PD, this model would serve as a useful platform for (i) the search of potential neuromelanin-related biomarkers for the early detection, diagnosis and/or progression of PD, and (ii) the screening of novel PD therapies aimed at modulating neuromelanin accumulation. SUMMARY OF THE INVENTION

Inventors have surprisingly found that a rodent that expresses the human tyrosinase gene in the brain, and more specifically in the SNPC, not only accumulates NM in its neurons, but also reveals physiological changes at the cellular level that correspond to those seen in the neurons of PD patients, and what is even more remarkable, it additionally displays motor dysfunctions and neurodegeneration typical of PD.

This is a striking result for several reasons. On the one hand, as stated above, there is a high degree of controversy surrounding the way NM is

synthesized/produced in the neuron. On the other hand, the accumulation of NM is not an established cause of neuronal dysfunction. Finally, the

accumulation of a pigment such as NM in the neurons of a rodent would, a priori, have unpredictable consequences in view of what is known in the field.

It has been remarkably found that the rodent of the invention does accumulate NM progressively with age as is the case of human subjects, that the accumulation takes place in the dopaminergic neurons (especially those of the SNPC) and further, that it is significant enough to surpass a certain pathologic threshold as it ultimately is linked with the appearance of neurodegeneration and motor dysfunction typically experienced by PD patients.

The rodent that is part of the invention can thus be used as a faithful preclinical model of PD, and be exploited as a screening tool for the search of potential neuromelanin-related biomarkers for the early detection, diagnosis and/or progression of PD, and for the testing of new therapies with disease- modifying potential that combat NM accumulation as a potential mechanism of action with which to treat PD.

Thus, a first aspect of the invention is a rodent that comprises the human tyrosinase gene, the gene being expressed in at least the dopaminergic neurons of the substantia nigra pars compacta.

A second aspect of the invention is a method of screening for therapeutic agents that prevent, delay or halt the development of neurodegenerative disease in the rodent of the first aspect of the invention, comprising: 1 ) exposing said rodent to at least one candidate therapeutic agent; and 2) assessing the effect of the at least one therapeutic agent on the progression of neurodegenerative disease in said rodent.

A third aspect of the invention is a method of screening for a biomarker for the early detection, diagnosis and/or prognosis of neurodegenerative disease in the rodent of the first aspect of the invention, comprising: 1 ) isolating from said rodent and a second reference rodent at least one sample; 2) determining the differential expression and/or level of a potential biomarker between said rodent and the second reference rodent, wherein if there is a difference in expression and/or level of the potential biomarker, then the potential biomarker is taken as a biomarker for the early detection, diagnosis and/or prognosis of neurodegenerative disease.

It is also part of the invention a nucleotide sequence or gene construct comprising the human tyrosinase gene under the control of or operably linked to a promoter of a tyrosine hydroxylase gene. The invention also

encompasses vectors comprising the above mentioned nucleotide sequence, as well as host cells in the rodent comprising said vectors or said nucleotide sequence

It is also a part of the invention a set of separate/independent nucleotide sequences, comprising: (a) a sequence that comprises the human tyrosinase gene operably linked to an inducible promoter, and (b) a nucleotide sequence comprising a neuron specific expression promoter (in particular a rodent tyrosine hydroxylase promoter) linked to a sequence the expression of which promotes expression of the adjacent gene, in this case human tyrosinase (a) by interaction with the inducible promoter.

All these gene constructs comprise sequences with the human tyrosinase gene sequence and also sequences promoting the expression of the human tyrosinase gene in neurons. By promoting it is to be understood that the expession is neuron specific or only achieved in some brain tissue types.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG.1 . Human-like NM production in the SNpc of AAV-hTyr-injected rats, (a) Left panel, adult Sprague-Dawley rats received a single unilateral stereotaxic injection with 2 μΙ of AAV2/1 -CMV-hTyr into the region immediately above the right SNpc. Right panel, by 4 weeks post-injection, 80% of ipsilateral DA SNpc neurons (TH, top right) were transduced with the tyrosinase gene (Tyr, middle right). The image in the bottom right panel is a composite of the top and middle images, (b) Similar to humans, accumulation of NM brown pigment was observed macroscopically in unstained ipsilateral SNpc of AAV-hTyr- injected rats at 4 weeks post-injection (darkened area, dashed line). No NM accumulation was observed in contralateral (control) hemisphere (identified by a puncture), as rodents normally lack this brain pigment, (c) Ipsilateral SNpc pigmentation in AAV-hTyr-injected rats corresponds to the presence of intracellular NM within SNpcA/TA neurons, as shown microscopically in a 30 μΐη-thick unstained section from the same brain. Whereas tyrosinase was widely expressed throughout the injected hemisphere, accumulation of NM was restricted to the SNpc and VTA, which mimics the pattern of NM distribution in humans, (d) By 8 weeks post-AAV-hTyr injection, ipsilateral SNpc neurons from injected rats exhibited intracellular NM levels similar to that of elder human brains, with NM occupying most of their cellular cytoplasm (Hematoxilin&Eosin staining), (e) As in humans, pigmented SNpc from AAV- hTyr-injected rodents can be detected (arrow) by NM-sensitive high-resolution T1 -weighted magnetic resonance imaging (MRI; data obtained ex-vivo from a mouse brain at 16 weeks post-AAV-hTyr injection), (f) Electron micrographs of NM from human LC of a 81 -year-old subject (top) and an AAV-hTyr-injected rat SNpc at 16 weeks post-injection (bottom), both exhibiting strikingly similar NM-containing autophagic-related vacuoles displaying NM (electron dense matrix) and characteristic lipid droplets (asterisks). Scale bar, 1 μηη (human), 500 nm (rat).

FIG.2. PD-like neuropathological features in AAV-hTyr-injected rats. By 16 weeks post-AAV-hTyr injection, ipsilateral rat SNpc (bottom panels) exhibit several neuropathological features typical of PD brains (top panels), including extracellular (E NM) and perivascular NM (P NM), neuronophagia (NP), Marinesco bodies (MB, arrows) and pale bodies (PB, LB precursors, arrowheads). Scale bars, 10 μηη (except b, 20 μηη).

FIG.3. SNpc dopaminergic neuronal dysfunction/degeneration in AAV-hTyr- injected rats. (Top panel - Forepaw akinesia). The x-axis represents weeks after the AW-hTyr injection and the y-axis represents the Contralateral forepaw contacts - CFC (% of total contacts). Progressive reduction of contralateral forelimb use in AAV-Tyr- injected rats, starting at 4 weeks post- AAV-hTyr injection and reaching statistical significance by 8 weeks and onwards, as assessed with the cylinder test. Animals were also tested prior to AAV-Tyr injections (basal) to exclude putative spontaneous asymmetry.

(Bottom panel - Number of SNpc Tyrosine Hydroxilase (TH)-positive neurons) The x-axis represents weeks after the AW-hTyr injection and the y-axis represents the number of SNpc TH-positive neurons (ipsilateral to the injected side) - SNpc TH+ . Stereological quantification of the number of TH-positive neurons in the SNpc of rats injected with the AAV-Tyr at different times post- injection (from 2 weeks to 2 years), indicating a progressive loss of TH- positive neurons. A/=6-8 animals per group. ^*p<0.05, compared to control animals (basal); #p<0.05, compared to 8 weeks and 12 months post-AAV- hTyr injection (one way ANOVA, Student-Newmann-Keuls post-hoc test).

FIG.4. NM accumulation in catecholaminergic brain structures of 12 month-old Tg-TH-hTyr mice. (Left and middle panels) Nissl-stained cryosections showing intracellular accumulation of NM (dark brown) in the cytoplasm of neurons from the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and locus coeruleus (LC) regions. (Right panel) Digoxigenin-based in situ hybridization for human tyrosinase showing expression of hTyr in SNpc, VTA and LC neurons of Tg-TH-hTyr mice.

