SPECIALIZED EXCITATORY SYNAPTIC PROTEIN BIOMARKERS OF PLASMA NEURONAL EXOSOMES FOR PREDICTION AND STAGING OF ALZHEIMER'S DISEASE

Abstract

Combining presynaptic proteins, neuronal pentraxin 2 (NPTX2) and neurexin 2 (NRXN2), with respective postsynaptic functional partners GluA4-containing glutamate receptor (AMPA4) and neuroligin 1 (NLGN1), and enhancing excitatory synaptic activities in areas of the hippocampus and cerebral cortex. As early damage of such excitatory circuits in Alzheimer's disease (AD) correlates with cognitive losses, plasma neuron-derived exosome (NDE) levels of these two pairs of synaptic proteins are quantified and serve as biomarkers. NDE contents of all four proteins decrease significantly in AD dementia and diminished levels of AMPA4 and NLGN1 correlate with the extent of cognitive losses. Prior to the onset of dementia, NDE levels of all but NPTX2 are significantly lower than those of matched control subjects and levels of all decline significantly with the development of dementia. Reductions in NDE levels of these excitatory synaptic proteins are indicators of cognitive losses and reflect progression of severity of AD.

Claims

1. Combined quantification of plasma neuron-derived exosome (NDE) levels of four synaptic proteins of excitatory central nervous system (CNS) pathways to determine risk of development of Alzheimer's disease (AD) and/or determine progression of cognitive loss, wherein said four synaptic proteins are (a) presynaptic proteins neuronal pentraxin 2 (NPTX2) and neurexin 2 (NRXN2), and (b) postsynaptic GluA4-containing glutamate receptor (AMPA4) and neuroligin 1 (NLGN1), and wherein said NDE levels for each synaptic pair of (i) said NPTX2 and said AMPA4 and (ii) said NRXN2 and said NLGN1, serve as an indicator of onset of AD, and of progression of cognitive functional losses.

2. Method of determining risk of developing Alzheimer's disease (AD) or measuring cognitive losses to indicate stage of AD using exosomal biomarkers comprising (a) obtaining from a test subject and combining excitatory presynaptic proteins neuronal pentraxin 2 (NPTX2) and neurexin 2 (NRXN2), and postsynaptic excitatory partners GluA4-containing glutamate receptor (AMPA4) and neuroligin 1 (NLGN1), (b) quantifying plasma neuron-derived exosome (NDE) levels for each synaptic pair of (i) said NPTX2 and said AMPA4 and (ii) said NRXN2 and said NLGN1, to provide biomarkers correlating with cognitive function, (c) comparing said NDE levels obtained in (b) with subsequently obtained NDE levels from said test subject or with NDE levels obtained from one or more control subjects to determine levels of cognitive loss indicative of presence of AD or progression of AD dementia.

3. Method of predictive testing or staging testing of a person for Alzheimer's disease (AD) comprising (a) manipulating a venous blood sample of a test subject to obtain synaptic proteins of neuronal pentraxin 2 (NPTX2), GluA4-containing glutamate receptor (AMPA4), neurexin 2 (NRXN2), and neuroligin 1 (NLGN1) therefrom, (b) quantifying neuron-derived exosome (NDE) levels for each synaptic pair of (i) NPTX2 and AMPA4 and (ii) NRXN2 and NLGN1, (c) comparing said NDE levels of each said synaptic pair of (b) with NDE levels of synaptic pairs of (i) NPTX2 and AMPA4 and (ii) NRXN2 and NLGN1 obtained from the same test subject at a different point in time or obtained from a control subject known to not have cognitive losses, and based on differences in the NDE levels between the compared synaptic pairs, determining presence of or progression of cognitive loss of the test subject.

4. The method of claim 3, wherein the NDE levels of the test subject are lower than the NDE levels for said synaptic pairs of said control subject.

