ALPHA-SYNUCLEIN IN PERIPHERAL BLOOD MONONUCLEAR CELLS AS BIOMARKER FOR SYNUCLEINOPATHY

Abstract

Disclosed is a rapid, non-invasive and highly specific and sensitive diagnostic assay for the identification of individuals with synucleinopathy and for measuring synucleinopathy progression or status. Test kits for diagnosis of an individual suspected of having synucleinopathy are also disclosed.

Claims

1. A method to diagnose synucleinopathy in a first subject comprising the steps of: a) detecting a lymphocyte expression level of Alpha-Synuclein or a monocyte expression level of Alpha-Synuclein in a first subject; b) comparing the lymphocyte expression level of Alpha-Synuclein or the monocyte expression level of Alpha-Synuclein with a second subject who does not have synucleinopathy wherein an increase in the expression level of Alpha-Synuclein in the first subject relative to the second subject indicates that the subject has synucleinopathy.

2. The method of claim 1 wherein said synucleinopathy is at least one selected from the group consisting of Parkinson Disease, Parkinsonism, dementia with Lewy bodies; and multiple system atrophy.

3. The method of claim 1 wherein said detecting step is detecting from a blood sample or a peripheral blood sample of said subject.

4. The method of claim 1 wherein said detecting step is detecting from a peripheral blood mononuclear cells sample collected from said first subject.

5. The method of claim 1 wherein a ratio of the expression level of Alpha-Synuclein in the first subject to the expression level of Alpha-Synuclein in the second subject is at least 1.5 to 1 for lymphocyte cells or at least 2 to 1 for lymphocyte cells.

6. The method of claim 1 wherein a ratio of the expression level of Alpha-Synuclein in the first subject to the expression level of Alpha-Synuclein in the second subject is at least 1.5 to 1 for monocyte cells or at least 2 to 1 for monocyte cells.

7. The method of claim 1, wherein the step of detecting is performed by flow cytometry.

8. The method of claim 1, wherein the step of detecting is performed with an anti Alpha-Synuclein antibody.

9. The method of claim 1 wherein said first subject or said second subject has Gaucher Disease or has Gaucher carrier status.

10. The method of claim 9 wherein said subject has at least one mutated GBA gene.

11. The method of claim 10 wherein the at least one mutated GBA gene encodes a glucocerebrosidase with a reduced glucosylceramide cleavage activity.

12. A method for monitoring the progression of synucleinopathy or synucleinopathy status in a subject comprising the steps of: a) detecting a first lymphocyte Alpha-Synuclein expression level or a first monocyte Alpha-Synuclein expression level from a first sample from said subject; b) determining the progression of synucleinopathy in the subject based on comparing said first lymphocyte Alpha-Synuclein expression level or said first monocyte Alpha-Synuclein expression level with a second expression level from a second sample from the same subject collected earlier or from a second subject with synucleinopathy; wherein a high first expression level relative to said second expression level indicates a more rapid progression of synucleinopathy and a low expression level relative to said second expression level indicates a less rapid progression of synucleinopathy.

13. The method of claim 12 wherein said synucleinopathy is at least one selected from the group consisting of Parkinson Disease, Parkinsonism, dementia with Lewy bodies; and multiple system atrophy.

14. The method of claim 12, wherein said detecting step is detecting from a blood sample or a peripheral blood sample of said subject.

15. The method of claim 12, wherein said detecting step comprises analysis by flow cytometry.

16. The method of Clam 12, wherein the step of detecting is performed with an anti-Alpha-Synuclein antibody.

17. The method of claim 12, wherein said subject has Gaucher Disease or has Gaucher carrier status.

18. The method of claim 17, wherein said subject has at least one mutated GBA gene.

19. The method of claim 18, wherein the at least one mutated GBA gene encodes a glucocerebrosidase with a reduced glucosylceramide cleavage activity.

20. A kit for the diagnosis of synucleinopathy in a subject comprising: a) a first reagent for the detection of Alpha-Synuclein expression a cell; and b) a second reagent selected from the group consisting of a positive control reagent and a negative control reagent.