DETAILED DESCRIPTION OF THE INVENTION

The human tyrosinase gene is a copper-containing enzyme that catalyzes the production of melanin and other pigments from tyrosine oxydation. In humans, it is encoded by the TYR gene. Its protein product is found in the Uniprot database with the entry P14679 (last update on 1 1 .06.2014). The sequence of the human tyrosinase gene used in the present invention is (SEQ ID NO 1 ):

GACCTTGTGAGGACTAGAGGAAGAATGCTCCTGGCTGTTTTGTACTGCCTGCTGTGG AGTTTCCAGACCTCCGCTGGCCATTTCCCTAGAGCCTGTGTCTCCTCTAAGAACCTG ATGGAGAAGGAATGCTGTCCACCGTGGAGCGGGGACAGGAGTCCCTGTGGCCAGCTT TCAGGCAGAGGTTCCTGTCAGAATATCCTTCTGTCCAATGCACCACTTGGGCCTCAA TTTCCCTTCACAGGGGTGGATGACCGGGAGTCGTGGCCTTCCGTCTTTTATAATAGG ACCTGCCAGTGCTCTGGCAACTTCATGGGATTCAACTGTGGAAACTGCAAGTTTGGC TTTTGGGGACCAAACTGCACAGAGAGACGACTCTTGGTGAGAAGAAACATCTTCGAT TTGAGTGCCCCAGAGAAGGACAAATTTTTTGCCTACCTCACTTTAGCAAAGCATACC ATCAGCTCAGACTATGTCATCCCCATAGGGACCTATGGCCAAATGAAAAATGGATCA ACACCCATGTTTAACGACATCAATATTTATGACCTCTTTGTCTGGATGCATTATTAT GTGTCAATGGATGCACTGCTTGGGGGATCTGAAATCTGGAGAGACATTGATTTTGCC CATGAAGCACCAGCTTTTCTGCCTTGGCATAGACTCTTCTTGTTGCGGTGGGAACAA

GAAATCCAGAAGCTGACAGGAGATGAAAACTTCACTATTCCATATTGGGACTGGCGG GATGCAGAAAAGTGTGACATTTGCACAGATGAGTACATGGGAGGTCAGCACCCCACA AATCCTAACTTACTCAGCCCAGCATCATTCTTCTCCTCTTGGCAGATTGTCTGTAGC CGATTGGAGGAGTACAACAGCCATCAGTCTTTATGCAATGGAACGCCCGAGGGACCT TTACGGCGTAATCCTGGAAACCATGACAAATCCAGAACCCCAAGGCTCCCCTCTTCA

GCTGATGTAGAATTTTGCCTGAGTTTGACCCAATATGAATCTGGTTCCATGGATAAA GCTGCCAATTTCAGCTTTAGAAATACACTGGAAGGATTTGCTAGTCCACTTACTGGG ATAGCGGATGCCTCTCAAAGCAGCATGCACAATGCCTTGCACATCTATATGAATGGA ACAATGTCCCAGGTACAGGGATCTGCCAACGATCCTATCTTCCTTCTTCACCATGCA TTTGTTGACAGTATTTTTGAGCAGTGGCTCCGAAGGCACCGTCCTCTTCAAGAAGTT TATCCAGAAGCCAATGCACCCATTGGACATAACCGGGAATCCTACATGGTTCCTTTT ATACCACTGTACAGAAATGGTGATTTCTTTATTTCATCCAAAGATCTGGGCTATGAC TATAGCTATCTACAAGATTCAGACCCAGACTCTTTTCAAGACTACATTAAGTCCTAT TTGGAACAAGCGAGTCGGATCTGGTCATGGCTCCTTGGGGCGGCGATGGTAGGGGCC GTCCTCACTGCCCTGCTGGCAGGGCTTGTGAGCTTGCTGTGTCGTCACAAGAGAAAG CAGCTTCCTGAAGAAAAGCAGCCACTCCTCATGGAGAAAGAGGATTACCACAGCTTG TATCAGAGCCATTTATAAAAGGCTTAGGCAATAGAGTAGGGCCAAAAAGCCTGACCT CACTCTAACTCAAAGTAATGTCCAGGTTCCCAGAGAATATCTGCTGGTATTTTTCTG TAAAGACCATTTGCAAAATTGTAACCTAATACAAAGTGTAGCCTTCTTCCAACTCAG GTAGAACACACCTGTCTTTGTCTTGCTGTTTTCACTCAGCCCTTTTAACATTTTCCC CTAAGCCC

For the sake of understanding, the following definitions are included.

The term "expresses" when referring to a gene, is to be understood here as the process by which the information stored in the DNA of the gene is used for the synthesis of a functional gene product (usually a protein, but can also sometimes be an RNA). In the case of the human tyrosinase, the expression of the human tyrosinase gene is to be understood as the production of the human tyrosinase enzyme. The term "progressively accumulates" as used herein refers to the

accumulation of NM along the lifetime of the rodent animal in such a way that the rodent of young age features a certain amount of NM in its dopaminergic neurons, the adults feature higher amounts than the young rodents, and the older animals are the ones featuring the highest amounts of NM, mimcking the progression of NM accumulation in humans, that is, the older the animal is, the higher the amount of intracellular NM.

The term "transduction" as used herein refers to the process whereby heterologous (foreign) DNA is introduced into a host cell via a viral vector. The aim of this process is for the piece of DNA to be integrated in the genome of the host cell so that the protein or RNA that the foreign DNA codes for is stably expressed in the host cell. In the case of the present invention, the transduction of the human tyrosinase gene is carried out with the aim of having the host cell (and especially the neurons of the SNPC of a rodent) stably expressing the h-tyrosinase gene.

The term "viral vector" as used herein refers to a virus that is used as a delivery device to insert heterologous genetic material into a host cell

(transduction).

The term "adeno-associated virus (AVV)" as used herein refers to a viral vector that infects both dividing and quiescent primate (and human) cells. Because they seem to lack any pathogenic effects, and usually integrate in the same place of the genome (the AWS1 site, in chromosome 19), this viral vectors can safely be used to transduce foreign DNA into human cells in gene therapy applications. The "AW-Tyr" of the present invention is an adeno- associated viral vector that bears the human tyrosinase gene. In particular, the full sequence of the transduction vector used in the present invention

(AW2/1 -CMV-TYR) is SEQ ID NO 2 ( the coding sequence for the human tyrosinase gene is highlighted in bold):

CAGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGG GCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCC AACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCT ACGTAGCCATGCTCTAGACATGGCTCGACAGATCTCAATATTGGCCATTAGCCATAT TATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGT ATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTT GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGAC CGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGC CAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACG GTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTT

GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT ACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCA TTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTC GTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTG GGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTAT

TGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGT CTCGAACTTAAGCTGCAGTGACTCTCTTAAGGTAGCCTTGCAGAAGTTGGTCGTGAG GCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAAC TGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTA CTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCT CTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCctcgacctcgagacgc gtgataaacttaagcttggtaccgagctcggatccactagtccagtgtggtggaatt ctgcagatatccagcacagtggcggccgctcgaccGACCTTGTGAGGACTAGAGGAA GAATGCTCCTGGCTGTTTTGTACTGCCTGCTGTGGAGTTTCCAGACCTCCGCTGGCC ATTTCCCTAGAGCCTGTGTCTCCTCTAAGAACCTGATGGAGAAGGAATGCTGTCCAC CGTGGAGCGGGGACAGGAGTCCCTGTGGCCAGCTTTCAGGCAGAGGTTCCTGTCAGA ATATCCTTCTGTCCAATGCACCACTTGGGCCTCAATTTCCCTTCACAGGGGTGGATG ACCGGGAGTCGTGGCCTTCCGTCTTTTATAATAGGACCTGCCAGTGCTCTGGCAACT TCATGGGATTCAACTGTGGAAACTGCAAGTTTGGCTTTTGGGGACCAAACTGCACAG AGAGACGACTCTTGGTGAGAAGAAACATCTTCGATTTGAGTGCCCCAGAGAAGGACA AATTTTTTGCCTACCTCACTTTAGCAAAGCATACCATCAGCTCAGACTATGTCATCC CCATAGGGACCTATGGCCAAATGAAAAATGGATCAACACCCATGTTTAACGACATCA ATATTTATGACCTCTTTGTCTGGATGCATTATTATGTGTCAATGGATGCACTGCTTG GGGGATCTGAAATCTGGAGAGACATTGATTTTGCCCATGAAGCACCAGCTTTTCTGC CTTGGCATAGACTCTTCTTGTTGCGGTGGGAACAAGAAATCCAGAAGCTGACAGGAG ATGAAAACTTCACTATTCCATATTGGGACTGGCGGGATGCAGAAAAGTGTGACATTT GCACAGATGAGTACATGGGAGGTCAGCACCCCACAAATCCTAACTTACTCAGCCCAG CATCATTCTTCTCCTCTTGGCAGATTGTCTGTAGCCGATTGGAGGAGTACAACAGCC ATCAGTCTTTATGCAATGGAACGCCCGAGGGACCTTTACGGCGTAATCCTGGAAACC ATGACAAATCCAGAACCCCAAGGCTCCCCTCTTCAGCTGATGTAGAATTTTGCCTGA GTTTGACCCAATATGAATCTGGTTCCATGGATAAAGCTGCCAATTTCAGCTTTAGAA ATACACTGGAAGGATTTGCTAGTCCACTTACTGGGATAGCGGATGCCTCTCAAAGCA GCATGCACAATGCCTTGCACATCTATATGAATGGAACAATGTCCCAGGTACAGGGAT CTGCCAACGATCCTATCTTCCTTCTTCACCATGCATTTGTTGACAGTATTTTTGAGC AGTGGCTCCGAAGGCACCGTCCTCTTCAAGAAGTTTATCCAGAAGCCAATGCACCCA TTGGACATAACCGGGAATCCTACATGGTTCCTTTTATACCACTGTACAGAAATGGTG ATTTCTTTATTTCATCCAAAGATCTGGGCTATGACTATAGCTATCTACAAGATTCAG