5. The method of claim 3, wherein the NDE levels for said AMPA4, said NLGN1 and said NRXN2 are each lower than a corresponding synaptic protein of said control subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIGS. 1A and 1B show the NDE levels of AMPA4 (FIG. 1A) and NPTX2 (FIG. 1B) in cross-sectional control and AD groups. Each point represents the value for a control subject or AD patient and the horizontal line in point clusters is the mean level for that group. MeanS.E.M. (S.E.M.=standard error of the mean) for control subject and AD patient values, respectively, are 2276180 pg/ml and 76668.0 pg/ml for AMPA4 and 2656343 pg/ml and 1250123 pg/ml for NPTX2. The significance of differences between values for control subjects and AD patients was calculated by an unpaired Students' t test; *=p<0.01 and **=p<0.0001.

[0008] FIGS. 2A and 2B show NDE levels of NLGN1 (FIG. 2A) and NRXN2 (FIG. 2B) in cross-sectional control and AD groups. Each point represents the value for a control subject or AD patient and the horizontal line in point clusters is the mean level for that group. MeanS.E.M. for control subject and AD patient values, respectively, are 189,49821,106 pg/ml and 33,1553305 pg/ml for NLGN1 and 83,3749132 pg/ml and 23,9302057 pg/ml for NRXN2. The significance of differences between values for control subjects and AD patients was calculated by an unpaired Students' t test; **=p<0.0001.

[0009] FIGS. 3A and 3B show correlations between NDE contents of excitatory synaptic proteins and cognitive function of AD patients in the cross-sectional set. Each point depicts the levels for one AD patient. The respective p values are 0.0020 for FIG. 3A and 0.0001 for FIG. 3B.

[0010] FIGS. 4A-4D show courses of decline in NDE levels of excitatory synaptic protein cargoes with worsening AD. Each point represents the value for a control subject or AD patient and the horizontal line in point clusters is the mean level for that group. MeanS.E.M. control subject, AD1 (preclinical) patient and AD2 (when dementia was diagnosed) patient values, respectively, are for FIG. 4A 169,87015,535, 58,7215451 and 15,8751405 pg/ml for NLGN1; for FIG. 4B 81,9689,306, 39,2422832 and 21,1501535 pg/ml for NRXN2; for FIG. 4C 2210188, 1476127 and 68644.1 pg/ml for AMPA4; and for FIG. 4D 2488281, 2195175 and 133076.2 pg/ml for NPTX2. The significance of differences between values for control subjects and AD1 patients was calculated by an unpaired Students' t test and for differences between values for AD1 and AD2 patients was calculated by a paired Students' t test; *=p<0.01 and **=p<0.0001.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Interactions of presynaptic proteins, neuronal pentraxin 2 (NPTX2) and neurexin2 (NRXN2) with respective postsynaptic functional partners GluA4-containing glutamate receptor (AMPA4) and neuroligin 1 (NLGN1) enhance or strengthen excitatory synaptic activities in areas of the hippocampus and cerebral cortex. The advantages of the invention involve the quantification of four synaptic protein mediators of selective excitatory neuronal impulses, where decreases in their levels predict very early the risk of development of AD, and permit easy repetitive staging of the severity of the neurodegeneration in AD by a simple blood-based test. As early damage of such excitatory circuits in AD correlates with cognitive losses, plasma neuron-derived exosome (NDE) levels of two pairs of these synaptic proteins are quantified and used as biomarkers for prediction and staging of severity of AD. NDE contents of all four synaptic proteins decrease significantly in AD dementia and diminished levels of AMPA4 and NLGN1 correlate with the extent of cognitive losses (FIGS. 1-3; total number of AD patients in both sets is 46). Prior to the onset of clinically evident dementia, NDE levels of all but NPTX2 are significantly lower than those of matched control subjects (FIG. 4, AD1) and levels of each of these synaptic proteins decline significantly with the development of dementia (FIG. 4, AD2). Reductions in NDE levels of these excitatory synaptic proteins are indicative of the extent of cognitive losses that have occurred and reflect the progression of severity of AD.

[0012] Testing and Patient Evaluation.

