METABOLIC MARKERS SPECIFIC TO NEURODEGENERATIVE DISEASES SUCH AS PARKINSON'S DISEASE AND USE THEREOF IN DIAGNOSIS
20230393152 · 2023-12-07
Assignee
- Universite Grenoble Alpes (Saint Martin d'Heres, FR)
- INSERM - Institut National de la Santé et de la Recherche Médicale (Paris, FR)
Inventors
- Sabrina BOULET (Voreppe, FR)
- Florence FAUVELLE (Grenoble, FR)
- David MALLET (Le Fontanil-Cornillon, FR)
Cpc classification
G01N2800/2835
PHYSICS
International classification
Abstract
An identification of novel combinations of specific metabolites for neurodegenerative diseases for use as biomarkers in the early diagnosis of neurodegenerative diseases.
Claims
1. An in vitro method for early diagnosis of a neurodegenerative disease from a biological sample of a patient comprising: a) the in vitro measurement in the biological sample of the expression levels of at least the following 6 metabolites: acetoacetate, betaine, β-hydroxybutyrate (BHB), creatine, pyruvate, and valine; b) comparison of the expression levels of the metabolites measured in step a) with those of a reference biological sample or with reference values; where a difference in the expression levels of the metabolites measured in step a) compared to those of the reference biological sample or to reference values is the indication that the patient is suffering from the neurodegenerative disease.
2. The method according to claim 1, wherein step a) further comprises the in vitro measurement of the expression level of at least one metabolite selected from the group consisting of alanine, lactate, DMSO2, glycine, serine, threonine, myoinositol, and leucine.
3. The method according to claim 1, wherein step a) comprises the measurement of the expression levels of the following metabolites: acetoacetate, betaine, β-hydroxybutyrate (BHB), creatine, pyruvate, and valine.
4. The method according to claim 1, wherein the neurodegenerative disease is Parkinson's disease.
5. The in vitro method for the early diagnosis of Parkinson's disease from a biological sample from a patient comprising: a) the in vitro measurement in the biological sample of the expression levels of the following metabolites: acetoacetate, betaine, β-hydroxybutyrate (BHB), creatine, pyruvate, and valine; b) calculation of Logit(P1) or Logit(P2) logistic regressions, respectively, according to the following formulas: −45.60+0.28EBHB−4.54EPyruvate−11.23EValine+7.05EAcetoacetate+2.85ECreatine−7.79EBetaine; or −42.78+0.16EBHB−3.31EPyruvate−12.01EValine+7.06EAcetoacetate+3.14ECreatine−8.27EBetaine, where EBHB, EPyruvate, EValine, EAcetoacetate, ECreatine and EBetaine correspond respectively to the measured expression levels of β-hydroxybutyrate (BHB), pyruvate, valine, acetoacetate, creatine and betaine, in the biological sample; and where a calculated value of Logit(P1) or Logit(P2) greater than 0.316 a.u. is an indication that the patient has Parkinson's disease; where a calculated value of Logit(P1) or Logit(P2) lower than 0.279 a.u. is an indication that the patient does not have Parkinson's disease; where a calculated value of Logit(P1) less than 0.316 a.u. is an indication that the patient does not have Parkinson's disease; where a calculated value of Logit(P2) greater than 0.279 a.u. is an indication that the patient has Parkinson's disease.
6. The method according to claim 4, wherein the patient's biological sample comes from a patient in the prodromal or de novo stage of Parkinson's disease.
7. The method according to claim 1, wherein the biological sample is a serum sample.
8. A biomarker for early diagnosis of a neurodegenerative disease comprising the combination of at least six of the following metabolites: acetoacetate, betaine, β-hydroxybutyrate (BHB), creatine, pyruvate, and valine.
9. The biomarker according to claim 7 further comprising the combination of at least one metabolite selected from the group consisting of alanine, lactate, DMSO2, glycine, serine, threonine, myo-inositol, and leucine.
10. The biomarker according to claim 8, comprising the combination of the following six metabolites: acetoacetate, betaine, β-hydroxybutyrate (BHB), creatine, pyruvate, and valine.
11. An in vitro use of a biomarker according to claim 8, as a biomarker of a neurodegenerative disease.
12. The use according to claim 11, wherein the neurodegenerative disease is Parkinson's disease.
Description
BRIEF DESCRIPTION OF FIGURES
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[0057] Mean±SEM, one-way ANOVA, followed by Tukey post-hoc test and correction for multiple comparisons. *: p≤0.05; **: p≤0.01; ***: p≤0.001.
[0058] a.u.: arbitrary units.
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EXAMPLES
Example 1: Materials and Methods
[0067] Scheme
[0068]
[0069] The second cohort of rats (
[0070] Monkeys (n=8) received intramuscular injections of MPTP (0.3 to 0.5 mg/kg) every 4-5 days for 3 weeks [77]. The repeated administration of low doses of MPTP has been used to mimic a moderate stage, with slow disease development, and the onset of moderate dopaminergic injury. Serum samples were collected at the start of the experiment and after the monkeys reached a stable parkinsonian state (
[0071] All serum samples were analyzed by .sup.1H NMR at 950 MHz and brain samples were subjected to HRMAS .sup.1H NMR at 500 MHz.
