WO2005116239A2

WO2005116239A2 - Diagnostic assays for neurodegenerative diseases

Info

Publication number: WO2005116239A2
Application number: PCT/GB2005/050075
Authority: WO
Inventors: Susan Adele Greenfield
Original assignee: Enkephala Limited
Priority date: 2004-05-28
Filing date: 2005-05-27
Publication date: 2005-12-08
Also published as: WO2005116239A3; GB0412041D0

Abstract

The invention provides a simple colorimetric assay method for screening for propensity for, or occurrence of, neurodegeneration in the brain. The assay relies on measuring enzymatic activity of a soluble monomeric form of acetylcholinesterase (G1-AChE) added to biological samples such as serum or plasma samples. Exogenous G1-AChE with such samples from patients with, for example, Motor Neurone Disease associated with amyotrophic lateral sclerosis, Parkinson's disease or Alzheimer's disease has selectively been found to exhibit significantly higher activity than in similarly treated control samples.

Description

Diagnostic Assays

Field of the invention The present specification relates to a simple in vitro method for screening and monitoring propensity for, or occurrence of, neurodegeneration in the central nervous system, especially the brain. More particularly, it provides such diagnosis based on measuring enzymatic activity of a soluble, monomeric, C-terminal truncated form of acetylcholinesterase (Gt-AChE) when added to biological samples such as serum samples. By use in combination with reference to other clinical indicators of neurological disorders, more specific diagnosis may be made, more particularly, for example, diagnosis of Motor Neurone Disease (TvIND) associated with amyotrophic lateral sclerosis (ALS), Parkinson's disease and Alzheimer's disease. This method of diagnosis is cheap and relatively painless, thus allowing routine, consecutive samples to be taken from patients, meaning that each patient can act as their own control. The diagnosis is thus sensitive and efficient. The particular value of the invention lies in providing an early and rapid screen for neurological disorders, which opens the way for improved control or prevention of disease progression.

Background to the invention Motor Neurone Disease (TvIND) presents in a variety of clinical forms and is characterised by the progressive death of key populations of neurons. As yet there is no cure for the disease, although the anti-excitotoxic agent Riluzole (2-amino-6- trifluoromethoxybenzothiazole) is the treatment of choice for reducing the speed of cell loss and alleviating the symptoms (Bensimon et al., New. Engl. J. Med. (1994) 330, 585-591; Turner et al., Pract. Neurology (2003) 3, 160-170). New drug treatments could be more successfully developed if there were means for more rapid diagnosis, which currently takes on average 18 months in the UK (www.mndassociation.org.uk). In any event, rapid diagnosis would allow patients to be started earlier on currently available medication. In the search for such a test, the inventor proposed exploration of an idea, following on from her previous published work on a novel role for acetylcholinesterase (AChE). AChE is now widely accepted as having novel, non-cholinergic functions, independent of its familiar catalytic action of the hydrolysis of acetylcholine. One such non-enzymatic function could be as a key signaling molecule in neurodegenerative disorders, such as MND, Parkinson's disease and Alzheimer's disease (Greenfield and Vaux, Neuroscience (2002) 113, 485-492). Moreover, one of the reasons for initially suggesting a novel function for AChE was that it is polymorphic, existing in a variety of asymmetric (collagen tailed) and symmetric 'globular' forms comprising different numbers of catalytic subunits (Massoulie, J., Neurosignals (2002) jj_, 130-143). Interestingly, the single subunit globular form ('G] ) increases significantly relative to the more usual globular tetramer ('G₄') in the brains of patients with neurodegeneration (Arendt et al. Neurochem. Int. (1992) 21, 381-386). Motor neurons secret AChE and this secretion actually precedes the neurotoxicity triggered by excitatory amino acids. In fact, the particular vulnerability of α motor neurons in MND might be due to their turnover of AChE and to autoantibodies to AChE seen in MND patients (Conradi and Ronnevi, Brian Res. Bull. (1993) 30, 369-371). Moreover in patients with MND, there are increased titres of IgG and IgA antibodies towards AChE (Sindhuphak et al. J. Neural. (1998) 86, 195-202), whilst in transgenic mice overexpressing AChE, there are aberrations in neuromuscular structure and pronounced amyotrophy (Andres et al. Neurochem, Int. (1998) 32, 449-456). It has now firstly been found that the soluble, monomeric G\ form of AChE can be used selectively as a probe in a simple and established colourimetric assay for acetylcholinesterase activity to detect a significant difference in serum samples between patients with ALS compared with controls and those with other forms of MND such as progressive muscular atrophy (PMA), primary lateral sclerosis (PLS) and ALS-dementia. Significantly higher activity in the assay, i.e. enhanced activity of exogenous Gt-AChE, has been observed with serum samples from patients with the ALS form of MND in comparison to control samples. Neither disease duration from symptom onset, nor time of sample donation, nor gender were significant factors. Those ALS patients taking riluzole at the time of sample donation had significantly higher scores still than those ALS patients not taking the drug. However, in a multivariate analysis this was not an independently significant factor. As indicated above, these observations have been extended to serum samples from patients with other neurological disorders previously implicated as associated with non-enzymatic function of AChE, notably Parkinson's disease and Alzheimer's disease. Niebrόj-Dobosz et al. in Folia Neuropathol. (1999) 37,107-112 previously reported increased AChE activity upon addition of serum ultrafiltrates derived from severe ALS patients to rat spinal cord homogenate. However, these studies provide no pointer to use of specifically the monomeric G\ form of AChE as a diagnostic probe as now proposed. Indeed, as further emphasized below, it has been confirmed that only G^AChE and not G /G_4-AChE is susceptible to activation by a factor in serum or plasma samples.