[0013] For the Cross-Sectional studies, 28 patients with early AD were retrospectively identified who had been evaluated extensively in the Clinical Research Unit of the U.S. National Institute on Aging (NIA, Baltimore, Md., USA) and 28 age- and gender-matched cognitively normal control subjects who had donated blood at the Jewish Home of San Francisco (JHSF) in the same time period as the patients (see Table 1(a) below). For the Longitudinal studies described herein, three patients were identified from the University of Kentucky Sanders-Brown Center on Aging and 15 patients from JHSF with moderate AD who had provided blood at two times: first when cognitively intact (AD1 in Table 1(b) below) and, secondly, 5 to 11 years later after diagnosis of dementia (AD2 in Table 1(b) below). Plasmas from 18 cognitively normal control subjects who were age- and gender-matched with the AD1 group were found at JHSF, that had been obtained in the same time period. All plasmas were identified, obtained and stored by the same methods and all plasmas were processed together with the same procedures by the same investigator. Plasmas from patients in the Longitudinal studies were analyzed without knowledge of the clinical data.

TABLE-US-00001 TABLE 1 Characteristics of AD Patients and Control Subjects Total Ages MMSE ADAS-cog Diagnosis Number Male/Female (Means S.E.M.) (Means S.E.M.) (Means S.E.M.) (a) Cross-Sectional Sets C 28 12/16 73.2 1.47 29.7 0.13 3.32 0.31 AD 28 12/16 73.1 1.44 25.6 0.83* 13.7 1.31* (b) Longitudinal Sets C 18 10/8 70.1 1.66 28.3 0.96 3.68 0.45 AD1 18 10/8 69.4 1.71 28.7 0.47 4.19 0.57 AD2 18 10/8 78.2 1.75 20.0 1.50* 17.6 1.64* C = Matched control subjects for respective study set. AD = Patients diagnosed with Alzheimer's disease. AD1 and AD2 are the groups of AD patients evaluated at two different times in the Longitudinal study, i.e., at a pre-clinical phase (AD1) and after conversion to moderate dementia (AD2), respectively. In the Longitudinal set, C is the control subjects matched to the AD1 patients. MMSE = Mini-Mental State Examination. ADAS-cog = AD assessment scale-cognitive subscale. The significance of differences between cognitive state (MMSE, ADAS-cog) values of the groups were calculated by an unpaired t test for C vs. AD in (a) and for C vs. AD1 in (b), and by a paired t test for AD1 vs. AD2 in (b). *= p < 0.001.

[0014] Patients with AD had mental status testing at the time of each blood sampling. Mini-mental state examination (MMSE) and the AD assessment scale-cognitive subscale (ADAS-cog) were conducted as described in The ADAS-cog in Alzheimer's disease clinical trials: psychometric evaluation of the sum and its parts, J. Neurol. Neurosurg. Psychiatry, Cano, S. J. et al., (2010) 81, 1363-1368 (which in its entirety is incorporated herein by reference). Cross-Sectional study patients from the NIA had amnestic mild cognitive impairment (MCI) or mild dementia from AD with high probability of AD and a Clinical Dementia Rating global score of 0.5 or 1.0 according to the Petersen and Dubois criteria as described in (1) Mild cognitive impairment as a diagnostic entity, J. Intern. Med., Petersen, R. C. (2004) 256, 183-194 and (2) Toward defining the preclinical stages of Alzheimer's disease: recommendation from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease, Alzheimer's Dement., Sperling, R. A. et al. (2011) 7, 280-292 (each of the above articles (1) and (2) being in its entirety incorporated herein by reference). AD2 patients from JHSF and the University of Kentucky had probable AD and mild-to-moderate dementia by NINCDS-ADRDA criteria and had a Clinical Dementia Rating global score of 1.0 at the time of the second blood collection as described in Research Criteria for the diagnosis of Alzheimer's disease: revising the NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and StrokeAlzheimer's Disease and Related Disorders Association) criteria, Lancet Neurol., Dubois, B. et al. (2007) 6, 734-746 (which in its entirety is incorporated herein by reference). All Cross-Sectional study patients with AD had abnormal cerebrospinal fluid (CSF) levels of amyloid -peptide (A) 1-42 and P-T181-tau that supported their diagnosis as described in Cerespinal fluid biomarker signature in Alzheimer's disease neuroimaging initiative subjects, Ann. Neurol., Shaw, L. M. et al. (2009) 65, 403-413 (which in its entirety is incorporated herein by reference).