[0072] Animals
[0073] Rats: the experiments were carried out on adult male Sprague-Dawley rats (Janvier, Le Genest-Saint-Isle, France), weighing approximately 300 g (7 weeks) at the start of the experiment, They were housed under standard laboratory and ethical conditions with an inverted light-dark cycle (12 p.m./light-dark cycle, with lights on at 7 p.m.) and with food and water available ad libitum.
[0074] Monkeys: the experiments were carried out on adult male Macaca fascicularis, weighing between 5 and 8 kg, aged 3 to 5 years and housed under standard conditions (light-dark cycle of 12:12 hours; 23° C.; 50% humidity).
[0075] The used protocols complied with the European Union Animal Protection Act 2010 and French Directive 2010/63, and were approved by the French National Ethics Committee (2013/113) No. 004 and by the local ethics committee C2EA84 and CELYNE C2EA.
[0076] Surgery
[0077] Bilateral injection of 6-OHDA: As described previously [18, 23], the rats received a subcutaneous injection of desipramine (15 mg/kg) 30 minutes before the injection of 6-OHDA in order to protect the noradrenergic neurons. All animals were then anesthetized by intraperitoneal injection of ketamine-xylazine (100-7 mg/kg), and placed in a stereotaxic frame (Kopf Instruments). The stereotaxic coordinates of the injection sites were as follows, according to the stereotaxic atlas of Paxinos and Watson [78] and in relation to the bregma: incisor bar placed at −3.2 mm: anteroposterior (AP)=−5.4 mm/lateral (L)=+/−1.8 mm/dorsoventral (V)=−8.1 mm. The animals received a bilateral injection of 2.3 μl of 6-OHDA (3 mg/ml) or physiological saline (NaCl 0.9%; control/sham), at a flow rate of 0.5 μl/min. For animals injected with 6-OHDA and presenting with transient starvation 2-3 days after the operation, supplementation with a high-calorie liquid diet and appetizing foods was started for 1-2 weeks, and stopped 10 days before serum collection. Animals that did not recover were excluded.
[0078] Bilateral injection of α-synuclein: As previously described [19], rats were anesthetized with 2.5% isoflurane and placed in a stereotactic frame (Kopf Instruments) in order to receive two bilateral injections of AAV-hA53Tα-syn (1 μl-7.0×10.sup.12 vg/ml) or AAV-GFP (7.0×10.sup.12 vg/ml, control/sham animals) in the SNc (AP=−5.1 and −5.6 mm/L=+1-2.2 mm/V=8 mm from the bregma). After being awakened, the animals were returned to the animal facility.
[0079] Behavioral Assessment
[0080] 6-OHDA rats: The rats were subjected to 2 behavioral tests, 3 weeks after the operation, while the 6-OHDA lesion was stabilized [79].
[0081] Operant self-administration of sucrose (motivational component): Rats were trained to self-administer a 2.5% sucrose solution in operant chambers (Med Associates, St Albans, VT, USA) containing an active lever, reinforced, for which pressing causes the delivery of 0.2 ml of a sucrose solution associated with a low-intensity light stimulus, and an inactive, unreinforced lever, for which pressing causes neither delivery of sucrose nor light stimulus. This task was performed with a fixed ratio of 1, with a single press of the reinforced lever resulting in the delivery of a reward. Each session ended when 100 rewards were obtained, or at the end of the allotted time (1 hour). The number of obtained rewards was counted for each session by the MED-PC IV software.
[0082] Pitch readjustment test (motor component): The animals, held by the posterior third of their body, were moved over a length of 90 cm by a straight and regular movement from left to right, and vice versa, along a smooth surface table. The number of forelimb adjustments made during movement was counted [80]. The test was carried out three times, by two different experimenters, blind (ignorance of the nature of the experimental groups).
[0083] MPTP monkeys: The severity of the parkinsonian state was assessed using a rating scale taking into account classic motor symptoms (bradykinesia, rigidity, tremors, freezing, posture and position of the arms), spontaneous activities (arm movements, spontaneous eye movements and activity in the housing cage) and other activities (vocalization, triggered eye movements and feeding) [81].
[0084] Histology: Immunolabeling of TH and Quantification of Denervation in 6-OHDA Rats.
[0085] Brain harvest: The rats were sacrificed by decapitation 1 day after the last session of sucrose self-administration and 1 hour after the last injection of Pra or Veh (that is to say 6 weeks after the injection of 6-OHDA or saline solution). The brains were immediately frozen in liquid nitrogen and stored at −80° C. They were then sliced using a cryostat thermostated at −20° C. Sections of the regions of interest were taken to carry out both the immunostaining of TH and to carry out the metabolomic study. For .sup.1HHRMAS NMR, thick sections of DS and Nacc tissue were pooled in inserts, and kept at −80° C. until NMR analysis.