Summary of the invention Accordingly, the present invention provides a method of determining whether a human or non-human mammalian individual has propensity for, or occurrence of, neurodegeneration in the central nervous system which comprises: (i) adding to a biological sample from said individual, such as a serum or plasma sample, a soluble, monomeric Gt-form of acetylcholinesterase (G\- AChE) and (ii) determining whether said exogenous Gt-AChE exhibits enhanced hydrolytic activity compared with exogenous Gt-AChE in control samples under the same conditions. This methodology can not only provide early, rapid indication of, for example, neurodegeneration in the brain, but as indicated above can be combined with observation or determination of other recognised clinical indicators to make more specific diagnosis of neurological disorders including MND associated with ALS, Parkinson's disease and Alzheimer's disease. In another embodiment of the invention, there is provided a method of screening for, or monitoring, an indicator of neurodegeneration in the central nervous system which comprises: (i) contacting a fraction of, or an element isolatable from, a biological sample with Gi-AChE, said sample being a sample from a patient with neurodegeneration and which is suitable for use in detecting said neurodegeneration by a diagnostic method as discussed above, and (ii) determining whether said Gt-AChE exhibits increased hydrolytic activity. The chosen sample may be, for example, a serum or plasma sample selected from patients with any of MND associated with ALS, Parkinson's disease and Alzheimer's disease. By "hydrolytic activity" of G\ -AChE will be understood hydrolytic activity as may be determined against an acetylcholine analogue substrate such as acetylthiocholine in a colorimetric assay.

Brief description of the figures

Figure 1: Effect of samples from control and non-neurodegenerative (non-ND) control patients (e.g. sciatica, cerebellum blastoma, chronic pain, temporal lobe abscess, hydrocephalis) on G^AChE and G /G₄-AChE activity. Dilution 1 is a 1 :4 dilution of G_\ or G /G₄ -AChE with buffer, dilution 2 is a 1 : 16 dilution of G_t-AChE or G /G₄-AChE with buffer, dilution 3 is a 1:64 dilution of Gi-AChE or G /G₄- AChE with buffer and dilution 4 is a 1 :256 dilution of G_t-AChE or G /G₄-AchE with buffer. Black bars = Gt-AChE control samples (n =27); White bars = G^AChE Non- ND samples; hatched bars = G /G₄-AChE control samples (n=2); grey bars = G /G₄- AchE non-ND samples (n=3)

Figure 2: Scatter plot of assay results with mean (short bar) and 95% CIs (long bar) superimposed. There is a significantly higher mean assay result in MND patients overall compared to controls (*p<0.001). Figure 3: Scatter plot of assay results with mean (short bar) and 95% CIs (long bar) superimposed for the MND clinical sub-groups. The ALS sub-group mean assay result is significantly higher than controls inpost hoc analysis (*p<0.05).