21. The method of claim 20 wherein said synucleinopathy is at least one selected from the group consisting of Parkinson Disease, Parkinsonism, dementia with Lewy bodies; and multiple system atrophy.

22. The kit of claim 20, wherein the positive control reagent comprises lymphocytes or monocytes from peripheral blood of a second subject which has at least one selected from the group consisting of synucleinopathy, Gaucher Disease and Gaucher carrier status.

23. The kit of claim 20, wherein the negative control reagent comprises lymphocytes and monocytes from peripheral blood of a third subject which does not have Gaucher Disease, Gaucher carrier status or synucleinopathy.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0022] FIG. 1 PBMCs from four cohorts NonGD-nonPD, GD-nonPD, NonGD-PD and GD-PD are assayed for Parkin using flow cytometry as mentioned in the methodology. In grey, are the control plots or background fluorescence from no-primary antibody tubes. Black filled plots indicate intracellular Parkin expression.

[0023] FIG. 2 PBMCs from four cohorts NonGD-nonPD, GD-nonPD, NonGD-PD and GD-PD are assayed for Alpha-Synuclein using flow cytometry. In grey, are the control plots or background fluorescence from no-primary antibody tubes. Black filled plots indicate intracellular Parkin expression.

[0024] FIG. 3 The mean fluorescence intensity for the Alpha-Synuclein plots is quantified relative to background fluorescence in each subject in FIG. 2. The absolute values are indicated at the bottom of each plot.

[0025] FIG. 4 PBMCs from subject GD-PD-1 from three visits over a period of 3 years are assayed at the same time for Alpha-Synuclein and the results from lymphocytes and monocytes are shown. At visit-1, the subject had very early signs of PD symptoms which progressively worsened to Hoehn and Yahr stages 1 and 4 at visit-2 and visit-3 respectively.

[0026] FIG. 5 PBMCs from subject GD-PD-2 from three visits over a period of 2 years are assayed at the same time for Alpha-Synuclein and the results from lymphocytes and monocytes are shown. Subject GD-PD-2 has been diagnosed with Hoehn and Yahr stage 2 at all three visits.

[0027] FIG. 6: The results from FIGS. 4&5 are quantified as relative expression of Alpha-Synuclein over the background fluorescence for each visit and plotted. The absolute relative values are mentioned at the bottom of the histograms. The Hoehn and Yahr stages for each subject and visit are also labelled at the bottom of the plot.

DETAILED DESCRIPTION OF THE INVENTION

[0028] This invention generally relates to methods, reagents and kits that are useful for the detection of synucleinopathy (e.g., Parkinson Disease). The invention can be used to identify patients that have Parkinson Disease (e.g., a diagnostic, prognostic or monitoring method) and to further understand the mechanisms of Parkinson Disease for the development of therapeutic strategies directed at preventing or treating this disease.

[0029] The present inventors have shown that Alpha-Synuclein is highly useful peripheral blood mononuclear cells biomarker to identify synucleinopathy (e.g., Parkinson Disease) especially in, but not necessarily in, Gaucher Disease or Gaucher Carrier Status patients.

[0030] More particularly, the present inventors compared the expression of two biomarkers Alpha-Synuclein and Parkin in the peripheral blood mononuclear cells of Gaucher Disease or Gaucher Carrier Status patients and show that the expression of Alpha-Synuclein shows distinct patterns which can be used to diagnose synucleinopathy (e.g., Parkinson Disease).

[0031] In one aspect of the invention, the cells used in the assay are lymphocytes and monocytes. One advantage of the present invention is that the cells, lymphocytes and monocytes, can be provided in the form of whole blood or peripheral blood or a fraction thereof from a patient and such samples can be collected with minimal invasiveness and inconvenience to the patient.