[0086] For TH immunostaining, 14 μm coronal sections of the striatal levels of interest were taken and stored at −20° C. Immunostaining was then performed as previously described [18,23]. Briefly, after fixation with 4% paraformaldehyde, striatal sections were incubated with an anti-TH antibody (mouse monoclonal MAB5280, Millipore, France, 1:2500) overnight at 4° C. Then, the sections were incubated with a biotinylated goat anti-mouse IgG antibody (BA-9200, Vector Laboratories, Burlingame, CA, USA; 1:500) and the immunoreactivity revealed by the avidin-peroxidase conjugate (Vectastain ABC Elite, Vector Laboratories Burlingame, CA, USA).
[0087] Quantification of the extent of striatal dopaminergic denervation was determined with the computerized image analysis system ICS FrameWork (Calopix, version 2.9.2, TRIBVN, Châtillon, France) coupled to an optical microscope (Nikon, Eclipse 80i). After drawing the masks of the three striatal levels of interest, the optical densities (OD) were measured for each striatal subregion (DS and Nacc). The ODs were expressed as percentages relative to the mean optical density obtained from the homologous regions of the operated control animals (intracerebral injection of physiological saline).
[0088] Progression of Parkinson's Disease (PD) Score in the 6-OHDA Model
[0089] In order to assess the progression of PD (PDP) in 6-OHDA rats, a scale called «PDP score», and combining 3 criteria, was developed: 1) criterion 1: assessment of neuropsychiatric symptoms (ie. behavior apathetic type), 2) criterion 2: assessment of motor symptoms (i.e. fine motor deficit), these two criteria also being used clinically in the scales allowing the diagnosis of PD, and 3) criterion 3: the level of dopaminergic denervation in the DS, available only after postmortem histological analysis in the patient. Each criterion was quantified for each animal, with values ranging from 0 to 4, as shown in
[0090] For the assessment of behavioral deficits in the self-administration and pitch readjustment tests (criteria 1 and 2, respectively), the performance of each animal was compared before and after the injection of 6-OHDA (or physiological saline (sham). A value of 0 for each of these two criteria corresponded to an absence of deficit, or to a deficit of less than 30%, i.e. the normal daily variability of the used tests. The following subcategories have been set as follows: 1=low deficit (30%-50%); 2=mean symptoms (50%-70%); 3=strong symptoms (70%-90%); 4=significant or total deficit (>90%).
[0091] Considering that motor symptoms appear after 70% loss of striatal dopaminergic neurons in patients with PD, the value of dopaminergic denervation in DS (criterion 3) was used to define the boundary between the prodromal phase and the clinical phase.
[0092] With regard to the PDP score obtained by adding the values of the 3 criteria, the animal mimicking PD was classified as: 1) asymptomatic rat with weak lesions but no behavioral symptoms (PDP score=1 to 2), 2) prodromal type animal, i.e. presenting only neuropsychiatric disorders and a small DS lesion (PDP score=3 to 7) and 3) clinical type animal, i.e. presenting neuropsychiatric disorders; motor symptoms and an extensive DS lesion (PDP score=8 at 12). Control (sham) animals were assigned a score of 0.
[0093] Given the low number of asymptomatic animals and the fact that they are of no clinical relevance, they were excluded from the study.
[0094] Human Cohorts
[0095] Blood samples from PD patients and corresponding comparison groups subjects were obtained through 1) the Parkinson's Disease Biomarker Program (PDBP) Consortium, supported by the National Institute of Neurological Disorders and Vascular Accidents National Institutes of Health (NIH-USA), and 2) Santa Lucia Foundation (Rome, Italy). All subjects (patients and comparison groups) gave written informed consent. Inclusion criteria for patients from the United States included recent diagnosis (<1 year) of PD according to the new Movement Disorders Society (MDS) criteria. For the Italian cohort, the diagnostic criteria were less restrictive and patients diagnosed for longer than those of the US cohort were included (0 to 3 years after diagnosis), In both cohorts, the patients had not received any antiparkinsonian treatment at the time of inclusion, did not have dementia or psychiatric disorders, or any other medical condition that could compromise the study. Inclusion criteria for comparison groups were: absence of neurological disease, absence of family history of movement disorders, and absence of specific medical conditions. The US cohort included 21 PD patients without antiparkinsonian therapy, 9 PD patients who received Pra treatment, and 30 age- and gender-matched comparison groups. The Italian cohort included 21 patients with PD and 23 age- and gender-matched comparison groups. All clinical and demographic information collected from the two human cohorts has been summarized in Table 1 below (data are represented by mean value±SEM, M: Male; F: Female; *2 patients of unknown sex),
TABLE-US-00001 TABLE 1 NIH Italy Control PD Control PD Age 61.4 ± 1.8 62.0 ± 2.1 57.26 ± 1.89 60.38 ± 2.00 Gender 16 M/14 F 9 M/12 F 9 M/14 F 14 M/7F*
[0096] NMR Experiments
[0097] HRMAS .sup.1H NMR of 6-OHDA rat brain samples: Just before HRMAS analysis, 10 μl of D.sub.2O was added to the inserts containing the brain biopsies, which were then sealed and placed in a 4 mm zirconium rotor.