Figure 4: Scatter plot of assay results with mean (short bar) and 95% CIs (long bar) superimposed for the revised El Escorial categories (excludes LMN-only forms of MND). The "possible" (which includes PLS patients) and "definite" ALS categories have significantly higher mean assay results than controls inpost hoc analysis (*p<0.05).

Figure 5: Scatter plot of assay results in MND patients with mean (short bar) and 95% CIs (long bar) superimposed. Those patients taking riluzole at the time of sample donation have a significantly higher mean assay result, compared to those unexposed to the drug (*p<0.05).

Figure 6: Preliminary data showing assay activity with serum samples taken from patients with Alzheimer's disease (AD; n = 32) and Parkinson's disease (PD; n=16) and other neurological, non-neurodegenerative conditions (non-ND; n = 14). Mid- grey bars = samples from AD and PD patients; dark grey bars = controls (AD controls = 32; PD controls = 27).

Detailed Description Gi-AChE as referred to herein may be native Gi-AChE, e.g. native human G^AChE or Gi-AChE of another mammalian species, e.g. mouse. It may be a variant of such a polypeptide which retains the ability to exhibit enhanced activity in the presence of a serum sample from a patient with MND associated with ALS. Such a variant may have at least 90% homology, preferably at least 95% homology, e.g. 97%, 98% or 99% homology in comparison to a native Gi-AChE. Such a variant may have one or more substitutions, e.g. one or more conservative substitutions. The Gi-AChE will preferably be produced recombinantly, e.g. as described in Marchot et al, Protein Sci. (1996) 5, 672-679. In one embodiment, recombinant Gi-AChE is employed which is truncated at the C-terminus compared to the corresponding G₄-AChE polypeptide from amino acid residue 549. In the following examples, such recombinant mouse Gi-AChE was employed expressed in a human cell line. For the purpose of a diagnostic assay of the invention, such Gi-AChE may be added to any type of biological sample which, when taken from a patient with MND associated with ALS, causes enhanced Gi-AChE activity compared with use of a control sample. It may be a sample of cerebrospinal fluid, but may more preferably be a serum sample or plasma sample. It is also envisaged that in other embodiments the test sample may be a urine sample or a saliva sample. The test sample may be derived from an asymptomatic patient. As indicated above, hydrolytic activity of Gi -AChE may be conveniently determined on an acetylcholine analogue substrate such as acetylthiocholine, for example using acetylthiocholine and the well-known colourimeteric assay method of Ellman et al. (1961, Biochem. Pharmacol. 7, 88-95); hereinafter referred to as the Ellman assay. Such an assay involves incubating test samples with acetylthiocholine and 5,5-dithio-bis(2-nitro benzoic acid) (DTNB). Any cholinesterase activity in the sample will cleave the acetylthiocholine yielding acetate and thiocholine. Thiocholine reacts with DTNB to produce the yellow anion of 5-thio-2-nitro-benzioc acid, whose absorbance may be determined at 405 nm.

The following examples illustrate the invention.

Examples

Example 1 : Use of Gi -AChE as a probe for MND in serum samples

I. Methods (i) Assay samples Blood samples were taken from MND patients and controls seen at a tertiary referral centre from 1990-2002 with informed consent, separated immediately and frozen only once before subsequent analysis. The diagnosis of MND was made by a consultant neurologist after exclusion of other conditions, including screening male patients with lower motor neuron presentations for the androgen receptor trinucleotide repeat sequence characterising the Kennedy syndrome. MND patients were separated into those with a diagnosis of amyotrophic lateral sclerosis (ALS), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS) or ALS- dementia complex (ALS-dementia). All were included in the analysis. All patients were classified at the time sample donation according to revised El Escorial (EE) revised criteria (Brooks et al. Neuron Disord. (2000) 293-299), i.e. possible, probable laboratory-supported, probable and definite ALS. This classification is based on the clinical likelihood of the condition being ALS, which is greatest when there are signs of both upper and lower motor neuron involvement, in several distinct anatomical territories. Whilst it is not a direct measure of disease progression or severity, the pre-revision EE category (Brooks, J. Neurol. Sci. (1994) 124 Suppl. 96- 107) at presentation has been shown to have prognostic significance in a separate analysis of this patient database (Turner et al., Other Motor Neuron Disord. (2002) 3, 15-21). LMN-only forms of MND, termed progressive muscular atrophy (PMA), do not form part of this classification, whereas UMN-only forms of MND, termed lateral sclerosis (PLS) are currently classified as "possible" in the revised system. Date of birth, gender, date of and age at symptom onset, and site of onset were recorded. The presence of a definite family history of MND was noted, and whether or not patients were taking riluzole at the time of sample donation. Riluzole is the only proven disease modifying therapy in MND. The disease duration in months (date of sample donation minus date of symptom onset) was calculated for all patients. For those patients with a recorded date of death, this parameter was also calculated as a proportion of total survival time (date of death minus date of symptom onset). This produced a scale from 0-1 of the point during the disease course that the sample was donated (0 at the time of symptom onset, 1 at the time of death). Approximately half of the control samples were obtained from spouses of