[0032] The method of the present invention is particularly useful because it can identify patients that have synucleinopathy in a rapid, in vitro assay that is minimally invasive and is capable of rapid diagnosis. The method is more sensitive and much less invasive than current methods of diagnosing synucleinopathy (e.g., Parkinson Disease). Moreover, prior to the present invention, the use of biomarkers in peripheral blood to diagnose synucleinopathy (e.g., Parkinson Disease) had not been described.

[0033] The experiments are performed as described in the Example Section of this disclosure.

Results:

[0034] When Parkin expression was assessed, compared to nonGD-nonPD subjects, all other three cohorts showed higher expression of Parkin, especially in monocytes (FIG. 1A-J). So, even though Parkin is elevated in GD and PD subjects compared to normal controls, Parkin alone may not be sufficient to be used as a biomarker.

[0035] We then looked at Alpha-Synuclein expression in lymphocytes and monocytes of the samples and quantified the results as relative Alpha-Synuclein expression over the background fluorescence for each sample (FIGS. 2&3). Alpha-Synuclein expression in control subjects, i.e., nonGD-nonPD group was comparable to background fluorescence in both lymphocytes and monocytes. In GD-nonPD group, even though the alpha-synculein expression is seen, it is still minimal and overlaps significantly with the background fluorescence. In PD subjects with no known GD symptoms or mutations, there seems to be a distinct peak in Alpha-Synuclein associated fluorescence. Similar to nonGD-PD group, GBA associated PD patients also showed elevated expression of Alpha-Synuclein in lymphocytes and monocytes. In addition, in monocytes on GD-PD patients, there seem to be a distinct subgroup of monocytes with elevated expression of Alpha-Synuclein. This pattern was unique to GD-PD group and not seen in any other control groups. However, when the quantity and pattern of both Alpha-Synuclein and Parkin are studied, GBA associated PD subjects show a distinct pattern.

[0036] We then looked at the Alpha-Synuclein expression in two GD-PD subjects with differing disease progression to see if the expression of Alpha-Synuclein can reflect the clinical changes over a period of time (FIGS. 4&5). The relative Alpha-Synuclein expression over time for GD-PD1&2 is quantified in FIG. 6. In GD-PD-1, where the subject had rapid progression of PD symptoms, we noticed an appearance of a second peak in the monocytes. This peak increases with time in visit-3. This indicated that number of cells with accumulation of Alpha-Synuclein increased with time in GD-PD-1 subject. This result was not seen as aggressively in GD-PD-2 whose PD symptoms did not progress as quickly as in GD-PD-1 (FIG. 5).

Significance and Claims:

[0037] Our results indicate, for the first time, that Alpha-Synuclein has a differential expression in cells of peripheral blood in subjects with GBA mutations and Parkinson's disease. This can be visualized by indirect immunofluorescence followed by flow cytometry which resulted in distinct pattern in subjects with PD and GBA associated PD. The assay utilizes easily accessible tissue type i.e., peripheral blood, minimally invasive method of sample collection and requires very little amount (1-5 ml) of peripheral blood. Therefore, we propose that this assay may be used to detect biomarkers (Alpha-Synuclein and Parkin) for diagnosis and disease progression for Parkinson disease in subjects. In one embodiment, these subjects has GBA gene mutations as well as other idiopathic PD and other synucleinopathies.

[0038] While specific embodiments are disclosed where the correlation between cell surface marker(s) or protein expression(s) and disease or disease risk is particularly useful, the assay may be applicable for diagnosis and risk assessment in patients without GBA gene mutations or previous disease state. Also, the methods of this disclosure are not limited to humans but is applicable to all mammals and animals such as commercially valuable livestock or domestic pets and also primates and other animals useful as an animal model of synucleinopathies (e.g., Parkinson Disease). The methods of the disclosure may be combined with other diagnostic and testing methods to achieve a synergistic and more reliable assessment of disease and/or disease risk.

REFERENCES

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[0041] Mazzulli et. al., Gaucher disease glucocerebrosidase and a-synuclain form a bidirectional pathogenic loop in synucleinopathies. Cell 2011; 146: 37-52.