[0098] Data acquisition: All HRMAS NMR spectra were acquired, as previously described [82], on a Bruker Advance Ill spectrometer (Billerica, USA) (IRMaGe, CEA Grenoble, France) at 500 MHz. The samples were rotated at 4000 Hz at the magic angle (54.7°) and the temperature was maintained at 4° C. for all experiments. One-dimensional spectra were acquired using a CPMG pulse sequence (TE=30 ms, 256 repetitions, 17 min). The residual water signal was pre-saturated for 1.7 s of relaxation.
[0099] Data processing: Quantification was performed with JMRUI software using quantification based on a quantum estimation procedure (QUEST) [83]. This procedure requires the use of a database of metabolites and a complete assignment of spectra. Nineteen metabolites were attributed and quantified: acetate, alanine, ascorbate, choline, creatine, gamma-aminobutyrate (GABA), glutamate, glutamine, glycine, glycerophosphocholine, glutathione, lactate, myo-inositol, N-acetylaspartate, phosphocholine, phosphocreatine, phosphoethanolamine, scyllo-inositol and taurine. The amplitude of the metabolite calculated by QUEST was normalized with respect to the total signal of the spectrum. CRLBs were calculated for each metabolite as an 2.5 estimate of the standard deviation of the fit.
[0100] .sup.1H NMR of Serum
[0101] Serum collection: For the rats, the blood was collected from the tail vein after 2 hours of fasting and under gaseous anesthesia with isoflurane (2%) and was stored in ice before being quickly centrifuged at 1600 g for 15 min at 4° C. The supernatant serum was collected and stored at −80° C. until the day of the NMR. The time before freezing never exceeded 30 min [84].
[0102] For monkeys, blood was collected from the saphenous vein after fasting for 12 hours, under anesthesia (atropine 0.05 mg/kg i.m. followed by Zoletil 15 mg/kg i.m.) and subjected to the same protocol as rat blood.
[0103] For humans, blood was collected after overnight fasting, stored at room temperature for 15-60 min before being centrifuged at 1200-1500 g at 4° C. for 10-15 min. The supernatant serum was then collected and stored at −80° C.
[0104] On the day of the NMR experiments, the samples were slowly thawed on ice and then quickly centrifuged to remove any cryo-precipitates. NMR tubes were filled with 60 μl of serum sample and 120 μl of 0.1 M phosphate buffered saline (PBS) in D.sub.2O (50% D.sub.2O, pH=7.4), and stored at 4° C. until acquisition by NMR.
[0105] Data acquisition: All animal and human serum samples were subjected to the same NMR protocol. .sup.1H NMR experiments were performed on a Bruker Advance III NMR spectrometer at 950 MHz (IBS, Grenoble, France) by using a 3 mm cryoprobe. Spectra were recorded using a CPMG pulse sequence. The residual water signal was pre-saturated for 2 seconds of relaxation.
[0106] Peak assignment was performed using the 2-dimensional homonuclear .sup.1H-.sup.1H (TOCSY) and heteronuclear .sup.1H-.sup.13C (HSQC) experiments, and databases. If necessary, addition of selected metabolites was used to clarify ambiguous identifications.
[0107] Data processing: The time signals were transformed by the Fourier method and phased manually with Topspin software from Bruker, version 3.6.2. Then, other pre-processing steps (baseline correction, alignment, segmentation) were performed using the online NMRProcFlow v1.2 software (http://nmrprocflow.org). Segments of ppm were made over the 0-10 ppm spectral range, excluding the residual water peak, macromolecule/lipid signals and other regions containing pollutions (at δ 5.0-4.7; δ 5.25-5.38; δ 2.2-2.0; δ 1.4-1.25; δ 1.15-1; δ 0.9-0.5). Each segment was normalized to the total sum of the segments.
[0108] Statistical Analysis
[0109] Multivariate analysis: Data from liquid NMR or HRMAS were imported into SIMCA v.14 (Malmo, Sweden) for multivariate statistics. Unsupervised principal component analysis (PCA) was first used for the overall visualization of the distribution of all samples, followed by OPLS to find the discriminating metabolites associated with a specific disease stage. For the latter, either a continuous variable (that is to say the PDP score), either a discrete variable (that is to say group membership) was used to label each sample, leading to OPLS-DA in the latter case. The total number of components was determined using the cross-validation procedure, which produces the R2Y and Q2 factors that indicate the goodness of fit and the predictability of the model, respectively. A model is considered robust and predictive when both factors are ≥0.5. The scores were plotted in 2D against the first two components of the OPLS models, while the weights were plotted in 1D to mimic an NMR spectrum but with positive/negative peaks indicating up-regulated/down-regulated metabolites, respectively. Indeed, in this «statistical spectrum», the correlation of each NMR variable with the continuous ranking variable (score), ranging from 0 to 1, was color coded, ranging from dark gray (high correlation) to light gray (low correlation). Metabolites with a correlation ≥0.5, considered the most discriminating, were subjected to univariate analysis. Analysis of variance of cross-validated predictive residuals (CV-ANOVA) was used to assess the significance of the model.