ACCCAGACTCTTTTCAAGACTACATTAAGTCCTATTTGGAACAAGCGAGTCGGATCT GGTCATGGCTCCTTGGGGCGGCGATGGTAGGGGCCGTCCTCACTGCCCTGCTGGCAG GGCTTGTGAGCTTGCTGTGTCGTCACAAGAGAAAGCAGCTTCCTGAAGAAAAGCAGC CACTCCTCATGGAGAAAGAGGATTACCACAGCTTGTATCAGAGCCATTTATAAAAGG CTTAGGCAATAGAGTAGGGCCAAAAAGCCTGACCTCACTCTAACTCAAAGTAATGTC

CAGGTTCCCAGAGAATATCTGCTGGTATTTTTCTGTAAAGACCATTTGCAAAATTGT AACCTAATACAAAGTGTAGCCTTCTTCCAACTCAGGTAGAACACACCTGTCTTTGTC TTGCTGTTTTCACTCAGCCCTTTTAACATTTTCCCCTAAGCCCctagagggcccgtt tatcggatcccggccggcggccgcTTCCCTTTAGTGAGGGTTAATGCTTCGAGCAGA CATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAA ATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTG CAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGA GATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCCGATA AGGGACTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACA AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGA GCGAGCGAGCGCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCC CAACAGTTGCGCAGCCTGAATGGCGAATGGAATTCCAGACGATTGAGCGTCAAAATG TAGGTATTTCCATGAGCGTTTTTCCGTTGCAATGGCTGGCGGTAATATTGTTCTGGA TATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTAC TAATCAAAGAAGTATTGCGACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACT CGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGTTCCTGTC TAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAG CACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAA GCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAG CGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGAT AGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGT TCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGA TTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACG CGAATTTTAACAAAATATTAACGTCTACAATTTAAATATTTGCTTATACAATCTTCC TGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTT TACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGA TAGCCTTTGTAGAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGC TAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCC GTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTC

TAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGG TCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAA TTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTGGAATCGCCTGATG CGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTC AGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCC

GCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTG ACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCG AGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATG GTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTG GTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCT GCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGA CCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGAT CGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTA GCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTT CTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAG CGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATC GTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATC GCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCA TATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAG ATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGA GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGC GTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATA CCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAG CGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGG AGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGG

GAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGA GCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAA

GCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAA TG

The term "a gene is operably linked to the promoter" as used herein refers to a functional linkage between two sequences, a first sequence which

corresponds to a promoter, and a second sequence, which in the invention at hand can be the hTyr gene (the DNA coding for human tyrosinase) or the gene coding for the tetracycline transactivator (tTA). The promoter sequence regulates transcription of the DNA corresponding to the second sequence. Generally, operably linked means that the two nucleic acid sequences being linked are contiguous and, when necessary, in the same reading frame.

The term "screening for therapeutic agents" as used herein refers to the use of the rodent that forms part of the invention for testing organic molecules, peptides, proteins, DNA, antisense oligonucleotide, small interfering RNA, therapeutic vaccines or any cell-based or radiation-based therapy for the treatment of any pathology related to the accumulation of NM, and especially PD.

The expression "screening for a biomarker" relates to the use of the rodent that forms part of the invention for determining whether a potential biomarker between said rodent and a second reference rodent, is differentially expressed and/or has different levels. If this is the case, then the potential biomarker is taken as a biomarker for the early detection, diagnosis and/or prognosis of neurodegenerative disease. The term "reference rodent" as used herein, refers to a rodent that is taken for comparison with the rodent of the invention (i.e. the rodent that expresses the human tyrosinase gene in at least the dopaminergic neurons of the substantia nigra pars compacta). The reference rodent can be a wild type rodent (that does not express the human tyrosinase gene) or a rodent of the invention which expresses human tyrosinase, but which is taken at a different developmental stage to the first rodent. The comparison allows defining which biomarkers (metabolites, proteins, etc.) express differentially between both rodents, and can therefore be used in the detection, diagnosis and/or prognosis of neurodegenerative disease.

The term "constitutive" as used herein, refers to a gene that is transcribed (and optionally also translated) continually and does not depend on the presence of an external stimulus in order to give its protein product. The term "inducible" as used herein, refers to a gene that is transcribed (and optionally also translated) only when there is a certain stimulus present in the environment. Thus, an inducible gene is not transcribed continually, as opposed to a "constitutive" gene. The stimulus can be, for instance, the presence of an effector molecule.

The term "Tet-Off" refers to a gene control system where the controlled genes can be activated (expressed) in the absence of tetracycline or one of its analogues (such as doxycycline - dox). In a Tet-Off system, the tetracycline transactivator (tTA) binds to Tet operators (TetO) in the absence of

tetracycline thereby activating transcription of the genes under the control of the described operators. In most Tet-Off systems, several repeats of such TetO sequences are placed upstream of a promoter. The coupling of several TetO sequences to the promoter is called a tetracycline response element (TRE), because it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter.

The term "tTA" as used herein, refers to a tetracycline transactivator protein of a Tet-Off system, able to bind TetO sequences. In a Tet-Off system, expression of TRE-controlled genes can be repressed by tetracycline and its derivatives, i.e., they bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of TRE-controlled genes.

The term "therapeutic agent" as used herein, refers to any organic small molecule, peptide, protein, DNA, antisense oligonucleotide, small interfering RNA,vaccine, radiation or combinations thereof that could be used to cure, slow down the progression of, or alleviate a neurodegenerative disease. In this particular case, it would modulate the accumulation of NM in order to prevent the initiation of PD or to slow down or halt its progression.

The term "transgenic rodent" as used herein refers to a rodent whose genome has been altered by in vitro manipulation, with the aim of inserting or deleting certain genetic material with the aim of driving a phenotypic change. In the present invention, the rodent genome is altered at the embryonic stage so that the animal expresses the human tyrosinase gene. This is typically achieved by pronuclear injection of an expression cassette into the male pronucleus of purified zygotes and reimplantation of the injected zygotes into pseudo- pregnant mouse females. The transgene randomly integrates into the mouse genome and its expression will therefore depend on the insertion position. In the present invention, the DNA fragment for microinjection in mice to generate the constitutive transgenic mice is (SEQ ID NO 3):

AGCTTGGCAAGAGCTGATTTTAAAAGGTCCTGGAGAACACAGGCCAGCTCCAGTGTC AGGCGCCCTCATTCCAACTCCAAAGTAATCGCCTGGCAAACAAGCACTGTCTAAATA TCACAGAGTGTGGGTCAGGCGCACCGCAGCCTGGAAATGTCAGTGGCGGGGGCCTCC TCCCAGCTCCAGGATTAGCCCAGGCACCTGAGCCACATGCATTTGCCTTCAGCCTGG CTCTAGCCCCAAGAACCCGAAGAGGCCTCAGGTGTGTTCTCACTGCCAGATCGTGGG ATTGGGTCCCTGGGGAAAGGTGTTGCCCGTGGGGTCAGACTGAGCCATAAGCGGCTG TAGCCTTTGGAATGACATACTTCCAAGTTCTCACTAGGAACCCGGAGTAGGGGCAGC CCCGTGGAGAAAGCCTGGCTGTCCCGGGCCCTCACTGCGAGATTACTCACCCTTGTG TCCTCCCTTGTTCTCTCACTTCTACAAAACCTGACCACTTGCTCAAATGGCAGTGAG AAGCTGCTGGGTGGGAACTGTTGCTACCATCTGCATGGGCTCTGGGGATGAGGTGGC CACAGCAGCAGCTGTCATGCTTGTTAGACACAAGGTTCCTTCACCCCATAATGGGGC TGGTGACATTGTCCTAAACAGAAGGGCTTTGCCAAGTAGAAGGATTATTGCTGGGTA AATAAGAGCCCTAAAGGTGGAGTTTTTGTCTTCCAGGAACCTTCTTTAAATCACTAG GAACTGCCCTTAGGTCAACTGTCTCGGATAAGTGCATCTAGTTTTAGATCAGTCCAA ACAGCCTCTATCAGTTCAAAGACCCCTACATAAAGTCTGTTGCTCTTCCACCATGCC CCCTCCACCTCTGAGATAGCCTTGGCAGTTTAATCTCCGATAAACCTTTCTTTCAAC TCACGGCATGTTCGTGTTTGGTGGGATCACTGTGGCCTCACAGTAGAGGATGTCCAG AGCTGTGTTTGGCTCTGTCCCTGGACGTTCCTCTCCCTTGTATTCGTTTTGTTTTGT TTTGTTTTGTTTTTTGTTTTTAATCAACCCAACACAAACTAGAGTCTTCTTGAAAGA GGGAACCTTAATTGATAAAATGCCATCTCCAGGCCTAGGAAGATGACTCAACAGTTA AGAGCACTGGCCACTTTTCCACAGGTCTTGGGTTTGATTCCCAGTCCCCACATGGTG GCTCACAACCAGTTCTAGTGGATCCAACTCCCTCTTCTGGCTTCTGCGGACACCAGA CATACAATTGGTATGTGGACACATGTAGGCAAGACATTCACATACATTGAAAACAAA