[0015] Blood and CSF sampling of patients and control subjects.

[0016] Ten ml of venous blood were drawn into 0.5 ml saline with EDTA (ethylene diamine acetic acid), incubated for 10 min at room temperature, and centrifuged for 15 min at 2500 g. Plasmas were stored in 0.25 ml aliquots at 80 C. CSF levels of P-T181-tau and A 1-42 were quantified by xMAP Technology (Luminex Corp., Austin, Tex., USA) using Inno-Bia AlzBio3 kits (Innogenetics, Ghent, Belgium).

[0017] Enrichment of plasma neuron-derived exosomes (NDEs) for extraction and ELISA (enzyme-linked immunosorbent assay) quantification of proteins.

[0018] Aliquots of 0.25 ml plasma were incubated with 0.1 ml thromboplastin D (Thermo Fisher Scientific, Waltham, Mass., USA), followed by addition of calcium- and magnesium-free Dulbecco's balanced salt solution with protease inhibitor cocktail (Roche, Indianapolis, Ind., USA) and phosphatase inhibitor cocktail (Thermo Fisher Scientific). After centrifugation at 3000 g for 30 min at 4 C., NDEs were harvested from resultant supernatants by sequential ExoQuick (System Biosciences, Mountain View, Calif., USA) precipitation and immunochemical enrichment with mouse antihuman CD171 (L1CAM neural adhesion protein) biotinylated antibody (clone 5G3; eBiosciences, San Diego, Calif., USA) as described in (1) Decreased synaptic proteins in neuronal exosomes of frontotemporal dementia and Alzheimer's disease, Faseb J., Goetzl, E. J., et al. (2016) 30, 4141-4148, (2) Dysfunctionally phosphorylated type 1 insulin receptor substrate in neural-derived blood exosomes of preclinical Alzheimer's disease, Faseb J., Kapogiannis, D., et al. (2015) 29, 589-596, and (3) Plasma Extracellular Vesicles Enriched for Neuronal Origin: a Potential Window into Brain Pathogenic Processes, Front. Neurosci., Mustapic, M. et al. (2017) 11, 278 (each of the above articles (1), (2) and (3) being in its entirety incorporated herein by reference). M-PER mammalian protein extraction reagent (ThermoFisher Scientific) lysates of NDEs, that contained protease and phosphatase inhibitors, were stored at 80 C.

[0019] NDE proteins were quantified by ELISA kits for human tetraspanning exosome marker CD81 (American Research Products-Cusabio, MA, USA), neuronal pentraxin 2 (NPTX2) and the GluA4 subunit of AMPA-type glutamate receptors (AMPA4) (American Research Products-Cloud Clone Corp.), and neurexin 2 (NRXN2alpha) and neuroligin 1 (NLGN1) (American Research Products-QAYEE-BIO).

[0020] The mean value for all determinations of CD81 in each assay group was set at 1.00 and relative values of CD81 for each sample were used to normalize their recovery.

[0021] Statistical Analysis.

[0022] A Shapiro-Wilks test showed that data in all sets were distributed normally. Statistical significance of differences between means for the Cross-Sectional study groups AD and C, and between the Longitudinal study groups AD1 and C were determined with an unpaired Student's t test, including a Bonferroni correction, and the significance of differences between means for the Longitudinal groups AD1 and AD2 were determined with a paired Student's t test (Prism 6; GraphPad Software, La Jolla, Calif., USA). Relationships between NDE content of a cargo synaptic protein and the corresponding cognitive level of an AD patient were evaluated by Pearson Correlation Coefficients.

[0023] Results.

[0024] AD patients in the Cross-Sectional study had cognitive scores consistent with mild cognitive impairment or mild dementia that were significantly different from the normal range of scores for the control subjects (see Table 1(a) above). The Longitudinal study subjects evaluated initially at their AD1 pre-clinical phase had normal cognitive scores which were no different than those of their matched control group (see Table 1(b) above). At the time of donation of the second blood sample, the Longitudinal study group was termed AD2 and had mild to moderate dementia and significantly worse cognitive scores than when they were at the AD1 phase.