MND patients with informed consent, and their date of birth and gender were also recorded. An additional group of control samples from healthy adult volunteers, taken as part of another study, were also included in the analysis. Non- neurodegenerative disease control samples (from patients with sciatica, cerebellar neuroblastoma, chronic pain, temporal lobe abscess, hydrocephalis or dystonia) were used to demonstrate the selectivity of Gi- AChE over AChE solution containing a mixture of dimer and tetramer (G /G₄) were collected by Professor Tipu Aziz at the Radcliffe Infirmary, Oxford. No demographic data was available for these control groups. This study was approved by the Ethics Committees of the Institute of Psychiatry, King's College Hospital.

(ii) Production and purification of Gi -AChE Recombinant mouse Gi-AChE was employed which was produced in stably- transfected human embryonic kidney (HEK) cells as described in Marchot et al., Protein Sci. (1996) 5, 672-679, and kindly provided by Professor Palmer Taylor (Department of Pharmacology, University of California, San Diego, CA, USA). Gi- AChE was generated from a cDNA with an inserted early stop codon leading to C- terminal truncation of the glycophospholipid-linked form of mouse AChE at amino acid 548. This truncation maintains the catalytic core, but truncates 37 amino acids from the nascent polypeptide and eliminates the signal for attachment of the hydrophobic glycophospholipid to the carboxy-terminal residue Gly 557 of the processed native enzyme. The cDNA for the Gi-AChE was operably-linked to a cytomegalovirus promoter in the plasmid pcDNA-3. The stably-transfected cell line was grown in 75 cm² sterile tissue culture flasks (37°C, 5% CO₂: 95% O₂) until 100% confluent. For harvesting, cells were passaged (1 :3) and placed into triple layer harvesting flasks and maintained in growth medium until 100% confluent. Cells were then washed with phosphate buffered saline and placed in serum-free 'ultraculture' medium (Biowhittaker, USA). Medium was harvested and centrifuged (10 minutes at 3000 rpm; 1499 g) and the resulting supernatant was analysed for cholinergic activity using the Ellman assay. Gi-AChE was purified from the harvested medium using a m- trimethylaminophenylamine-coupled sepharose affinity resin (Taylor and Jacobs, Mol. Pharmacol (1974) 10, 93-107). The affinity resin was added to the harvested medium and stirred for 8 hours at 4°C. To ensure binding of the Gi-AChE in the harvested medium to the resin, an Ellman assay was performed repeatedly until over 80% binding was achieved. The resin was loaded onto the column and rinsed with bicarbonate buffer (1 ml/hr). To elute the Gi-AChE, decamethonium bromide was employed. Each fraction (40 drops = 1 ml) was measured for AChE content by the Ellman assay and the active fractions subjected to dialysis using a 14-16 KDa cut-off dialysis tubing. The purity was assessed using gel filtration with UV detection.