AAGCCTGTTGAGAGAAAATATCTCCATCAGATTTACCTAGAAGCAAATCTGTATGCA TTTTCTTGCTTAATGGTTGGTATGAGAGGGCCCAGCCCACTGTGGGTGGTAACCATA GGCTGGTGATCCTGGGGTGCATAGAAAGGCAAGTTGGGAGAGGCACTTTGCAATGTC CCTTCATAGTCTCAGCTTAAGTCCCTGCCTCCAGGCTCCCACCCTGACTTTTCCCAG TGATGGACAGTGACCAGGACATGTAAGCCAAATAAACTCTCTCCTTCCCAAGTGACT

TTTGGTCCTGGTGTTTATTACAGCAACAGAAATCAAGCTAGGACACTGCTTTTCTTT TTACTTCATATACTTCGATCATGTGTGTTTGTATGTGATTAGCAGTATGGAAGCCAG AGGACAGACTAGAATATTGGTTCTTTGATGCATTGGGAGAATTTCCTTCCTGGGGGC TAGGAGGAGCATGGAGGAGCAGAGACTGGACAACACTTTGGCCTACCAATTGTAGCC CTTCCCTAAGACATCAAGGCCAACTGCAATCAGGGCAGACCATCATACAGCATGTGG GGAGGAAGGATTGAGTACTTCAATGAGGGTCCCATGACTCTATCCCCTCCATCCATG CTGGGCGGTCATCAGTCAACAAGCCAAGCTGAGAAATCCTCTGCAGGCTTAACTCAA GCGAAGTCATGAAGTCAAGATATTGGTTGTTTGGCTTGGAGAATAGTGCTATGTAAC AGCCGAGTCCACCTTGCCTTGAGACCATATCCCACACTGGCCTGGAGCTCGCCAGGT AGGCTACACTGCTTTTAGCTGAGTCCTAGGGATCCTCCTGCCTCTGCCTCCTCACTG CTGGGATTAGAAACATGCACCACCACACCTGTCTACATGTCTTCTAGGAATCAGACT AGGCCCATATGCTGTGTAAAAAGAACTCTGTGGACTGAGTTACCTCTGTACACTACA GAGCAAGTATTTAACTGGGGGCTGGCTTACAATTTCAGAGGCTTAGTCCATTACAAT AAGGGCAGGGAGCATAGCAGTGTGCAGAATCTGAGAGCTACATCCTGATCTGGAGAG AGAAAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGACAGA CAGACAGACACTGAACCTGGCATGGACTTTTAAAACCCCAATAACATGCCCACTCCA GCCAGGCCACACCCACTCTGACAAGGCCACAGTTCCTAACACTTCTCAAATAGTGCC ACTCCTCAGTGACTACGCATTCAAACACAGCAGCCACACTAGGTAACTATGAGGCCG CAAAGCCACGGGATGGGAGCCCTCTCCACCTGGAGAAGCCTACCCTAATGACAGAGG TGATAGGGTTATTTCTCCTGAGTTCCCCAGACTGGACCCCAAACACATTAAAGTCTG CTCTAAACCAAGCCAAGCTGCCCCTGGGCCTCCCCTGCCAATGTCCTCCACACATGC AGAAGCTACACCTCCTTTGGACATCTCTTCAGTTCAGGGGGACAAAGCTCCAGAGTC TGCTGCTGTCCCATCTCCTGTCCCTGCCCATAGGCATCCTCCATAGGCAGGGCCCTA GAAGTTTTTCTACTGTGGCTTGGCACATGTGAGGCAAGTGAGACTGTAAGACTATCC CTGCTAAATGCTGGGGCCACTGTAACTCTGCTAACACAGCCCCGGGCCATGCTGTGG GGAGCAGAAGCCACCTTGTAGATGAGAAGAGAGGACTATACACCACCACAAACTGCA AGAAAGAAAGCACTCCCATTCGGAGCAGGAACCACTGCCCCTCCCTACCACAGGATG GGAGGGGGACCACCACCCACAGGCTCCAGTGCTTCCGTGGGCCTGGGGTGGGTAGGC AGCCGTCTTGCAATTAGACATGATTGCTGTCACATCACCACCGTGGTCCTTGTGAGT GACTAATGGGAACTGAATGTCACTCTTATTGCTTTTACTGGCTAATTTGTCAGACTG TCAAGTACTCTGGGAGGTGGCACGGAGGGTATTACGTGACTCACTGTGCACAGGTTG GGAGAGGCAAGGGCCTTAGGGGGGACAGAGGGAAGCAGGAATACCAGAGAGGACCAG

GGGTCTGACCCATGACATTTTCGGAAAGTCTGAGGGGCATGGGAGCTGAGGTTACCC CACCTGTCACATATCGATGGCTCTGAGTTCCTCTAGGCCCTTCTGTGTACACACATG CTTATATGAAGGCTTGGGGATATAGAACACATGCCCTGCCCTGCATGTGCATATAGA CACAATGGCATGTGCATAAGGCATGTACATGTGCATGCACATGTGCACATACATGTA GTGTATTGCATTCAGACCAAATGAGGTTAGATATAGATCATGGGTCCCTGGCCTGGA

CACTCATGTAGCCAGCCACTAGTGAATTTGGCATGACACCTCTCTATCATCTCAGGG TTGGTGTGTAGGTTGCCAGGTTGGTGTGGGTTGCCAGGTGACATAGGTGTAGCTGTG AGCCTCTGCTAATGTGAGGCTAGTGTTGAGCGCTTCAGAGAAGCCTGGTTCTAATGC TGACTTCCTGTTGGCTAGGTCTAACAGAGGGACAGGCTCTCTAACCTGGACTCACTC ACTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGTCTCT CTGTCTCTCTGTCTCTCTGTCTCTTGCCTTAGTTGCACTCTGGCTATTGGTCCTCCC TCCTGGTGACCACCCTGACCCAGGCCCCTTGGCCTTTGAGAACGCTACACACCTAAC TAAAGTCATCTCCTACTAGAAGCTCAGAGAAGGGCCAGCCATCTCTCAGCAGATGGA GAGTTCTTTAGAAGCACCAGAGCTCCTGAGAGCTGCCACGAGTCACTCCCAGGAATC ACCGTGAAGCTAGGGAAATCAAGGCACAAGGTACGAGAGGCTGAGCCACGCTGGGGC TCTCTTACCTGTAGACAGTGAATCTCCAGGAATAAGGCAGGCAGAGAAGCAGTGCTG GTCTTACTAGCCAAAGGTGGAAGTGGTTTAGGGGAGCGAGGAGGGTGGGCTGCCTGG AAGATACTCTGGACCTGAAGGCTTGGTGGAAGTGGGGAGCCAGGGTAGAGGAAGCAG ACAGGTGGGGGCTCCTTGGGGAGAGAGGGCCTAGAGCCTAGGTTGAGACGGGGGCTG ATCAGCAGCTCTTGCCTCTGGTCTGACTTTCAACTGCCCAATTATCCCTAAGTGTCT CCTATCGACCGACCGCAGCTGGTGCTGTCCGGTACGTGTTCTGAGTCTACAGCCCCG AGGCTGCTGCTCCATCCGATGGCCTCGTTAGGGCTAATTGCTCTGGCATTTGGGCCT GATGAGGACAAGAATGGCTGGACCCTACCAGATGTCAGGGAGCTTCATTCTCTTTCC AGTCAGATTGGTGAGCTGTGTCAGGGACAAAGGGCCCTGCGGTCTCACTGAGCATCA GGTCCCTGCTTTACAACCCATCTCAGTCTCCTGGTGGCAGGGAAAAGCAACCTCACT CTCCCTTCTGTCTTGGGCTCACCCTGCACAGCACTTAGGCAACTGGTCATGATGTCT TGACCAACTTAAATCCAAGAGAAGCTCCCACTGCCTTTCAGGGTATGTCAGTTGATG ATTTCATGGACCTGCATCTCTGTGAGCAGGAACACTGACCCCAGAGCCCATCTGGTG ACAGGATTCTTGAGACCCTGGCATCATGTGTTTCTATAGAAAGCGATTTGGGTGGAC TTGTCAAAGTGGGTGGGGTATGTGTTTGCGTGTGTGTGTGTGGGGGTCTATAGGTAT AGCATGTTTGTGAATGCATGAACATATTATAATACATGTATGCCTATGCGATTCTGT TTGTATGTGTATATCTGTGTTCGCATATGTTTGTGTGTGCATGTGGCTGCTCCTATG TATACTTGTGTATGTATTTGTGTGTCTGTGTCTCCATGTATAAGCACTGTGAATCTG TATATCTGTATGTGCATACGTTTGTGTGTCTATGTGTCTATGTATATACCATTGTGT ATATACATTTGTGTGTGCCAATCTCTCTCTGAATGTATCTGTATATGGCCATATGTG TACATGTATGTCTTTATACATGTGTGTATGCCTATGTATGTCCTCATATATGTGTGG TCATGTGTCTCTGTGTGTACATGCTTCTGCGTGTCTATGGTAGAAGGTCATGATGCT