[0025] NDE levels of both synaptic proteins of the two sets were significantly lower than those of the matched control subjects (see FIGS. 1A-1B and FIGS. 2A-2B). Values for the NLGN1-NRXN2 pair were much higher than values for the AMPA4-NPTX2 pair and showed much less overlap with the values of the control subjects. For NLGN1 and NRXN2, respectively, only four and five control subject values were in the range of those for AD patients (see FIGS. 2A-2B). There were significant inverse correlations between ADAS-cog scores and NDE levels of AMPA4 and NLGN1, but not of NPTX2 or NRXN2 (see FIGS. 3A-3B). Similarly, there were significant positive correlations between MMSE scores and NDE levels of AMPA4 (r=0.621; p=0.0004) and NLGN1 (r=0.525; p=0.0053), but not of NPTX2 or NRXN2.

[0026] For the Longitudinal series of AD patients in the AD1 preclinical phase, NDE levels of AMPA4, NLGN1 and NRXN2, but not NPTX2, were significantly lower than those of the matched control subjects (see FIGS. 4A-4D). At the AD2 stage of mild to moderate dementia five to 11 years later, NDE levels of all four synaptic proteins had decreased significantly for the group and in every patient compared to their levels at the AD1 phase.

[0027] The methods described herein permit quantification of meaningful levels of both members of the two sets of excitatory synaptic proteins and demonstration of significant differences between levels in AD patients and control subjects as well as between pre-clinical and clinically apparent stages of AD (see FIGS. 1A-1B, 2A-2B, and 4A-4D). There are four primary differences between the invention described herein and findings for the group of broadly distributed synaptic proteins described in the Background of the Invention above.

[0028] The first is distinctive functions in specific excitatory synapses of the hippocampus and areas of the cerebral cortex. Presynaptic complexes that include NPTX2 are secreted into excitatory synapses of pyramidal neurons of the hippocampus and cerebral cortex, bind specifically with the AMPA4 and thereby mediate enhanced synaptic transmission in these circuits. Presynaptic NRXN2 and the postsynaptic adhesion protein NLGN1 interact trans-synaptically in these excitatory synapses of the hippocampus and cerebral cortex to also ensure structural stability and enhanced synaptic functions.

[0029] The second distinguishing feature of these two protein pairs is their localization in areas that are affected very early in A where the NRXN2-NLGN1 pair also may contribute directly to pathogenesis through binding and selective concentration of neurotoxic oligomers of A peptides, such as A1-42 as described in Interaction of amyloid-beta (Abeta) oligomers with neurexin-2-alpha and neuroligin 1 mediates synapse damage and memory loss in mice, J. Biol. Chem., Brito-Moreira J. et al. (2017) 292, 7327-7337.

[0030] The third difference between two of these functionally excitatory synaptic proteins and numerous other NDE cargo proteins implicated in AD is the correlation between cognitive scores and the levels of AMPA4 and NLGN1 (see FIGS. 3A-3B). This type of correlation, that suggests value for NDE levels of these proteins as staging indicators of AD clinical severity, is shared only by the more broadly distributed synaptic proteins synaptopodin, synaptotagmin and synaptophysin but not by a wide range of other NDE cargo proteins.

[0031] Finally, the fourth distinguishing feature of these two synaptic protein pairs is a striking progressive decrease in all of their NDE levels as patient clinical status declines from normal cognition in the pre-clinical stage to dementia with overt AD (see FIGS. 4A-4D). This progressive reduction in NDE level with declining clinical status was seen only for synaptotagmin, GAP43 and to a much lesser extent for synaptopodin, but not for synaptophysin or neurogranin of the more broadly distributed set of synaptic proteins.

[0032] Without being bound by theory, diminished NDE levels of cargo proteins may reflect lower neuronal concentrations or less efficient loading of some proteins into NDEs as the disease progresses. There also is potential involvement of greater post-loading proteolysis or distribution in the CNS.

[0033] Additional test data is set forth in Table 2 as to a clinical time-course of appearance of AD-associated abnormal proteins in plasma NDEs.