(iii) Production and purification of G - AChE G₄-AChE, from a commercial stock of Electrophorus electricus AChE

(Sigma-Aldrich Company Ltd.; Type V-S, 1000 U/ml) was purified using a procainamide (Sigma, St. Louis, MO) -ECH Sepharose 4B (Amersham Pharmacia, Uppsala, Sweden) affinity column (Ralston et al, 1985). Briefly, the washed affinity column was loaded with AChE in PBS at 12 ml per hour in a cold room and left to recycle overnight. The column was then washed again with 20 mM Na HPO₄ + 5 mM EDTA, pH 7.2 (solution x) and solution x + 400 mM NaCl, and AChE was eluted with 400 mM TEA in solution x and 10 mM decamethonium in solution x. The eluate was dialysed overnight at 4°C using a 10-kDa molecular weight cut-off dialysis cassette (Perbioscience, Cheshire, UK) against solution x. The dialyzed AChE from affinity chromatography was applied to a column of Superdex 200 (PC 3.2/30) with a bead bed volume of 2.4 ml (Pharmacia Biotech, Uppsala, Sweden) and eluted in ascending mode. The affinity chromatography sample was monitored for protein at absorbances of 214 and 280 nm to assess the molecular species of cholinesterase present. The affinity column eluted fractions were also assessed for protein concentration and AChE concentration.

(iv) Measurement of AChE activity The cholinergic activity of samples was tested using the Ellman assay

(Ellman Biochem. Pharmacol. (1961) 7, 88-95). Briefly, samples were, where appropriate, diluted in buffer, and then 20 μl of control or disease human serum was added to 80 μl of either Gi-AChE (or G /G₄-AChE in control experiments) or buffer. 25 μl of each was then added to separate wells of a 96-well microtitre plate, in triplicate. 175 μl of Ellman reagent was then added to each well. Absorbance was read at 405 nm over a 20 minutes time-span using a Molecular Devices plate reader (Alpha Laboratories Ltd., Hampshire, UK). Analysis was performed using the equation below to detect the change in cholinergic activity due to the sample.

Assay score = ((sample + Gi-AChE) - (sample alone + Gi-AChE alone)) / (Gi-AChE alone)* 100

(v) Statistical analysis Calculations were performed using SPSS (version 10, SPSS Inc., Chicago,

Illinois). Chi-square analysis was used to test significant differences between categorical variables such as gender. Significance differences between groups for continuous variables was tested using ANOVA. Where there were more than two groups, post-hoc analysis with Dunnett C test (equal variances not assumed) was performed. Linear regression was used to measure correlation between continuous variables in the ALS patients. Multivariate linear regression was used to exclude potentially confounding variables across all subjects. P<0.05 was considered significant for all results.

II. Results

(i) Selectivity of Gr- AChE as probe Samples from control (non-disease) patients and patients with non- neurodegenerative conditions increased the activity of Gi-AChE in comparison to G /G₄-AChE, at all dilutions tested. The test showed selectivity for Gi-AChE

(Figure 1)

(ii) Patient demographics Assay results from 119 MND patient and 70 control serum samples were available for analysis. Within the MND patients, the disease sub-groups included

ALS (n=100), PMA (n=8), PLS (n=7) and ALS-dementia (n=4). Mean age at sample donation was 57 years (range 22-81; SD 13); 55% were male (n=65), and

45% (n=54) female. Mean age at symptom onset was 55 years (range 22-79; SD 13). Location of symptom onset was bulbar in 32% (n=38), upper limb in 35% (n=41) and lower limb in 34% (n=40). There was a definite family history of MND in 12% (n=14) of cases. At the time of sample donation, the mean duration of disease was 21 months (range 3 -121; SD 18). At the time of the analysis, 47% of the patients (n=56) had died. Of these patients, samples were donated at a mean proportion of 0.58 of the total disease duration (range 0.16-1.00; SD 0.22). For those MND cases covered by one of the revised El Escorial clinical categories (n=l 11), 48 cases were EE "definite" (43%), 37 were "probable" (33%), 12 were "probable laboratory supported" (11%) and 14 were "possible" (13%) (7 of the latter group were PLS cases). Thirty-five patients (30% of the ALS group) were taking riluzole at the time of the sample donation - the drug history was missing from a single patient. Demographic data were missing in 32 of the control samples. Of the other 38 controls, mean age at sample donation was 59 years (range 32-77; SD 12); 32% were male (n=12) and 68% female (n=26). Where known, gender was significantly different between patient and control groups (Pearson Chi-square 6.1 , p<0.02) with relatively more females in the control group.