GGTTGAAAGTGGCCTTTGAGCCTAGGTTGTAGAGGCATGTGTTCCAAAATGCACATA GGTTTCCATAGTGCCCAAAGCTATGGGGTAGATCTGGGGTCCTTGCTCCAAGACCAA GGATCAGGACACCCCCTAGTCCTCTGCCTCCTTAGTTCCATGATGTCCTGGGCAGGG AGGTCCATATCAGCAGGGTGCATACAGGATAGCACAGCAGCCTCCACACCAGCAACA CTGATGCCAGGGAGTGGGTGAGCTGTCATTATGGACAATTAAGCACCGTGCTGAGAG

AGAAAGGCCCCGTGTGCTCCCCGTCTCTGCAGGCCTAATCGCTGTGGTGACAGAGGA GGGTAATTATCAGGACGGCTGTGAATGGCACAGGCATTACAGGGGTGTAAAGGGCTC AGGAAGGAGGGAGGGCCTGCTATGCCCCCAGCACGCATCTGAGCCCAGAGCAGGCAA TGGCCCACTGCACTGGCCTTGCGCTTCAGATCAGCCCCCTTTCTTAACTGCTAGGGG ATGCTTCCCAATCACTCCTCTAGGCTCTGCGGCTTGGCTTCCAGCCTGTACGCTGTC CAGAGAGCCTTCAAAGCCTCACTTCGACCAACCAGAAGCCTCTCGTCAGCCCTGCCC TGACCTCGTGTGCCTCTTCAAAGTGAGATTTAGCAGCTGCAGCTGGGGGTGCCTGAG CCCCACTCATGCTGTCTTCCTTGAAGACAGAAGTGTTGGGAGCTGAGGACCTGGGCC GTATGATCCAGAGAAGTAGTGTGCTTCTGGGTCTCAGCTCTCCCTTCTGCAGAATGG GTCTGTCTGAAATGGAAAGGCAGGTGCCCCTCTGCAGGGCCTAATCTGAGTCGCCAT GAGTGGTTAAAAGATCCAGCTTGTCTGTGGGTGAGCTTTGAGAGGAGGCAGGGACCT CTAGCATGGAACAGGGCTGAGTCCTGGAAAGCTGACCAAGGGCAGGCCTAAGAGGCC TCTTGGGATTCTTCTCATCAAAAAGGGCATGGGACACAGCTAAAGCGTCCAGGGCTC CTCTGTGCCCACAGATGCCTTAGATCTTGGCACAATGTAGTCAGCCAGCTCCGTGTG TGTGTGTGTGTGTTTGCATGTATCTCACAGACAGTGCACAATGGCCTGGATGTGAAC AGAGGCAAGAGTCTGGGCCAGCAGTTGTCTCCCAGGAGGGTCCAAAGACATCGTATT TTCAAGTTTAGGCCAGGTGCTCACTTGGGTGAGCTCAGACACAGACAAAGGTCTGGA GAGCACACATTCCCCACCCCCACCCAGCTCCTATGCAAGCACCTCCAGCCGAGACAA GAAAACGAATTAAAAAGCAATATTTGTGTCAGCGTAAGACATTTGCCGAAAGGTTAA ATCCACACTCGTGGTGCTGCACAGCAGCCCCCTGTGCAGGATTTGTTAGGCACAGCT CCCTCCTACCCCGTGCCACCTGAGCAAATGCCAGGCTGGGTGGGCTGGAACCAGCCT GGGCTTGCCTCACCCTGGAATCCCCAGCACCCTCCAAAGGAGGACCCTGGGAGTGGG CATAGACGCCCTTCAGGTGTGGGCAACAGCCCCCAGTCCTCAGGATGAAAGGCTAAG GTGCAGCCAGCTCTGCCTTCACGGTGGGAATGTCTCTATGTGAGCCCTTTCTGGGCT GTGAAGAACGCTCTGAGAAGGGTCCTGGGACCCTGGATAGGCCAGAGCTGTGCTGGG CATGTAGAGACAGGAGTGGGCTAAAGCAGCAAAGGCACTGACCAAGGAAGAGTTCAG AGAGGAGCGTGGAATATGGGGAGGGGTTCATAGTAAGAGAGAGCAGGCAGTGGAGAG TAAATAGTCACTGAGCCGGGGTTTATGGGGTTTGTAGGAGCTTACTCAGAGAAAGTA GATGAGAGATGCCATGCCAGTCTGAGTATCACAGAGCCCCAGGCTCTCCTGGGAACG GAACTGTGAGGGCCAGAAGGTCAGCAAGGGAGGTTAGGGAGAGTTCCTTTTGTACTG ACTCAGCATTTATCCTGCTCCCAGGGGGCAATGGGGGCCAGTGAGGGATGCAGAGCA AGGCAGTGATGTGGCAGGCAGTTCCTGTTGTGAAAGAGCTGGGAAGGGAGCGGGCTG

GGCCTGGTACGTACAGCAGGCCATTTCTGAGGGTCCGAGTGCTGTCTAGGAGGTGCA GTGAGACTTCAGTGATCAGCCAGAACAGAAGCTAAGCGGGGTGGGGACTGCGAGTTC AGGCTTCTGGGTCTTGCAAATATCCAGAATGCTAAATCCTCAGAACCCCAGGGTGGC CATTTTCAGAGTGGGTTTTGTCCTTTGGGCACTTGTGCAGACTCCAATATCCAGAGG GATAAGGATGGTACTCTTCAGTACCCTTAGTGAGAGGACACTTTTCTCTGAAGGGCT

TGAATGTGCCGAGCCATTACCTGAAGGAAGGAAATGACTCCAGGGACATAGGATGGG CCCAGCACAACTCACCTGCTACAGAGAAAGGTCCCCTCCCTGGTCTCCTTAGAGATC CTGTTTCCCTGGCTGAGGAAGCTAGGGTGGATCTTTGTGTAAGTGGGTGTGGATGCT AACTGGAAAACAAAAGGTCACTTACTGTTAGACCTCGGGGTACCATGGAAGAGATGA TCACTGAGTGTGCCCTTACATGGGGACCAGCTGAGAATGGGGCTACCACTAGCTCGA GACCATGATACAGGGAATAAGTGTGCATTTGGGGGTAGGGAGTGGCTCAGAATACTC TTAACCAAAGCAGAGGTTTGCTCCCACAGGAAGGTGAGGTCAGAAGGCCTTAGGGAG CTGCCAGGGGCTAGGGTTGGCACCATCTCCCAGGCTGTGTCTTTAAGGAGATGATAA TCAGAGGGATAGAACCTTGCAAAAGTGGGCCAGTCTTGGGAATACTATAGAGGAATA GCCTTCTGGAACATTCTGTGTCTCATAGGACCTGCCTGGGGATCCAGCCCCAGTGCC AGCACATATACCGACTGGGGCAGTGAATAGATAGTACACTTTGTTACATGGGCTGGG GGGAACATGGCCCATGTCCTGGAGGGGACTTTATGACAGACATCCAAAAATCCAGTG AGAGGGCTTCTAGATTTGTCTCCAAAGGTTATAGTTCTAACATGAGCCCTTAGGAAA TCCAGCATGGTTCTCCCTGTGTGCCCTGGTTTGGTTAGAGAGCTCTAGCGGTCTCCT GTCCCACAGAATACCAGCCAGCCCCTGCCCTACGTCGTGCCTCGGGCTGAGGGTGAT TCAGAGGCAGGTGCCTGTGACAGTGGATGCAATTAGATCTAATGGGACGGAGGCCTT TCTCGTCGCCCTCGCTCCATGCCCACCCCCGCCTCCCTCAGGCACAGCAGGCGTGGA GAGGATGCGCAGGAGGTAGGAGGTGGGGGACCCAGAGGGGCTTTGACGTCAGCCTGG CCTTTAAAGAGGGCGCCTGCCTGGCGAGGGCTGTGGAGACAGAACTCGGGACCACGa attctgcagatatccagcacagtggcggccgctcgaccGACCTTGTGAGGACTAGAG GAAGAATGCTCCTGGCTGTTTTGTACTGCCTGCTGTGGAGTTTCCAGACCTCCGCTG GCCATTTCCCTAGAGCCTGTGTCTCCTCTAAGAACCTGATGGAGAAGGAATGCTGTC CACCGTGGAGCGGGGACAGGAGTCCCTGTGGCCAGCTTTCAGGCAGAGGTTCCTGTC AGAATATCCTTCTGTCCAATGCACCACTTGGGCCTCAATTTCCCTTCACAGGGGTGG ATGACCGGGAGTCGTGGCCTTCCGTCTTTTATAATAGGACCTGCCAGTGCTCTGGCA ACTTCATGGGATTCAACTGTGGAAACTGCAAGTTTGGCTTTTGGGGACCAAACTGCA CAGAGAGACGACTCTTGGTGAGAAGAAACATCTTCGATTTGAGTGCCCCAGAGAAGG ACAAATTTTTTGCCTACCTCACTTTAGCAAAGCATACCATCAGCTCAGACTATGTCA TCCCCATAGGGACCTATGGCCAAATGAAAAATGGATCAACACCCATGTTTAACGACA TCAATATTTATGACCTCTTTGTCTGGATGCATTATTATGTGTCAATGGATGCACTGC TTGGGGGATCTGAAATCTGGAGAGACATTGATTTTGCCCATGAAGCACCAGCTTTTC TGCCTTGGCATAGACTCTTCTTGTTGCGGTGGGAACAAGAAATCCAGAAGCTGACAG