[0042] Giraldez-Perez et. al., Models of alpha-synuclein aggregation in Parkinson's disease. Acta Neuropathol Commun. 2014; 2: 176.

[0043] Swan and Saunders-Pullman, The association between beta-glucocerebrosidase mutations and parkinsonism. Curr Neurol Neurosci Rep., 2013; 13.

[0044] Kawahara et al., Alpha-Synuclein aggregates interfere with Parkin solubility and distribution. J Biol Chem 2008; 283:6979-6987.

[0045] Eblan M J, Nguyen J, Stubblefield B, et al. (2005) Glucocerebrosidase mutations in brain samples from subjects with parkinsonism. Mol Genet Metab. 84: 217-217.

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[0047] Guttman M, Burkholder J, Kish S J, Hussey D, et al. (1997) 11C RTI-32PET studies of the dopamine trasporter in early dopa-nave Parkinson's disease: implications for the symptomatic threshold. Neurology 48: 1578-1583.

[0048] Morrish P K, Rakshi J S, Bailey D L, Sawle G V, Brooks D J. (1998) Measuring the rate of progression and estimating the preclinical periods of Parkinson's disease with [18 F] dopa PET. J Neurol Neurosurg and Psychiatry. 64:314-319.

[0049] All patents, patent applications, and references cited in this disclosure are incorporated by reference into this disclosure.

EXAMPLES

Example 1

Subjects

[0050] Study included four cohorts: 1) Patients and carriers of Gaucher disease with confirmed disease causing mutations in GBA gene who have developed Parkinson disease symptoms (GD-PD), 2) Patients and carriers of Gaucher disease with no known Parkinson disease symptoms (GD-nonPD), 3) Patients with diagnosed Parkinson disease and no known GBA mutation or GD symptoms (nonGD-PD) and 4) Subjects with no known PD or GD symptoms (NonGD-nonPD).

Example 2

Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

[0051] PBMCs are extracted from 3-5 ml peripheral blood using Ficoll-paque (GE health care). 2-4 ml of whole blood is diluted 1:2 using Phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS) and overlayed onto Ficoll solution in 15 ml leucosep tube. The tubes are centrifuged at 2000RCF for 10 minutes with no brakes. The layer containing PBMCs is transferred into a fresh 15 ml tube and washed with PBS+2% FBS. The cells are then resuspended in cell freezing medium (50% RPMI+40% FBS+10% DMSO) and stored at 150 C. in a freezer until their use.

Example 3

Immunostaining

[0052] The cryopreserved PBMCs were thawed at 37 C. for 2 minutes, washed and resuspended in PBS+2% FBS and used for immunostaining. Approximately 510.sup.5 cells per tube were fixed and permeabilized using Fix & Perm Cell Fixation and Cell Permeabilization kit (Thermo Fisher Scientific) as per manufacturer's instructions. Rabbit anti-human-Alpha-Synuclein and Rabbit-anti-human-Parkin antibodies (catalog numbers 701085 and PA5-13399 respectively, from Thermo Fisher Scientific) were added to individual tubes for intracellular staining for 20 minutes at room temperature. No primary antibody was added to the control tubes. The cells were then washed with 2 ml of PBS+2% FBS. The tubes were centrifuged to remove wash buffer. The cells were resuspended in PBS+2% FBS containing Goat anti-Rabbit antibody conjugated with Alexa fluor 647 which acted as secondary antibody and incubated at room temperature for 20 minutes. The cells were then washed in PBS+2% FBS and acquired on Flow cytometer (BD accuri). The results were analyzed using FCS Express 6 software (Denovo software). Using FSC vs SSC scatter plot, lymphocytes and monocytes were gated and were analyzed for subsequent Alpha-Synuclein expression or Parkin expression using scatter plots and histogram overlays.

[0053] Although the disclosure has been described with reference to various example embodiments, it should be understood that various modifications can be made without departing from the spirit of the disclosure. Accordingly, the scope of the disclosure should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all cited articles and references, including patent applications and publications, are incorporated herein by reference for all purposes.