TABLE-US-00002 TABLE 2 Years before dementia AMPA4 NPTX2 NLGN1 NRXN2 Alzheimer's 681 42.1** 1324 72.3** 15,618 1353** 20,822 1488** diagnosis (n = 19) 5-8 yrs (n = 19) 1105 90.8** 1800 117* 34,763 2586** 28,240 1638* 12-14 yrs (n = 14) 1523 162* 2144 203* 63,695 6283* 52,193 5287* 18-20 yrs (n = 8) 2051 140** 2697 274* 108,147 11,777 61,094 7018.sup.ns Healthy controls 2811 118 3698 217 166,917 14,988 80,305 8958 (n = 19) Years before dementia P-T181-tau REST Synaptophysin Synaptopodin Alzheimer's 196 4.85* 96.2 4.01** 259 21.4** 1301 76.8** diagnosis (n = 19) 5-8 yrs (n = 19) 176 7.57** 308 18.1** 3974 542** 2609 193** 12-14 yrs (n = 14) 119 6.01* 576 43.7.sup.ns 9407 590 4838 335.sup.ns 18-20 yrs (n = 8) 83.0 3.99.sup.ns 768 72.1.sup.ns 13,523 994.sup.ns 4522 585.sup.ns Healthy controls 92.0 3.83 625 50.6 13,070 1190 4916 341 (n = 19)

[0034] Table 2 is a clinical time-course of appearance of AD-associated abnormal proteins in plasma NDEs. Each value is the mean pg/mlS.E.M. in plasma NDE extracts of 19 healthy controls matched by age and sex with 19 patients diagnosed with AD by conventional criteria, and in NDE extracts of 19, 14 and 8 of the same patients from plasmas obtained, respectively, 5 to 8 years, 12 to 14 years and 18 to 20 years before their diagnosis of AD. The level of significance of differences between mean values relative to mean values from the next earlier time period (paired t test) or for the 18 to 20 year group relative to mean values for the matched healthy controls (unpaired t test) are shown by the symbols: **=p0.0001, *=p0.01, =p0.05 and ns=not significant. AMPA4, GluA4-containing glutamate receptor, NPTX2, neuronal pentraxin 2; NLGN1, neuroligin 1; NRXN2, neurexin 2; REST, RE1-silencing transcription factor.

[0035] Plasma mean NDE levels of the two pre-synaptic proteins, NPTX2 and NRXN2, and two post-synaptic proteins, AMPA4 and NLGN1, of human excitatory brain pathways affected in AD are significantly lower at each time point measured than at the earlier time point leading up to the time of clinical diagnosis of AD. For three of the proteins, but not for NRXN2, the mean NDE level 18 to 20 years before diagnosis of AD is significantly lower than the corresponding NDE level of matched healthy controls. Thus, progressively decreasing plasma NDE levels of these synaptic proteins reflect the increasing severity of preclinical CNS damage by AD, that is already detectable at 18 to 20 years before clinical diagnosis of AD. In contrast, none of the plasma NDE mean levels of the other four recognized biomarkers of AD is distinguished from those of matched healthy controls at 18 to 20 years before clinical diagnosis of AD. Further, only the plasma NDE levels of P-T181-tau and synaptophysin are significantly different at 12 to 14 years than at 18 to 20 years before clinical diagnosis of AD, whereas these differences are significant for all four of the synaptic proteins of the brain excitatory pathways. Thus, these synaptic proteins of human excitatory brain pathways are shown to be the earliest available blood-based biomarkers of pre-clinical AD in living humans. The ability to provide a determination in a living human being of risk or likelihood of AD in the living human being and to determine the progression of AD and cognitive loss is significant for providing early treatment of AD to slow or delay progression of the disease.

[0036] The exemplary embodiments herein disclosed are not intended to be exhaustive or to unnecessarily limit the scope of the invention. The exemplary embodiments were chosen and described in order to explain the principles of the present invention so that others skilled in the art may practice the invention. As will be apparent to one skilled in the art, various modifications can be made within the scope of the aforesaid description. Such modifications being within the ability of one skilled in the art form a part of the present invention and are embraced by the appended claims.