GAGATGAAAACTTCACTATTCCATATTGGGACTGGCGGGATGCAGAAAAGTGTGACA TTTGCACAGATGAGTACATGGGAGGTCAGCACCCCACAAATCCTAACTTACTCAGCC CAGCATCATTCTTCTCCTCTTGGCAGATTGTCTGTAGCCGATTGGAGGAGTACAACA GCCATCAGTCTTTATGCAATGGAACGCCCGAGGGACCTTTACGGCGTAATCCTGGAA ACCATGACAAATCCAGAACCCCAAGGCTCCCCTCTTCAGCTGATGTAGAATTTTGCC

TGAGTTTGACCCAATATGAATCTGGTTCCATGGATAAAGCTGCCAATTTCAGCTTTA GAAATACACTGGAAGGATTTGCTAGTCCACTTACTGGGATAGCGGATGCCTCTCAAA GCAGCATGCACAATGCCTTGCACATCTATATGAATGGAACAATGTCCCAGGTACAGG GATCTGCCAACGATCCTATCTTCCTTCTTCACCATGCATTTGTTGACAGTATTTTTG AGCAGTGGCTCCGAAGGCACCGTCCTCTTCAAGAAGTTTATCCAGAAGCCAATGCAC CCATTGGACATAACCGGGAATCCTACATGGTTCCTTTTATACCACTGTACAGAAATG GTGATTTCTTTATTTCATCCAAAGATCTGGGCTATGACTATAGCTATCTACAAGATT CAGACCCAGACTCTTTTCAAGACTACATTAAGTCCTATTTGGAACAAGCGAGTCGGA TCTGGTCATGGCTCCTTGGGGCGGCGATGGTAGGGGCCGTCCTCACTGCCCTGCTGG CAGGGCTTGTGAGCTTGCTGTGTCGTCACAAGAGAAAGCAGCTTCCTGAAGAAAAGC AGCCACTCCTCATGGAGAAAGAGGATTACCACAGCTTGTATCAGAGCCATTTATAAA AGGCTTAGGCAATAGAGTAGGGCCAAAAAGCCTGACCTCACTCTAACTCAAAGTAAT GTCCAGGTTCCCAGAGAATATCTGCTGGTATTTTTCTGTAAAGACCATTTGCAAAAT TGTAACCTAATACAAAGTGTAGCCTTCTTCCAACTCAGGTAGAACACACCTGTCTTT GTCTTGCTGTTTTCACTCAGCCCTTTTAACATTTTCCCCTAAGCCCctagagggccc gtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtt tgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcc taataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctgggg ggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgct ggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagg gggtatcccccacg

As mentioned above, a first aspect of the invention is a rodent that comprises the human tyrosinase gene, the gene being expressed in at least the dopaminergic neurons of the substantia nigra pars compacta. In a particular embodiment of the first aspect of the invention, the rodent progressively accumulates NM in said neurons along its lifetime.

In a particular embodiment of the first aspect of the invention, the human tyrosinase gene is the human tyrosinase gene of SEQ ID NO 1 .

In another particular embodiment of the first aspect of the invention, the human tyrosinase gene has been transduced with a viral vector. Thus, the rodent is a rodent transduced with a viral vector comprising the human tyrosinase gene.

The former embodiment can be reformulated into a method of obtaining the rodent of the first aspect of the invention, comprising transducing the human tyrosinase gene via a viral vector which is injected into the brain of the rodent. In another particular embodiment of the first aspect of the invention, optionally in combination with any of the embodiments above or below, the viral vector is an adeno-associated viral vector. In another particular embodiment of the first aspect of the invention, the adeno-associated viral vector, is the adeno-associated viral vector of SEQ ID NO 2.

In another particular embodiment of the first aspect of the invention, the rodent is a transgenic rodent.

In another particular embodiment of the first aspect of the invention, in the transgenic rodent, the expression of the human tyrosinase gene is

constitutive.

In another particular embodiment of the first aspect of the invention, in the transgenic rodent, the human tyrosinase gene is operably linked to the promoter of a rodent tyrosine hydroxylase.

In another particular embodiment of the first aspect of the invention, the transgenic rodent departs from an embryo that has been injected with the construct comprising SEQ ID NO 3. In particular, the construct is the construct consisting of SEQ ID NO 3.

The former embodiment can be reformulated into a method of obtaining the rodent of the first aspect of the invention, by using the following protocol: a) designing a gene construct comprising SEQ ID NO 3; b) microinjecting the gene construct in the masculine pronucleus of a rodent fertilized ovus; c) culturing the obtained ovus; d) transplanting the latter into the oviduct of a female pseudopregnant rodent; c) selecting, among the offspring, those animals which contain the cDNA coding for the transgene. The former embodiment can be reformulated into a method of obtaining the rodent of the first aspect of the invention, by using the following protocol: a) designing a gene construct consisting of SEQ ID NO 3; b) microinjecting the gene construct in the masculine pronucleus of a rodent fertilized ovus; c) culturing the obtained ovus; d) transplanting the latter into the oviduct of a female pseudopregnant rodent; c) selecting, among the offspring, those animals which contain the cDNA coding for the transgene. In another particular embodiment of the first aspect of the invention, the expression of the human tyrosinase gene is inducible.

In another particular embodiment of the first aspect, the transgenic rodent is an inducible double transgenic rodent derived from mating a first parent rodent comprising the gene coding for the tTA transactivator operably linked to the promoter of a rodent tyrosine hydroxylase, and a second parent comprising the gene coding for the human tyrosinase operably linked to a promoter under the control of the target sequence for the tTA transactivator.

The former embodiment can be reformulated into a method of obtaining the rodent of the first aspect of the invention, by using the following protocol: a) generating a first parent rodent comprising the gene coding for the tTA transactivator operably linked to the promoter of a rodent tyrosine

hydroxylase; b) generating a second parent rodent comprising the gene coding for the human tyrosinase operably linked to a promoter under the control of the target sequence for the tTA transactivator; c) mating both parent rodents to obtain the double transgenic rodent. In another particular embodiment of the first aspect, optionally in combination with any of the embodiments above or below, the rodent is selected from the group consisting of rat and mouse.

As also mentioned above, a second aspect of the invention is a method of screening for therapeutic agents that prevent, delay or halt the development of neurodegenerative disease in the rodent of the first aspect of the invention, comprising: 1 ) exposing said rodent to at least one candidate therapeutic agent; and 2) assessing the effect of the at least one therapeutic agent on the progression of neurodegenerative disease in said rodent.

In a particular embodiment of the second aspect of the invention, the neurodegenerative disease is associated with neuromelanin accumulation.

In another particular embodiment of the second aspect of the invention, the neurodegenerative disease is associated with a-synuclein accumulation.