(iii) Assay results When Gi-AChE specifically was used as a probe, the mean assay activity was significantly higher in the MND patient sample than controls (214 vs. 173; p<0.001 ; Figure 2). The age of the samples from time of donation to time of assay was 3.3 years for controls (range 0.8-7.4; SD 1.5), and for the ALS patients 3.0 years (range 0.5-11.6; SD 2.8), which was not significantly different between the two groups. However, increasing sample age correlated significantly with reduced assay result across all subjects (p<0.03). There was no significant effect of gender on assay results across all samples. In a sub-group analysis of the different clinical forms of MND, only the ALS group result was significantly different from controls (< 0.05). The means plot showed very high results in the PLS group and correspondingly low results in the ALS- dementia group, but these were no significantly different from controls in the post-hoc analysis (Figure 3). In a sub-group analysis for the revised El Escorial categories, the "possible" and "definite" ALS groups were significantly different from controls (p< 0.05) (Figure 4). The significance of the "possible" group was abolished when the PLS patients were excluded from the analysis. Site of symptom onset had no significant effect on assay result, even when amalgamating limb-onset into one category versus bulbar-onset. The presence of a family history of MND was not a significant factor. The age at symptom onset was not a significant predictor of assay result, nor was the time of sample donation as a proportion of the entire disease course for those deceased patients. Those MND patients taking riluzole at the time of sample donation had significantly higher mean assay result than those not taking the drug (234 vs. 204; p<0.05; Figure 5). In a multivariate linear regression analysis of disease category (MND or control), riluzole use, and sample age, versus assay result, only the disease category variable remained significant (p<0.004), excluding other confounding effects, including that of riluzole. Furthermore, a difference in activity was not seen when doses of exogenous riluzole alone, at comparable concentrations to those in plasma (0.005-2 mg/ml; Groeneveld et al. Neural. (2003) 61_, 1141-1143) were added directly to the assay reagents in the absence of serum.

Example 2: Use of Gi- AChE as a probe for Parkinson's disease and Alzheimer's disease in serum samples In an expanded study, the effect of serum from patients with Alzheimer's disease (AD) and Parkinson's disease (PD) on the activity of recombinant mouse Gi- AChE was compared to that observed with control samples. Significantly higher Gi- AChE activity was observed for AD and PD samples compared with controls as shown in Figure 6: AD (n=32), AD controls (n=32); PD patients (n= 16), PD controls (n= 27), control samples from patients with other neurological, non- neurodegenerative conditions (n=14).

Claims

CLAIMS:

1. A method of determining whether a human or non-human mammalian individual has propensity for, or occurrence of, neurodegeneration in the central nervous system which comprises: (i) adding to a biological sample from said individual a soluble monomeric Gi-form of acetylcholinesterase (Gi-AChE) and (ii) determining whether said exogeneous Gi-AChE exhibits enhanced hydrolytic activity compared with exogeneous Gi-AChE in control samples under the same conditions.

2. A method as claimed in claim 1 wherein said sample is from a human.

3. A method as claimed in claim 1 or claim 2 which is combined with observation or determination of other clinical indicators to make specific diagnosis of a neurological disorder.

4. A method as claimed in claim 3 wherein said neurological disorder is selected from Motor Neurone Disease (MND) associated with amyotiophic lateral sclerosis (ALS), Parkinson's disease and Alzheimer's disease.

5. A method as claimed in any one of claims 1 to 4 wherein said sample is a serum or plasma sample.

6. A method as claimed in any one of claims 1 to 5 wherein a recombinant Gi- AChE is employed which is truncated at the C-terminus compared to the corresponding G₄-AChE polypeptide from amino acid residue 549 (T₅₄₈-Gι-AChE).

7. A method as claimed in any one of claims 1 to 6 wherein said determining is carried out by using a colorimetric assay in which cleavage of acetylthiocholine is detected.

8. A method of screening for, or monitoring, an indicator of neurodegeneration in the central nervous system which comprises: (i) contacting a fraction of, or an element isolatable from, a biological sample with Gi-AChE, said sample being a sample from a patient with neurodegeneration and which is suitable for use in detecting said neurodegeneration in accordance with claim 1, and (ii) determining whether said Gi-AChE exhibits increased hydrolytic activity.

9. A method as claimed in claim 6 wherein said biological sample is a serum or plasma sample from a patient with a neurological disorder.

10. A method as claimed in claim 8 or claim 9 wherein said sample is from a patient with a neurological disorder selected from MND associated with ALS, Parkinson's disease and Alzheimer's disease.