[0110] Metabolites significantly varying in at least three of the four studied models (6-OHDA, α-synuclein, MPTP and human) were subjected to multiple logistic regression to assess their relevance as a potential predictive biomarker of PD at using the R software (version 3.6.1—R core team). The regressions, including cross-validation, were performed for prodromal type animals (6-OHDA and α-synuclein prodromal type rats). Clinical-type animals were used for external validation (clinical-type 6-OHDA and α-synuclein rats, and all primates). The same analyzes were performed for the US patient cohort. The Italian patient cohort was used for external validation. All combinations of retained metabolites were tested, and ROC curves were then generated for each of them to assess the goodness of fit using the area under the curve (AUC) and the optimal threshold that maximizes sensitivity and specificity.
[0111] Univariate Analysis:
[0112] All univariate analyzes were performed using Graphpad Prism 8 software (San Diego—USA). All results were expressed as mean values±SEM with a significance level set at 0.05.
[0113] 6-OHDA models: For the self-administration tests and the step readjustment tests, a univariate analysis of the effect of Pra was performed by applying a one-way repeated measures (MR) ANOVA followed by a Sidak post-hoc test.
[0114] For histological and metabolomic data, a one-way ANOVA followed by Tukey post hoc test with correction for multiple comparisons was performed.
[0115] α-synuclein models: For the longitudinal study performed in α-synuclein animals, some values were missing, due to artifacts during NMR measurement, Data were therefore analyzed by fitting a proposed mixed model using a compound symmetry covariance matrix, and were fitted using the restricted maximum likelihood (REML) method. This model was followed by a Tukey post hoc test.
[0116] MPTP monkeys: Given the small number of animals, the non-parametric Mann-Whitney test was used.
[0117] Humans; Data were log-transformed given the non-Gaussian distribution of metabolite levels. First, t-tests were performed individually in the two different cohorts. Then a two-way ANOVA was applied with i) origin of cohort and ii) group (comparison group or PD) as factors, followed by Sidak post hoc test.
Example 2: Results
[0118] The 6-OHDA Rat Model Makes it Possible to Study the Different Stages of Parkinson's Disease
[0119] Although, in humans, the diagnosis of PD relies on a scale that incorporates assessments of neuropsychiatric and motor symptoms [22], its definitive confirmation is based on postmortem histological assessments, primarily of neuronal loss in the nigrostriatal pathway leading to DS denervation. To characterize 6-OHDA rats for clinical disease progression, a score, called the Parkinson's disease progression score (PDP), based on the same type of criteria as those used in humans, ie. the neuropsychiatric component—assessed by self-administration performance (motivation)—, fine motor skills—assessed by the performance of the step readjustment test—, and the extent of the DS lesion—assessed by the post-mortem histological analysis was assigned to them. Based on this score, animals with PD were divided into two categories, namely prodromal type animals and clinical type animals.
[0120] Tyrosine hydroxylase immunoreactivity (TH-IR) revealed that bilateral injection of 6-OHDA into the SNc led to partial nigrostriatal dopaminergic injury resulting in dopaminergic denervation in the DS and to a lesser extent in the Nacc (
[0121] With respect to fine motor skills, all animals showed a similar number of paw readjustments before 6-OHDA/physiological saline injection, which was not changed after 6-OHDA injection, except in the clinical type group where it was significantly reduced (
[0122] Co-Evolve Serum Metabolic Signatures with PD Progression in 6-OHDA Rats
[0123]
TABLE-US-00002 TABLE 2 .sup.1H .sup.13C Metabolite Groupe ppm ppm Multiplicity 3- γ-CH.sub.3 1.19 24.4 d hydroxybutyrate half α-CH.sub.2 2.30 49.2 dd half α-CH.sub.2 2.39 49.2 dd β-CH 4.15 m Acetoacetate 3.44 s 2.27 s Acetate CH.sub.3 1.91 25.9 s Acetone CH.sub.2CO 2.22 32.9 s Alanine CH.sub.3 1.47 18.8 d α-CH 3.77 53.5 q Albumin lysyl ε-CH.sub.2 2.88 42.0 t ε-CH.sub.2 2.95 41.9 t ε-CH.sub.2 3.01 42.0 t Arginine γ-CH.sub.2 1.64 γ-CH.sub.2 1.69 β-CH.sub.2 1.89 δ-CH.sub.2 3.23 43.2 t Aspartate half β-CH.sub.2 2.66 q half β-CH.sub.2 2.80 dd Betaine CH.sub.2 3.9 s CH.sub.3 3.26 s Cholesterol C18 (in HDL) 0.66 m C18 (in VLDL) 0.68 C2 and C27 0.83 25.2 m Choline N (CH.sub.3).sub.3 3.21 56.7 s 3.51 4.06 Citrate half CH.sub.2 2.53 d half CH.sub.2 2.66 d Creatine CH.sub.3 3.03 s CH.sub.2 3.92 s Creatinine CH.sub.3 3.04 s CH.sub.2 4.05 s Dimethylamine CH.sub.3 2.71 s Ethanol CH.sub.3 1.17 21.