In another particular embodiment of the second aspect of the invention, the neurodegenerative disease is PD.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" and its variations encompasses the term "consisting of. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

EXAMPLES

A) Material and Methods Generation of vectors

The pcDNA4_TYR construct containing the coding sequence of the human tyrosinase gene (TYR, 2.082 bp) (Hasegawa et al. "Increased dopamine and its metabolites in SH-SY5Y neuroblastoma cells that express tyrosinase" J. Neurochem. 2003, vol. 87, pp. 470-475) was used as a parent construct to generate the adeno-associated viral (AAV) vector and the transgenic mice. To generate the AAV2/1 -CMV-Tyr (SEQ ID NO 2), the human TYR was subcloned from the pcDNA4_TYR construct into an AAV of serotype 2/1 , commonly used to transfect distinct areas of the brain. The AAV encodes an internal ribosome entry site (IRES) under the control of cytomegalovirus immediate-early (CMV-IE) promoter and contains a SV40 polyadenylation signal, flanked by two inverted terminal repeats (ITRs). CMV-null AAVs of

SEQ ID NO 4 were used as control (its sequence found below). AAVs were generated and purified by the Viral Vector Production Unit (UPV) of the Center of Animal Biotechnology and Gene Therapy (CBATEG) at the Universitat Autonoma de Barcelona (Barcelona, Spain). Titers obtained were in the range of 1 .2x10¹² to 2.5x10¹³ vector genomes per ml (vg/ml).

SEQ ID NO 4:

CAGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGG GCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCC AACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCT ACGTAGCCATGCTCTAGACATGGCTCGACAGATCTCAATATTGGCCATTAGCCATAT TATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGT ATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTT GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGAC CGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGC CAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACG GTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT ACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCA TTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTC GTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTG GGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTAT TGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGT CTCGAACTTAAGCTGCAGTGACTCTCTTAAGGTAGCCTTGCAGAAGTTGGTCGTGAG GCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAAC TGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTA CTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCT CTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGccagcacatgactgaca ctcggaagcacagaacaaagggtaggacggtgcccatgggtcaggctgtagccacgc caccctttccaccctgtcctagccagaggcagcaatgtgctccatacagatcctcct aacacacccacactgtcggtccccagcacgcagatgcccgacagccccttaggcaaa tggcttagctgactgccccaccacacgccgtcgccatgcagtccagtggggagtcgg aggcagcctccttcctgcctctcctcggcctgcacgtgtccccccaccaggcagaga cccttctacaccccgggtgtctgcggtcacatcgcggtggggcatgcagctgttggc cttcgagcatgttttgttttccttggccagtgtctccagagaaacgcacgtgggttt gtgtccagcggtccatctctgcaacagttgttcctttgggattggatgctaggaggt cacgggagaggtgtccatccaaagcagtgtctgtgtcacacactgtccccacacaca gggccacctctgcacagactcccccgactcgattctgggcacagagctcagtgacct tccagagactgccacgaaccggtgatgcctccacgcttgagacatcctgaccgcagg gcccaaggcgcactggctcagggggtgacagtgaggggtctgcaaacagactgctga tgctcaacccggccgctgccgagctgtgtgacttgggcacgtcacttaacctctctc ggcctctgtctcctcccggggataagagtagtagcacctgcttcccggggctgtgag gatccagtgggacgtataggaactagcgaggcaccggcagttgggtcagagctactg ttgtcacttcacaaggcattttcttcaacagcaagtcggaaatctcatgagcctaag gcagaatccacctgtggcctctggttacaacccacaggactgaaaatccttccagcc acagcaactggtgaatttcctggtcaattgccacaagtcatgagctgaaccccactt gagtttcagttcaggcagaactctagagacgactagggcaagctagacagcgactgc agagccttttgttgcagcgtgagcagtcctcagctgttgacatcactggggagcaaa cgaggaccaggagcggtgaaaggacagtgtctgctgcagattgtcgtagcacccaag gaacactccagaaagcctcctaagcagtaacaagtgtggcaaggtgtagcccagcca acagtggcatctgcgaggcgtcccctccttcctcccactaccccgtataccctggga cctgtgcactgaaggactcattctaaaggctgtgcccctgcagccgccagcctcact cactggctgcctgtgccagctagagatttctttcctctgaggctggctgagaggacc actccagtttcctggcccatccagcaaagaagatacacatcatgcacgtgtaaaatg aggaaccggtttattgaacagcttaaggagagcaaaaatagtggctttagctacatt ttttacacactgagcaggaaagtctaaaccatcccgttcccctgtaccccaaagaga acagggcttgctggaggccagtgccaagggcggagtcgtgctcgcagcagacttgaa ttaaccccatgtaggccggcgagcagttgcccgcgtgaaaacaccaccctcttctcc tggctgagaagatcaaagctctttttttaccctcttttcagcaaaggacctatttgt tttcaggcaggaggatgttaaacttgcagcctctgacacacggtggaacctgcagtg cttggagaaacggcacgcacacgtgaaaacatcatgcctactccaaagccttcttgt tgctggcaggagggaagcttgagactttcccacgcatagtcgtgacccgcgtggccg tttctgctctcagcaacattctctagtgttccggcttcaagcagcgcttgtcaggtt tgaagctagcctcgacctcgagacgcgtgatatcggatcccggccggcggccgcTTC CCTTTAGTGAGGGTTAATGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGG ACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGC TATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTG CATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTA

AAACCTCTACAAATGTGGTAAAATCCGATAAGGGACTAGAGCATGGCTACGTAGATA AGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCA CTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGAC GCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCCAGCTGGCGTAAT AGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAA

TGGAATTCCAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCGT TGCAATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAG TTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCGACAACGGT TAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACAC TTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTT TAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCTCGTCAAAGCAAC CATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCA GCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTT CCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTG ATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGG AGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTA TCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAA AAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTCTA CAATTTAAATATTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGATTATCAA CCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTG TTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAAT AGCTACCCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGATGG TGATTTGACTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTACCTACACATTACTC AGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAAT AAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTT AGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTA TGATTTATTGGATGTTGGAATCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGC GGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAG TTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTG CTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAG AGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTA TTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTT CGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG TATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGC CTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAG

TTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAG AGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGT GGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACAC TATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGAT GGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCG

GCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCAC AACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCC ATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGC AAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGG ATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGG TTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAG GCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAG CATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTT CATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAA ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAA GGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTA ATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGC ACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAG CTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGC GGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTC CTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCT GTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACG ACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCG CCTCTCCCCGCGCGTTGGCCGATTCATTAATG

To generate tissue-specific transgenic rodents, the 9 Kb upstream rat tyrosine hydroxylase (TH) promoter was cloned into the pcDNA4_TYR construct using the restriction sites Hindi 11 and EcoRI. The TH_pcDNA4_TYR construct was then amplified in XL1 -Blue bacteria and isolated using the NucleoBond Extra Midi Kit (Macherey-Nagel). 10 μg of construct DNA were sequentially digested with the enzymes Hindi 11 and BciVI and run overnight in a 1 % agarose gel electrophoresis to isolate the 1 1 Kb DNA fragment to be injected. This expression cassette, a lineal DNA fragment containing the rat tyrosine hydroxylase (TH) promoter, the human TYR gene and a polyadenylation site, was purified by electroelution.

To obtain inducible rodents two different constructs are required: (i) parent construct pcDNA4_TYR containing the TYR gene and a tetracycline- responsive element (tet-O); (ii) a tetracycline transactivator (tTA) construct (pcDNA6/TR_TH) containing the rat TH promoter. The expression cassettes are excised by enzymatic restriction and purified by agarose electrophoresis followed by electroelution. Sterotaxic injection of the AAV-Tyr vector.

Overexpression of human tyrosinase (Tyr) was induced by unilateral stereotaxic injection of 2 μΙ of the AAV2/1 -CMV-Tyr (1 ,70E+13 vg/ml) into the substantia nigra (SN) of adult Sprague-Dawley rats [stereotaxic coordinates: - 5.2 (AP), 2 (L) and -7.6 (DV)] or 1 μΙ in C57BL/6 mice [stereotaxic coordinates: -2.9 (AP), 1 .3 (L) and -4.2 (DV)]. Control rats/mice were injected with an empty viral vector (AAV2/1 -CMV-null, 2,48E+13 vg/ml) or with vehicle (sham). At different times post-injection (2, 4, 8, 16 weeks and 6, 12, 18 y 24 months) animals were subjected to behavioral tests and processed for further characterization.

Generation of transgenic rodents Constitutive transgenic mice and rats.

Transgenic embryos were generated by microinjecting the expression cassette into zygote pronuclei that were afterwards transferred into

pseudopregnant recipient females. Transgenic lines were generated using mice of a mixed genetic background (C57BL6/J and SJLF2), or rats of the Sprague Dawley strain following the same protocol. Founders were identified by Southern blotting of tail DNA using transgene specific probes. Each individual line was then backcrossed with mice or rats from the same strain and the expression level of the litters assessed by real time PCR or

quantitative Western blotting. 3 lines (high, medium and low) were used to determine expression specificity in different tissues and for further

characterization.