t CH.sub.3COH 3.65 q Fatty acids CH.sub.3(CH.sub.2)n 0.84 16.5 m (mainly LDL) (CH.sub.2)n 1.27 32.2 m Fatty acids CH.sub.3CH.sub.2CH.sub.2C═ 0.86 m (mainly VLDL) CH.sub.2CH.sub.2CO 1.57 27.4 m CH.sub.2CH.sub.2CH.sub.2CO 1.29 m Fatty acids Ch.sub.3CH.sub.2 0.93 21.05 m CH.sub.3CH.sub.2(CH.sub.2)n 1.24 34.4 m CH.sub.3CH.sub.2(CH.sub.2)n 1.26 25.2 m CH.sub.2 1.26 19.2 m CH.sub.2 1.30 m CH.sub.2CH.sub.2C═C 1.68 29.2 Fatty acid CH.sub.2C═C 2.00 29.7 m CH.sub.2CO 2.22 3
.3 m C═CCH.sub.2C═C 2.72 28.1 m CH═CHCH2CH═CH 5.26 10.
m CH═CHCH2CH═CH 5.29 132.2 m Formate CH 8.45 s Fructose 3.99 m 4.01 dd Fucose/β- 4.54 d Galactose Glucose H2 3.24 7
.9 t H4 3.40 72.4 t H4 3.41 72.4 t H5 3.4
78.
m H3 3.48 78.5 t H2 3.53 74.3 q H3 3.71 75.6 t half CH.sub.2—C6 3.72 63.5 q half CH.sub.2—C6 3.76 63.4 m H5 3.82 74.2 ddd half CH.sub.2—C6 3.84 63.4 m half CH.sub.2—C6 3.89 63.5 dd H1 4.64 98.7 d H1 5.23 94.9 d Glutamate half β-CH.sub.2 2.04 m half β-CH.sub.2 2.12 m half γ-CH.sub.2 2.34 33.9 m half γ-CH.sub.2 2.3
m 3.74 m Glutamine 2.08 2.09 half β-CH.sub.2 2.11 29.7 m half γ-CH.sub.2 2.44 33.9 m 2.4
57.4 m 3.74 Glycerol half CH.sub.2 3.5
65.8 q half CH.sub.2 3.65 65.6 q C.sub.2—H 3.87 74.6 m Glyceropho- 3.22 s sphocholine NCH.sub.2 3.66 68.7 m OCH.sub.2 4.29 62.2 m Glycerol 4.06 backbone PGLYS and CHOCOR 4.22 TAGS 5.20 Glycine CH.sub.2 3.55 44.3 s Histidine 3.09 dd 3.98 dd H4 7.04 s H2 7.75 s Isoleucine δ-CH.sub.3 0.93 13.9 t β-CH.sub.3 1.00 17.15 d half γ-CH.sub.2 1.24 half γ-CH.sub.2 1.46 1.96 3.65 Lactate CH.sub.3 1.32 22.7 d CH 4.11 71.2 q Lactose 3.55 3.
6 3.97 4.45 Leucine δ-CH.sub.3 0.95 d δ-CH.sub.3 0.96 d 1.66 m 1.70 42.7 m 1.73 m α-CH 3.71 Lysine γ-CH.sub.2 1.43 m γ-CH.sub.2 1.49 m δ-CH.sub.2 1.72 m β-CH.sub.2 1.88 m β-CH.sub.2 1.91 m 3.02 t 3.74 t Mannose 4.89 d 5.18 d Methanol CH.sub.3OH 3.35 s Methionine 2.
4 31.4 t Methionine 2.15 s Myo-inositol 4.05 3.27 t N-acetyl- NHCOCH.sub.3 2.04 24.7 s glycoprotein 1 N-acetyl- NHCOCH.sub.3 2.07 25 s glycoprotein 2 Phenylalanine half β-CH2 3.26 α-CH 3.97 H2, H6 7.31 d H4 7.35 m H3, H5 7.40 m Proline γ-CH.sub.2 1.98 m γ-CH.sub.2 2.01 m half β-CH2 2.05 m half β-CH2 2.34 m half δ-CH2 3.33 m α-CH 4.12 m Succinate 2.39 s Threonine γ-CH.sub.3 1.31 d α-CH 3.55 d β-CH 4.23 m Trehalose 3.40 74.3 Tyrosine 6.88 d H2, H6 7.18 d Valine CH3 0.98 19.4 d CH3 1.03 20.6 d α-CH 2.26 m β-CH 3.60 63.4 d Urea NH.sub.2C═ONH.sub.2 5.77 Xylose 3.41 78.6
indicates data missing or illegible when filed
[0124] The graph of the OPLS scores, made with the NMR data and the PDP scores for each animal (i.e. the «6-OHDA serum OPLS model») shows a clear gradation of the dot colors, from «cold» (white) on the left to «warm» (dark gray) on the right. This indicates that the metabolic profile is changing along with disease progression (
[0125] Metabolic Profiles of Brain Tissue in the 6-OHDA Rat Model Strengthen Blood Findings
[0126] The «High Resolution Magic Angle Spining» (HRMAS) .sup.1H NMR” spectroscopy was used to study the metabolic profiles of intact brain biopsies (DS and Nacc) of 6-OHDA animals, sampled at the same level as sections used for histology to quantify dopaminergic denervation. A representative spectrum of a DS biopsy from a 6-OHDA rat is shown in
[0127] The OPLS analysis constructed with the Nacc data (i.e. the «OPLS 6-OHDA Nacc model») showed the same trend, although less clear (
[0128] Although some metabolites such as pyruvate and citrate are not detected in tissue samples, due to their immediate transformation upon death (post-mortem effect), these experiments show dysregulations of alanine and lactate levels common to serum and tissue samples.
[0129] Pramipexole Modifies Serum and Tissue Metabolic Dysregulations Induced by 6-OHDA
[0130] It was checked whether the serum metabolic profile was altered by a classically used clinical dopamine agonist, Pramipexole (Pra), known to improve only neuropsychiatric symptoms when used at low dose in animal models [23,24]. As expected, after 15 days of subchronic Pra administration, deficits in the self-administration task measured in 6-OHDA rats were fully or partially ameliorated in prodromal-type and clinical-type animals, respectively. On the other hand, the performance of Veh-treated rats was not improved (
[0131] With respect to serum metabolic profiles, to visualize simultaneously the effect of 6-OHDA lesion and treatment, metabolite levels of prodromal-like animals were normalized to the level of control (sham) animals (lesion effect, solid bar) and metabolite levels of Pra-treated prodromal-type animals were normalized to Veh-treated prodromal-type animals (Pra effect, hatched bar). In prodromal animals, alanine BHB and pyruvate levels increased after injury (
[0132] Unlike serum, for which multiple longitudinal samples could be collected from the same animal, brain samples could only be collected at the end of the experiment, when all animals had received a Pra or Veh chronic treatment. Therefore, control (sham) animals treated with Veh were used as a reference to normalize metabolite levels for prodromal type animals treated with Veh (Lesion effect, solid bar). The metabolite levels of Pra-treated prodromal-type animals were normalized relative to prodromal-type animals given Veh (Pra effect, hatched bar). As in serum, administration of Pra caused a decrease in some metabolites previously increased by 6-OHDA lesion. That was true for alanine in DS and Nacc, and for lactate in Nacc in prodromal type animals (
[0133] Overall, significant alterations in serum and tissue metabolites were measured in the 6-OHDA rat model and associated with the different phases of the disease and, interestingly, were detected as early as the prodromal stage. Moreover, some of these disturbances were partially reversed by chronic administration of Pra, which also corrected the neuropsychiatric deficits.
[0134] In order to test the specificity of the biomarkers found in the 6-OHDA model regarding disease progression and pathophysiological mechanisms, the serum metabolomics study was extended to two other animal models of PD.
[0135] 6-OHDA, α-Synuclein Rats and MPTP Monkeys Share Serum Metabolic Disturbances
[0136] First, using the same strain of rat (Sprague Dawley) as for the 6-OHDA study, an α-synuclein rat model was selected, in which overexpression of human A53T alpha-synuclein was induced in SNc using adeno-associated viral (AAV) vectors. This targeted expression leads to progressive neurodegeneration in the targeted region, which makes it possible to follow the different stages of PD in the same animal [19], namely the prodromal-type phase around 3 weeks and the clinical-type phase around 10 weeks after the injection.
[0137] An OPLS-DA model was used to assess whether a specific metabolic signature was associated with these different phases (i.e. the «serum α-synuclein OPLS-DA model»). Sham animals (control injected with a comparison group virus encoding GFP) showed no difference between the three different time points 0, 3 and 16 weeks after injection (
[0138] These results revealed similar metabolic disturbances between the two rat models of PD, which are associated with the disease progression. To go further, a metabolic analysis was carried out in a non-human primate model of PD, e.g. the MPTP model, which mimics the clinical phase of PD and shows greater homology with humans [25].
[0139] The OPLS-DA model shown in
[0140] Taken together, these results reveal common metabolic alterations associated with PD progression in three different animal models of PD, performed in two different species. Thus, to validate the clinical relevance of these markers, these preclinical results were compared with those obtained in samples from patients with PD.