Inducible transgenic mice and rats

The expression cassettes corresponding to the tTa and the tet-O elements are microinjected in the pronuclei of Sprague Dawley rats or hybrid mice to produce two different lines of transgenic animals: (i) one transactivator line expressing the activator (tTA) under the control of the tissue-specific TH promoter and (ii) a responder line containing the tetracycline responder element (tet-O) that controls the expression of the downstream human tyrosinase gene. Conditional double-transgenics are generated by crossbreeding tet-O founder males with tTA founder females (or the other way round), and the litters backcrossed to obtain a stable line. Under normal tetracycline or doxycycline free conditions, the double-transgenic progeny express tyrosinase with a tissue specific pattern determined by the TH promoter (including dopaminergic neurons from the substantia nigra pars compacta and noradrenergic neurons from the locus coeruleus). In the presence of doxycycline, added to food or drinking water, the system is activated resulting in the suppression of the transgene over-expression.

All founder rodents are generated at the Center of Animal Biotechnology and Gene Therapy (CBATEG) at the Universitat Autonoma de Barcelona

(Barcelona, Spain). Rodents are housed in specific pathogen-free conditions, with ad libitum access to water and food, in the Animal Facility of the Vail d'Hebron Research Institute (VHIR).

All experimental procedures were approved by the Animal Experimentation Ethics Committee (35/14 CEEA) at the VHIR (Barcelona, Spain).

B) Results

Adult Sprague-Dawley rats received a single unilateral stereotaxic injection with 2 μΙ of either the adeno-associated viral (AAV) vector containing the human tyrosinase gene under the control of the CMV promoter (AAV2/1 -CMV- Tyr), the empty viral vector (AAV2/1 -CMV-null) or the vehicle (sham) into the right substantia nigra pars compacta (SNpc) (FIG. 1 a, Left panel). Delivery of AAV2/1 -CMV-Tyr produced widespread expression of cytoplasmic tyrosinase in dopaminergic TH-immunoreactive neurons in ipsilateral substantia nigra

(SN) and ventral tegmental area (VTA) (FIG. 1 a, Right panel). Similar to humans, accumulation of NM-like brown pigment could be observed macroscopically in the injected hemisphere 4 weeks post-injection in absence of any kind of staining (FIG. 1 b). No NM accumulation was observed in the contralateral (control) hemisphere, since rodents normally lack this brain pigment. Whereas tyrosinase was widely expressed throughout the injected hemisphere, accumulation of NM was restricted to the SNpc and ventral tegmental area (VTA). This accumulation pattern mimics NM distribution in human brain and indicates that NM synthesis requires enzymes or other components from dopamine metabolism. Ipsilateral SNpc pigmentation in

AAV-hTyr-injected rats corresponds to the presence of intracellular NM within SNpc/VTA neurons (FIG. 1 c). By 8 weeks post-AAV-hTyr injection, intracellular NM reached levels similar to that of elder human brains, with NM occupying most of the cellular cytoplasm of SNpc neurons (FIG. 1 d). As in humans, pigmented SNpc from AAV-hTyr-injected rodents can be detected by NM-sensitive high-resolution T1 -weighted magnetic resonance imaging (FIG. 1 e). Ultrastructurally, NM from AAV-hTyr-injected rats is indistinguishable from human NM (FIG. 1f). By 16 weeks post-AAV-hTyr injection, NM-containing SNpc neurons from AAV-hTyr-injected rats exhibit several neuropathological features typical of PD brains, such as extracellular and perivascular NM, neuronophagia, Marinesco bodies and pale bodies (FIG. 2)

To determine whether progressive NM accumulation in SNpc neurons could lead to nigro-striatal dopaminergic dysfunction/neurodegeneration, AAV-Tyr- injected rats were subjected to motor behavioral tests, such as the cylinder test, and neuropathological analyses, by stereological cell counts of dopaminergic TH-positive SNpc neurons. The cylinder test is designed to evaluate forelimb use asymmetry by quantifying the number of forelimb contacts against the wall within an open-top, clear plastic cylinder while rearing. The number of contacts with the forelimb ipsilateral or contralateral to the injection site is expressed as a percentage of total contacts.

Animals were tested both prior to AAV-Tyr injections, to exclude putative spontaneous asymmetry, and regularly every 4 weeks after AAV-Tyr inoculations. Rats were then euthanized at different time-points from 4 weeks to 16 weeks post-injection and their brains removed and processed for histochemical examination. Progressive reduction of contralateral forelimb use was observed in AAV-Tyr-injected rats starting at 4 weeks post-injection and reaching statistical significance by 8 weeks and onwards (FIG. 3, Top panel). Behavioral deficits in AAV-Tyr-injected rats were associated to progressive loss of TH-positive neurons in the ipsilateral SNpc starting at 8 weeks post- injection, compared to non-injected rats (FIG. 3, Bottom panel).

Progressive NM accumulation can also be seen in catecholaminergic brain structures, including the SNpc, VTA and LC, of 12 month-old Tg-TH-hTyr mice (FIG. 4).

As can be seen from the results discussed, it has been clearly proven that the rodents expressing the human tyrosinase gene in the SNpc not only show remarkable neurodegeneration at the cellular level (FIG.3, Bottom panel), but also early-on motor dysfunctions typical of PD (FIG.2, Top panel).

Thus, it is concluded that the rodent of the present invention can be employed as a faithful model of the progression of PD in humans, and therefore as a promising in vivo screening tool for the search of novel biomarkers and therapies to control and combat the disease.

REFERENCES CITED IN THE APPLICATION

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Tief, K., et.al. "Tyrosinase, the key enzyme in melanin synthesis, is expressed in murine brain" Eur. J. Biochem. 1996, vol. 241 , pp. 12-16

Xu, Y., et.al. "Tyrosinase mRNA is expressed in human substantia nigra" Brain Res. Mol. Brain Res. 1997, vol. 45, pp.159-162

Greggio E., et. al. "Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease" J. of Neurochem. 2005, vol. 93, pp. 246-256

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Claims

1 . A rodent that comprises the human tyrosinase gene, the gene being expressed in at least the dopaminergic neurons of the substantia nigra pars compacta.

2. A rodent according to claim 1 , wherein the rodent progressively

accumulates neuromelanine (NM) in said neurons along its lifetime.

3. The rodent according to claims 1 -2, wherein the human tyrosinase gene is the human tyrosinase gene of SEQ ID NO 1 .

4. The rodent according to any one of claims 1 -3, wherein the human tyrosinase gene has been transduced with a viral vector.

5. The rodent according to claim 4, wherein the viral vector is an adeno- associated viral vector.

6. The rodent according to claim 5, wherein the adeno-associated viral vector is the adeno-associated viral vector of SEQ ID NO 2.

7. The rodent according to any one of claims 1 -3, wherein the rodent is a transgenic rodent.

8. The transgenic rodent according to claim 7, wherein the expression of the human tyrosinase gene is constitutive.

9. The transgenic rodent according to claim 8, wherein the human tyrosinase gene is operably linked to the promoter of a tyrosine hydroxylase.

10. The transgenic rodent according to any one of claims 8-9, departing from an embryo that has been injected with the construct comprising SEQ ID NO 3

1 1 . The transgenic rodent according to claim 7, wherein the expression of the human tyrosinase gene is inducible.

12. The transgenic rodent according to claim 1 1 , which is an inducible double transgenic rodent derived from mating a first parent rodent comprising the gene coding for the tTA transactivator operably linked to the promoter of a rodent tyrosine hydroxylase, and a second parent comprising the gene coding for the human tyrosinase operably linked to a promoter under the control of the target sequence for the tTA transactivator.

13. A method of screening for therapeutic agents that prevent, delay or halt the development of neurodegenerative disease in the rodent according to any one of claims 1 -12, comprising:

1 ) exposing said rodent to at least one candidate therapeutic agent; and

2) assessing the effect of the at least one therapeutic agent on the progression of neurodegenerative disease in said rodent.

14. The method of screening for therapeutic agents according to claim 13, wherein the neurodegenerative disease is associated with neuromelanin accumulation.

15. The method of screening for therapeutic agents according to any one of claims 13-14, wherein the neurodegenerative disease is associated with a- synuclein accumulation.

16. A method of screening for a biomarker for the early detection, diagnosis and/or prognosis of neurodegenerative disease in the rodent according to any one of claims 1 -12, comprising:

1 ) isolating from said rodent and a second reference rodent at least one sample;

2) determining the differential expression and/or level of a potential biomarker between said rodent and the second reference rodent, wherein if there is a difference in expression and/or level of the potential biomarker, then the potential biomarker is taken as a biomarker for the early detection, diagnosis and/or prognosis of neurodegenerative disease.