[0141] Serum Metabolic Signatures of Newly Diagnosed Parkinson's Patients and Animal Models of PD Show Similarities
[0142] Each cohort was first analyzed individually, ie. newly diagnosed parkinsonian patients (n=21 in each cohort) compared to age- and sex-matched comparison groups (n=30/23 NIH/Italy).
[0143] With the exception of some metabolites, e.g. alanine (data not shown), the variations observed were consistent between the 2 cohorts, as shown in Table 3. The latter presents the main individual and collective changes in metabolites between patients with PD and controls in the Italian and United States (NIH) cohorts, analyzed individually by t-test and collectively by two-way ANOVA, followed by a Sidak post hoc test. *: p≤0.05; **: p≤0.01; ***: p≤0.001; ns: not significant.
TABLE-US-00003 TABLE 3 Italy NIH 2 ways Anova Change p Change p Cohorts inter- (%) value (%) value Origin Case action Acetoacetate 17.26 0.05 28.12 0.12 ** ** ns Betaine −9.74 0.35 −26.27 0.01 ns ** ns BHB 28.28 0.24 103.23 0.09 *** * ns Leucine 5.26 0.37 11.14 0.14 *** 0.08 ns Valine −11.04 0.13 −14.61 0.04 *** * ns
[0144] For example, a marginal increase in BHB and acetoacetate was observed in Italian PD patients (+28.3%, p=0.24 and +17.2%, p=0.05 respectively), and was even higher in patients from the United States (+103.2%, p=0.09 and +28.1%, p=0.12).
[0145] In order to increase the statistical power, the two cohorts were pooled (PD n=42, comparison group n=53). Nevertheless, since the variance of the data was mainly affected by the origin of the sample, that is to say Italy or the United States, independently of the case (PD or comparison group), a two-way ANOVA was performed with i) cohort origin and ii) case (comparison group or PD) as factors to extract PD-only information. Table 3 illustrates the significant metabolites for the case factor, with no interaction between the origin cohort and the case. The increase in BHB and acetoacetate, observed in each cohort, appeared significant when pooled, A significant decrease in betaine in the serum of patients with PD was also detected, as in all animal models. In addition, a significant decrease in valine was observed as in the 6-OHDA rat model. Finally, a decrease in leucine was observed. Additionally, the analysis of a small number of serum samples from other newly diagnosed and treated with Pra 0.5-100 mg/day (n=9) revealed a trend towards normalization of BHB and betaine levels compared to untreated PD patients, as observed in rats 6-OHDA (
[0146] Serum Metabolites as Potential Markers for the Early Diagnosis of Parkinson's Disease
[0147] To assess the potential of the identified metabolites to distinguish prodromal type animals from control (sham) animals, multiple logistic regression models were developed, based on all possible combinations of levels of acetoacetate, betaine, creatine, BHB, pyruvate and valine, grouping prodromal-type 6-OHDA and α-synuclein rats.
[0148] The best regression model, evaluated by the ROC curves (see materials and methods), retained the six metabolites, using a regression based on the following predictive algorithm (p: 1.107×10.sup.−15):
Logit(P)=log(P/(1−P))=−54.72+0.49BHB+0.39Pyruvate−0.42Valine+0.06Acetoacetate+0.52Creatine−0.10Betaine
[0149] The ROC curve had an area under the curve (AUC) of 0.936. By applying the optimal threshold of 0.618 (sensitivity 0.85, specificity 0.88) (
[0150] Regarding human samples, the United States (NIH) cohort was used to build a model because i) it is the largest cohort and ii) it includes only recently diagnosed patients, unlike the Italian cohort, which includes patients with longer PD duration. The latter was used as an external validation cohort. Here again, the best model retained the six metabolites combined with the following regression algorithm (p: 1.95×10.sup.−4): Logit(P.sub.1)=log(P.sub.1/(1−P.sub.1))=−45.60+0.28BHB−4.54Pyruvate−11.23Valine+7.05Acetoacetate+2.85Creatine−8.27Betaine.
[0151] The following ROC curve had an AUC of 0.88. The optimal threshold was 0.316, and the corresponding sensitivity and specificity were 0.957 and 0.714, respectively (
[0152] The United States (NIH) cohort minus 2 unretained patients was also used to build a model. Here again, the best model retained the six metabolites combined with the following regression algorithm (p: 1.95×10.sup.−4):
Logit(P.sub.2)=log(P.sub.2/(1−P.sub.2))=−42.78+0.16BHB−3.31Pyruvate−12.01Valine+7.06Acetoacetate+3.14Creatine−7.79Betaine.
[0153] The following ROC curve had an AUC of 0.87. The optimal threshold was and the corresponding sensitivity and specificity were 0.952 and 0.714, respectively (
[0154] Finally, multiple logistic regression was performed using serum data from all MP animals (prodromal and clinical type) from all models using the same six metabolites and was evaluated with the following ROC curve showing an AUC of (
[0155] In total, the precision of the biomarker developed from the same six serum metabolites was at least 82.6% regardless of the species.
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